Hybridoma marked m, and a monoclonal antibody secreted by this hybridomas


(57) Abstract:

The invention relates to biotechnology and immunology, and can be used in immunohistochemical analysis to determine the localizationd. Hybridoma obtained by immunization with purified hamster mousedand transfectional mousedCho cells with the subsequent merger of silanation with NS-1 myeloma cells and screening of target cells. Hybridoma produces a monoclonal antibody reactive with a-subunit of rat integrin (d). The invention allows to determine the localizationdin the tissues. 2 C. p. F.-ly, 4 Il., table 1.

This application is a continuation of part application of U.S. No. 08/362652, filed December 21, 1994, which is pending, which is a continuation of part application of U.S. N 08/286889, filed August 5, 1994, which published as U.S. patent 5470953 dated November 28, 1995, which in turn is a continuation of part application of U.S. N 08/173497, filed on December 23, 1993, which published as U.S. patent 5437958 from August 1, 1995.

The present invention relates to the cloning and expression of polynucleotides encoding a new human2integranova subunit, denoted by d, CD11b, and CD11c. The present invention also relates to polynucleotides isolated from other species that exhibit homology with human coding sequences.

Integrins are a class of membrane-associated molecules that are actively involved in cell adhesion. Integrins are transmembrane heterodimeric, including subunit in non-covalent connection with the subunit. Up to this time have been identified at least fourteen subunits and eight subunits [Review Springer, Nature 346:425-434 (1990)] . Usually subunit are able to associate with more than one subunit, and heterodimer swap common subunit, were classified as subselect inside integranova population.

One class of human integrins, which hinders the expression in white blood cells, characterized by total2subunit, as a result of this cell-specific expression of these integrins is generally referred to as leucocythemia integrins, Leu-CAMs or LaMontagne. Because of the General2subunit alternative designation of this class represents a2integrins. 2subunit (CD18) was selected earlier in the Association is t.al., Cell 48: 681-690 (1987). The official WHO nomenclature heterodimeric proteins belong to the CD11a/CD18, CD11b/CD18 or CD11C/CD18; in the normal range they belong to LFA-1, Mac-1 or Mol and R or Leu M5, respectively [Cobbold, refer to LFA-1, Mac-1 or Mol and R or Leu M5, respectively [Cobbold, et.al., in Leukocyte Tiping 111, McMichael (ed), Oxford Press, p.788 (1987)]. It was demonstrated that human2integranova subunits, CD11a, CD11b, and CDHc, migrate in terms of recovery electrophoresis with facile molecular weights of approximately 180 kD and 155 kD and 150 kD, respectively, and were cloned DNA-t encoding these subunits [CD11a, Larson, et.al., J. Cell Biol. 108: 703-712 (1989); CD11b, Corbi, et.al., J. Biol.Chem. 263: 12403-12411 (1988) and CD11c Corbi, et.al. EMBO J. 6:4023-4028 (1987)]. Putative homologues of human2integranova and circuits that are defined by using the approximate similarities in molecular weight, were previously identified in other species, including monkeys and other primates [Letvin, et.al., Blood 61:408-410 (1983)], mice [Sanchez-Madrid, et.al., J. Exp.Med. 154:1517 (1981)] and dogs [Moore, et.al., Rissue Antigens 36:211-220(1990)],

It was shown that the absolute molecular weight homologs from other species varied significantly [see, for example, Danilenko et.al., Tissue Antigens 40:13-21 (1992)], and the lack of consistent information on the, certificirovannim in other species. However, between different types of observed changes in the number of elements in the protein family. Suppose, for example, that has been allocated more IgA isotopes in rabbits than in humans [Burnett, et. al., EMBO J. 8:4041-4047 (1989) and Schneiderman, et. al. , Proc.Natl.Acad.Sci (USA) 86:7561-7565 (1989)]. Similarly, people were previously identified at least six options metallothionine protein [Karin and Richards, Nature 299: 797-802 (1982) and Varshney, et. al., Mol.Cell.Biol, 6:26-37, (1986)], while the mice are in the presence of only two such variants [Serle, et.al, Mol.Cell.Biol. 4: 1221-1230 (1984)]. Therefore, the existence of many elements of the protein family in one form does not necessarily imply that the relevant elements of the family exist in other species.

In a particular context2integrins in dogs was observed that mostly dog2the part opposite to human CD18, is capable of forming a dimer with such a large number of subunits, four potentially distinct subunit [Danilenko, et.al., in print]. Antibodies generated impunitivnoj mouse with dog splenocytes led to monoclonal antibodies which have immunoprecipitate proteins, temporarily designated as canine homologues in human splenocyte antibody San, learned and immunoprecipitates fourth link dog subunit, is also capable of assotsiatsii 2subunit, but has a unique molecular weight and restrictionon in expression in subnasale differentiated tissue macrophages.

Antibodies generated by the immunization of hamsters murine dendritic cells, resulted in two antiintegrin antibody [Metlay, et.al., J. Exp. Med. 171: 1753-1771 (1990)], One antibody, E, immunoprecipitation mostly heterodimer with subunits having an approximate molecular weight of 180 kD and 90 kD, in addition to small bands in the region of molecular weight of 150-160 kD. The second antibody, N418, precipitable other observed heterodimer, having an approximate molecular weight of 150 kD and 90 kD. Based on the studies by blocking cell adhesion has been hypothesized that the antibody HE learns mouse part, the opposite of human CD18. While the molecular weight N418 antigen confirmed the recognition of the murine homologue of human CD11c/CD18, further analysis showed that murine antigen detects the distribution pattern of the fabric, which is not consistent with that observed for human CD11/CD18.

the example, glycosylation or splice variant) of a previously identified canine or murine subunit. Or these antigens may represent a unique dog and mouse integranova subunit. In the absence of specific information relating to the primary structure, there can be a distinction among these alternatives.

People CD11a/CD18, is expressed on all leukocytes. CD11b/CD18 and CD11/CD18 limited essentially in the expression on monocytes, granulocytes, macrophages cells and natural killer (NK), but CD11/CD18 found on certain types of b-cells. In General, CD11a/CD18 is dominated by lymphocytes, CD11b/CD18 on granulocytes and CD11/CD18 on macrophages [see review Arnaout, Blood 75:1037-1050 (1990)]. The expression of the circuits, however, is variable relative to the state of activation and differentiation of individual cell types [see review, Larson and Springer, Immunol. Rev. 114:181-217(1990)].

The inclusion of integrins in human immune and inflammatory responses has been demonstrated with the use of monoclinal antibodies, which are capable of blocking adhesion2integrin-associated cells. For example, CD11a/CD18, CD11b/CD18 and CD11/CD18 are actively involved in the binding of the natural is [Nourshargh, et.al., J. Immunol. 142:3192-3198 (1990)], granulocyte-independent leakage of plasma [Arford, et.al., in the print], and leukocyte adhesion to the vascular endothelium [Price, et.al., J. Immunol 139:4174-4177 (1987) and Smith, et.al., J. Clin.Invest. 83: 2008-2017 (1989)]. The fundamental role2integrins in immune and inflammatory reactions becomes apparent in the clinical syndrome referred to as leukocyte adhesion deficiency (LAD), where clinical manifestations include recurrent and often life-threatening bacterial infection. LAD is a consequence of heterogeneous mutations in the subunit [Kishimoto, et.al., Cell 50:193-202 (1987)] and the severity of the disease is proportional to the degree of deficiency in expression 2subunit. Education full integranova of heterodimer weakened2mutation [Kishimoto, et.al., above].

Interestingly, it was shown that at least one antibody specific for CD18, inhibits human immunodeficiency virus type 1 (HIV-1) syncytium formation in vitro, although the exact mechanism of this inhibition is unclear [Hildreth and Orentas, Science 244:1075-1078 (1989)]. This observation is consistent with the discovery that the fundamental protivorechitto CD11a/CD18, SAM-1, is a surface receptor for a large group rhinoviral serotypes [Greve, e is liteline reactions emphasizes the need to develop a more complete understanding of this class of surface proteins. Identification of yet unknown elements of this subselect and their protivorechathie and generation of monoclonal antibodies or other soluble factors that can alter the biological activity2integrins, will provide a practical way for therapeutic intervention in2integrin-associated immune and inflammatory responses.

In one aspect the present invention provides a new purified and isolated polynucleotide (e.g., DNA and RNA transcripts, both types of coded and non-coding strands) encoding a new human2integranova subunit, dand their variants (i.e., deletion analogs of merger or substitution), which have a binding and/or immunological properties inherent ind. Preferred DNA molecules of the invention include cDNA, genomic DNA, and fully or partially synthesized DNA molecules. Now the preferred nucleotide DNA is continue as SEQ ID NO: 1, encoding the polypeptide SEQ ID NO:2, is also Provided recombinant plasmid and viral DNA constructs (expression design), which includedcoding sequences, wheredID the th regulatory element or elements.

The present invention also provide for the separation and purification of mouse and rat polynucleotide, which show homology to polynucleotides coding for human d. Preferred murine polynucleotide seems to continue in SEQ ID NO:52, and the preferred rat polynucleotide is to continue in SEQ ID NO:54.

As another object of the invention to provide a prokaryotic or eukaryotic cell hosts, tranformirovanie or transfection DNA sequences of the invention that expressyourdpolynucleotide or its variants. Cells-the hosts of the invention are particularly useful for large scale production.dpolypeptide, which can be allocated either from the host cell or from the medium in which it is grown a host cell. Cell owners who expressyourdthe polypeptide on their intracellular membrane surface are also useful as immunogens in productiond- specific antibodies. Preferably, the cells of the host, transfectiondwould cotransfection with akrasia2integranova subunit to allow Poveradpolypeptides, fragments and variants. Preferreddthe polypeptide appears to continue in SEQ ID NO:2. Newdthe products of the invention can be obtained in the form isolated from natural sources, but similar productsdvariants are preferably produced using recombinant procedures, including cells-the hosts of the invention. Fully glycosylated, partially glycosylated and fully deglycosylated formdthe polypeptide can be generated by modifying the host cell selected for recombinant production and/or processing after the selection. Optionsdpolypeptides of the invention may include water-soluble and not soluble in waterdpolypeptides, including analogues, where one or more amino acids are delegated or replaced: 1) without loss, and preferably with enhancement of one or more of the biological activities or immunological characteristics specific ford; or 2) with the specific failure of a specific ligand/receptor function binding or signalizovania. Also provide a composite polypeptide, wheredamino acid sequence expressyou have modified biological, biochemical and/or immunological properties compared withdwild-type. Considered the analogue of the polypeptide with additional amino acid (e.g. lysine or cysteine) residue, which facilitates the formation of multimer.

The present invention also addresses the polypeptides and other ones of molecules that are specifically associated withd. Preferred binding molecules include antibodies (e.g. monoclonal or polyclonal antibodies), protivorechathie (for example, membrane-associated and soluble forms) and other ligands (e.g., molecules of natural origin or synthetic molecules), including those ligands that competitive binddin the presence of admonoclonal antibodies and/or specific protivorechathie. Binding molecules are useful for cleaningdpolypetides and identification of cell types, which Expressd. Binding molecules are also useful for modulating (i.e., inhibiting, blocking or stimulating) in vitro binding or signal transduction activitiesd.

Provided also tests for identificat the participating solutions scintillation proximal analyses, di-hybrid srinilaya analyses and the like.

The in vitro tests to identify antibodies or other compounds that modulate the activitydmay include, for example, immobilization dor the natural ligand, which bindsdfindable tagging neimmunizirovannah binding partner, incubating the binding partners together and determining the effect of test compounds on the number of related tags, where the reduction in the associated label in the presence of the test compound compared to the amount of bound label in the absence of the test compound indicates that the test agent is an inhibitor ofdbind.

Another type of analysis for the identification of compounds that modulate aimogasta betweendand the ligand includes immobilizationdor its fragment on a solid substrate (or impregnated agent) coated with a fluorescent agent, labelling the ligand compound capable of exciting the fluorescent agent, contacting the immobilized dwith labeled ligand in the presence and absence of the alleged modulating seeding connection which affects the emission light of the fluorescent agent in comparison with the emission light of the fluorescent agent in the absence of a modulating compound. Ordthe ligand can be immobilized, anddcan be labeled in the analysis.

The invention is another method for identifying compounds that modulate the interaction betweendand ligand, including the transformation or transfection of appropriate host cells design DNA, containing the reported gene, under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, Express in cells of the host's first hybrid DNA sequence encoding a first component part or alldand either the DNA binding domain or activating domain of the transcription factor, which is not introduced in the first hybrid, assess the impact of the proposed modulating compounds on the interaction betweendand the ligand by detecting binding of the ligand with the second hybrid DNA sequencedin a specific cell host by measuring the production of the reported gene product in the absence of modulating the GaLI4 TRANS-activation domain, LacZ reported gene and the yeast cell is the master.

A modified version of the above analysis can be used in the selection of polynucleotide encoding a protein that binds ddue to the transformation or transportirovaniia appropriate host cells design DNA, including the reported gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expression in cells of the host's first hybrid DNA sequence that encodes a first component part or alldand either the DNA binding domain or activating domain of the transcription factor, expression in cells of the owners of the library of second hybrid DNA sequence encoding a second hybrid part or all of the allegeddbinding proteins and the DNA binding domain or activating domain of the transcription factor, which is not introduced in the first hybrid detection linkdbinding proteindin a specific cell host by detecting the production of the reported gene product in the cell host, and the selection of the second hybrid DNA sequences encodingdbinding protein from a particular also discusses invention. Methods of production of hybridomas that secrete monoclonal antibodies are well known to specialists in this field. Hybridoma cell line can be generated after immunization of an animal treateddwaysdor cells which Expressdor its variants on the extracellular membrane surface. Immunogenic cell types include cells that Express adin vivo, or transfection prokaryotic or eukaryotic cell line that normally are not exposed to normal expressiondin vivo. Preferred antibodies of the invention now are secreted by the hybridomas designated A, 169C, 170D, 170F, T, H, N, A, V, S, E, 188F, 188G, 188I, K, 188L, M, 188N, R, 188R, T, A, S, 195D, E, N, A-1, A-2, A-3, A-4, A and M.

Detected value of the information entered by disclosure of the DNA and amino acid sequenced. In one episode of the examples discloseddCDNA sequence makes possible the allocation of human dthe genomic DNA sequence comprising transcriptionally controls for genomic sequences. The author also discusses the identificationdRomney DNA and DNA-t heterologous species may be achieved by standard techniques of DNA/DNA hybridization with strict conditions, using all or part of thedcDNA sequence as a probe for screening of the entire library. Or the polymerase chain reaction (PCR) using oligonucleotide primers that are designed based on the known cDNA sequence may be used to amplify and identify genomicdcDNA sequences. Synthetic DNA you, encodingdpolypeptide, including fragments and other variants can be obtained by conventional synthetic methods.

Information about the DNA sequences of the invention also makes possible the development, through homologous recombination or strategies "knockout" [see, for example, Kapecchi, Science 244:1288-1292 (1989)], to obtain rodents that fail to Express functionaldthe polypeptide or that Express a variantdthe peptide. These rodents are useful as models for studying activitiesdanddmodulators in vivo.

DNA and amino acid sequences of the invention also make possible the analysisdepitopes that are actively involved in protivorechathie binding, as well as epitopes, which are most likely the regulation of the th signal transduction, also being considered by the invention.

DNA of the invention is also useful for the detection of cell types that expressyourdthe polypeptide. Standard techniques of DNA/RNA hybridization, which usedDNA for detection dRNA can be used to identify thedtranscription within cells, as well as changes in the level of transcription in response to internal or external agents. Identification of agents that modify transcription and/or translationdcan be evaluated for potential therapeutic or prophylactic purposes. DNA of the invention also makes possible in situ hybridizationdDNA in cellular RNA to determine the cellular localizationdspecific transfers within complex cell populations and tissues.

DNA of the invention is also useful for identifying inhuman polynucleotide sequences which show homology to human dthe sequences. The possession of superhumandDNA sequences allows for the development of animal models (including, for example, transgenic fashion is or polyclonal antibodies, specificdthat can be used in immunohistochemical analysis for localizationdin subcellular provisions or individual cells within tissues. Immunohistochemical analyses of this type are particularly useful when used in combination with in situ hybridization for localization anddmRNA and polynucleotide productsdGena.

Identification of the cell types that Expressdcan have significant spur for the development of therapeutic or preventive agents. It is assumed that the products of the invention, referred todcan be used in the treatment of diseases where macrophages are an essential element of the disease process. Animal models for many pathological conditions associated with makropoulou activity, have been described in the literature. For example, in mice mikrofalowe refill in sites of chronic and acute inflammation was reported Jutila, et. al., J. Leucocyte Biol. 54:30-39 (1993). In rats Adams, et.al.[Transplantation 53:1115-1119 (1992) and Transplantation 56: 794-799 (1993)] describes a model for the transplanted arteriosclerosis after heterotrophy abdominal cardiac holographically transplantation. Rosenfeld, et.al., [Arteriosclerosis 7:9-23 is anitelea cholesterol. Henenberg, et. al., [Diabetologia 32:126-134 (1989)] reported spontaneous development of insulin-dependent diabetes in BB rats. Yamada, et.al. [Gastroenterology 104: 759-771 (1993)] have described-induced inflammatory bowel disease, chronic granulomatosis colitis rats after injection of streptococcal peptidoglycan-polysaccharide polymers. Cromartie, et.al. [J. Exp. Med. 146: 1585-1602 (1977] and Schwab, et.al., [Infection and Immunity 59:4436-4442 (1991)] reported that injection of rats protein streptococcal cell wall resulted in the arthritic condition characterized by inflammation of the peripheral joints and the subsequent destruction of the joint. Finally, Huitinga, et.al. [Eur. J. Immunol., 23:709-715, (1993)] described experimental allergic encephalomyelitis, a model for multiple sclerosis in rats Lewis. In each of these modelsdantibodies, otherdbinding proteins or soluble formdthe attenuation of the status of the disease, mainly due to the inactivation makropoulou activity.

The invention is provided a pharmaceutical composition for the treatment of these and other disease States. Pharmaceutical compositions are prepared with the purpose of inhibiting the interaction betweendand its ligand(s) and include various soluble the intracellular or extracellular modulatorsdbinding activity, and/or modulators dand/ordthe ligand polypeptide expression, including modulators of transcription, translation, post-translational processing and/or intracellular transport.

The invention also deals with the treatment of disease States in which implicit dbinding or localized accumulation of cells that expressyourdwhere a patient suffering from said disease state, provided the amount of the pharmaceutical composition of the invention sufficient to modulate levelsd, binding, or modulating the accumulation of cell types, which Express d. The method of treatment according to the invention is suitable for the conditions of the disease, such as, but not limited to, diabetes type 1, atherosclerosis, multiple sclerosis, asthma, psoriasis, pneumonia, acute respiratory disorders and rheumatoid arthritis.

Numerous other aspects and advantages of the present invention will be apparent upon consideration of the following description, with reference to the drawings, where:

Fig. 1A - 1D include the balance of human aminokislot is seen in the following examples, concerning the allocation of cDNA clone encodingdfrom a human spleen cDNA library. More specifically, Example 1 illustrates the use of a dogTM1antibodies in the attempt to discover the homologous human protein. Example 2 details the cleaning dogTM1and N-terminal sequencing of the polypeptide to design oligonucleotide primers for PCR amplification dogTM1the gene. Example 3 is directed to large-scale purification dogTM1for internal sequencing to construct additional PCR primers. Example 4 describes the use of PCR and internal primers sequences for amplification of fragment dogTM1the gene. Example 5 is directed to the cloning of a humand-coding cDNA sequence. Example 6 describes hybridization analysis Northern blotting of human tissue and cells for expressiondthe mRNA. Example 7 details the design of the humandthe expression plasmids and transfection of COS cells with obtaining plasmids. Example 8 describes the ELISAdexpression in transfection COS cells. Example 9 describes the FACS analysis of COS cells, transfection humanddstructures of expression in the ovary cells of Chinese hamsters. Example 12 is directed to CDI8-dependent bindingdwith intracellular adhesion molecule ICAM-R, with ICAM-R mutant protein and complementum fact iC3b. Example 13 describes the scintillation srinilaya assays for identifying inhibitors or enhancers (i.e., modulators)dligand/inteligent binding interactions. Example 14 is directed to the construction of the expression plasmids that encode soluble formsdand analyses linking products expression. Example 15 relates to the production ofd-specific polyclonal sera and monoclonal antibodies. Example 16 describes the analysisdthe tissue distribution, expressiondin peripheral blood leukocytes anddthe expression in inflammatory and non-inflammatory synovial membranes using anti-dpolyclonal synovial membrane. Example 17 describes the allocation of the rat cDNA sequences that show homology to human dgene sequences. Example 18 relates to the design of the ratdplasmids the expression of full length ratdTitel full length and I tomanovich integral proteins. Example 19 is aimed at highlighting mouse cDNA sequences that show homology to human dgene sequences. Example 20 describes the allocation of additional mousedcDNA clones used to confirm sequence analysis. Example 21 refers to hybridization the in situ analysis of various mouse tissues to determine the tissue and cell specific expression of the alleged murine homologue in the humand. Example 22 describes the generation of expression constructs that encode estimated murine homolog of the humand. Example 23 is aimed at designing a "knockout" mouse, where the gene encoding the estimated murine homolog of the human d, is destroyed. Example 24 describes the allocation of the rabbit cDNA clones, which show homology to humandcoding sequences. Example 25 describes animal models of human disease cases where the modulationdat therapeutic susceptibility. Example 26 describes the expressiondin animal models of disease States.

Example 1

Attempts to detect human homology dog is but the cross-reactivity in human leukocytes of peripheral blood in an attempt identification of the human homologue dog TM1. Cell preparations (usually 1106cells were preincubator undiluted supernatant of hybridoma or purified mouse IgG-negative control antibody (10 μg/ml) in ice in the presence of 0.1% of sodium azide. The binding of the monoclonal antibodies was detected by subsequent incubation FI-paired horse anti-mouse IgG (Vector Laboratories, Burlingame, CA) with 6 μl/ml Stained cells were fixed with 2% weight/about, paraformaldehyde in phosphate buffer solution (PBS) and analyzed using a Facstar Plus fluorescently-activated cell sorter (Becton Dickenson, Mountain View, CA). Typically analyzed 10,000 cells using logarithmic amplification for fluorescence intensity.

The results indicate that CA.N not subjected to cross-interaction with surface proteins expressed on leukocytes in human peripheral blood, whereas control cells neoplastic canine lymphocytes of peripheral blood in principle, all showed a positive reaction toTM1.

Since the monoclonal antibody Sa.N specific dog subunit, was not subjected to cross-interaction with the human homologue, the particular human gene, if he existed.

Example 2

Affinity purification dogTM1for M-teminalia sequencing.

DogTM1. was subjected to affinity purification to identify the N-terminal amino acid sequence to design oligonucleotide sample/primer. In short, antiTM1. monoclonal antibody Sa.N has been combined with Affigel 10 chronographically resin (Bio Rad, Hercules, CA), and protein was selected due to specific interaction of the antibody protein. The antibody was subjected to conjugation with resin according to the Protocol Bio Rad at a concentration of approximately 5 mg of antibody per ml of resin. After the conjugation reaction, the excess antibody was removed and the resin was blocked by three volumes of 0.1 M ethanolamine. Then the resin was washed with a solution of phosphate saline buffer (PBS) in the amount equal to thirty volumes of the column.

Twenty-five grams of one dog spleen were homogenation in 250 ml of buffer containing 0.32 M sucrose in 25 mm Tris-HCl, pH 8.0, ProcessName inhibitors. Nuclear and cellular debris were subjected to precipitation by centrifugation at 1000 g for 15 minutes. Membranes were collected in the form of lumps of supernatant (50 mM NaCl, 50 mm borate, pH 8.0 with 2% NP-40) and preincubation for 1 hour in ice. Then insoluble material was concentrated in the form of a lump by centrifugation at 100000 g for 60 minutes, 10 ml of cleared lysate was transferred into a 15 ml polypropylene test tube with 0.5 ml Sa.N-conjugated with Affagel 10 resin described above. The test tube was preincubation overnight at 4oWith rotation, and the resin is successively washed with D-PBS solution with a volume equal to 50 volumes of the column. Then the resin was transferred into a test tube for microcentrifuge and subjected to boiling for 10 minutes in 1 ml of Laemmli sample buffer (not restored), containing 0.1 M Tris-HCl, pH 6.8, 2% SDS, 20% glycerol and 0.002% bromophenol blue. The resin was concentrated in a lump by centrifugation and unloaded; the supernatant treated 1/15 volume-mercaptoethanol (Sigma, St.Louis, MO) and plotted on a 7% polyacrylamide gel. Selected proteins were transferred to Immobilon PVDF membrane (Millipore, Bedford, MA) as follows.

Gels were washed once in deionized water, Millipore-filtered water and balanced within 15-45 minutes in 10 mm buffer transfer 3[cyclohexylamino]-1-propanesulfonic acid (CASP), pH 10.5 with 10% methanol. Immobilon membrane was moistened meta is the postponement was performed using a transfer apparatus Biorad at 70 for 3 hours. Immobilon membrane was removed after transfer and stained in filtered 0.1% R250 camazine for 10 minutes. Membranes were decolorized in a 50% solution of methanol/10% acetic acid three times, each time for 10 minutes. After bleaching, the membrane was washed in filtered water and dried in the air.

Detected protein bands of approximately 150 kD, 95 kD, 50 kD and 30 kD. Probably the band of 50 kD and 30 kD appeared due to contamination of the antibody. N-terminal sequencing was then tried on both bands 150 kD and 95 kD, but 95 kD protein was blocked, preventing sequencing. Protein band 150 kD was cut from the membrane and directly sequenced with an Applied Biosystems (Forster City, CA) Model 573A protein sequencing machine in accordance with the manufacturer's instructions. The resulting amino acid sequence represented in SEQ ID NO:5, using only the writing of amino acid symbols.


The identified sequence included FNLD characteristic sequence of subunits integranova family [Tamura, et.al., J. Cell.Biol. 111:1593-1604(1990)].

Design of primers and attempt amplification dogTM1sequences.

Of information about N-those who completely degenerated oligonucleotide; 6)"Patmer" partially degenerated oligonucleotide and (c)"Guessmer" degeneratively oligonucleotide based on the codon usage of a mammal. These samples are provided below as SEQ ID NO: 6, 7, and 8, respectively. Symbols of nucleic acids in accordance with 37 C. F. R. p. 1.882 for these and all other nucleotide sequences shown here.

< / BR>
Based on the data sequence corresponding clones were not detected using these oligonucleotides in some not very stringent conditions of hybridization (in sevral low stringency hybridizations) in the cDNA library of canine spleen/macrophages peripheral blood, cloned in ZAP (Stratagene, La Jolla, CA).

Four other oligonucleotide primer, designated 5' Deg, 5' Spec 3' Deg and 3'Spec (presented in SEQ ID NO: 9, 10, 11 and 12, respectively, where Deg indicates degenerated and Spec indicates degeneratively) were subsequently designed based on the established N-terminal sequence for attempts extracapillary dogTM1sequences using PCR from ragovoy library DNA, purified from platelets lysate Stratagene library described above.

< / BR>
TM1oligonucleotide primers were evaluation of what was hybridisable with sequences, flanking polylinker region in Bluescript fahmida found in ZAP.

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PCR ekstragirovanie was performed in Taq buffer (Boehringer Mannheim, Indianapolis, IN) containing magnesium, with 150 ng of library DNA, 1 μg of each primer, 200 μm dNTPs and 2.5 units of Taq polymerase (Boehringer Mannheim) and the products were separated by electrophoresis in 1% agarose gel buffer Tris-acetate-EDTA (TAE) with 0.25 μg/ml of acidini bromide. DNA was transferred to Hybond (Amersham, Arlington Heights, IL) membrane with a tampon in overnight in 10X SSPE. After transferring the immobilized DNA was denaturiruet 0.5 M NaOH, 0.6 M NaCl, neutralized 1.0 M Tris-HCl, pH 8.0, 1.5 M NaCl, and washed 2X SSPE before UV crosslinking using a Stratalinker (Stratagene) suturing device. The membrane was preincubation in pre hybridization buffer (5X SSPE, 4X Denhardts, 0.8 SDS, 30% formamide) for 2 hours at 50oWith stirring.

Oligonucleotide samples 5' Deg, 5' Spec 3' Deg and 3'Spec (SEQ ID NO: 9, 10, 11 and 12, respectively) was in the state using the Boehringer Mannheim kinase buffer with 100-300 MX P32-dATP and 1-3 units of polynucleotide kinase for 1-3 hours at 37oC. Not included tag was removed with Sephadex G-25 (Pharmacia, Piscataway, NJ) chromatography using 10 nm Tris-HCl, pH 8.0, 1 mM EDTA (TE) buffer and released from the column rastvor 42oWith stirring and washing again with a finite simple washing with 1X SSPE/0.1% SDS at 50oC for 15 minutes. Then the blot was subjected to exposure on Kodak X-Omat AR film for 1-4 hours at -80oC.

The oligonucleotides 5' Deg, 5' Spec 3' Deg and 3'Spec was hybridisable only with PCR products from reactions in which they were used as primers, and were incapable of hybridization, as expected, with PCR products from reactions in which they were used as primers. Thus, it was concluded that not one of the PCR products was not specific toTM1because there was no product, hybridizing with all relevant samples.

Example 3

Large-scale affinity purification dog TM1for internal sekhonyane.

In order to provide additional amino acid sequence for primerno design dogTM1was cleared for internal sequencing. Three slice frozen spleen (approximately 50 g each) frozen cells from two partial spleens from adult dogs were used to generate protein for internal sequencing. Fifty grams of salute is assault 1 volume of buffer, containing 4% NP-40, and the mixture was then gently stirred for at least one hour. The resulting lysate was cleared of large debris by centrifugation at 2000 g for 20 minutes and then filtered either through Corning (Corning NY) prefilter, or through Corning 0.8 micron filter. Next, the lysate was cleared by filtration through Corning 0.4 micron filtration system.

Splenic lysate and antibody conjugated to Affigel 10 resin described in Example 2 were combined with the volume ratio of 150:1 in 100 ml aliquot and preincubator overnight at 4oWith shaking. The lysate was removed after centrifugation at 1000 g for 5 minutes, combined with the greater part of the antibody conjugated to Affigel 10 resin, and preincubation during the night, as described above. Aliquots of the absorbed resin were then combined and washed with 50 volumes of D-PBS/0.1% tween-20 and the resin was transferred into a 50 ml Biorad column. The absorbed protein was suirable from the resin 3-5 volumes of 0.1 M glycine (pH 2.5); collected fractions of approximately 900 ml and neutralized with 100 μl of 1 M Tris buffer, pH 8.0. From each fraction were removed 15 ál aliquots and boiled in an equal volume of 2X Laemmli buffer 1/15 volume of 1 M dithiothreitol (DDT). These samples were subjected to electrophoresis on what rubrene, using a set of Daiichi (Enprotech, Natick, MA) according to the Protocol suggested by the manufacturer. Fractions that contained the highest amounts of protein were combined and subjected to concentration under vacuum. The remaining solution was diluted 50% restoring Laemmli buffer and tested at 1.5 mm 7% polyacrylamide gels in Tris-glycine/SDS buffer. Protein was transferred from gels to Immobilon membrane using the procedure described in Example 2, using the transfer device Hoefer.

The protein band corresponding to dog TM1were carved out of 10 PVDF membranes, and received approximately 47 µg of total protein. Strips were bleached in 4 ml of 50% methanol for 5 minutes, air-dried and cut into pieces of 1 x 2 mm Pieces of the membrane were immersed in 2 ml of 95% acetone at 4oC for 30 minutes with periodic turning and then air-dried.

To proteolytic cleavage of the protein associated with membrane 3 mg CYANOGEN bromide (CNBr) (Pierce, Rockford, IL) were dissolved in 1.25 ml of 70% formic acid. Then this solution was added to the tube containing PVDF pieces of the membrane, and the test tube preincubation in the dark at room temperature for 24 hours. Then ant (S2) was removed and added to the previously obtained supernatant (S1). To the United supernatant (S1 and S2) were added to two ml of Milli Q water and the solution liofilizovane. PVDF pieces of the membrane were subjected to drying in a nitrogen atmosphere and again proektirovanii 1.25 ml of 60% acetonitrile, 0.1% tetracarboxylic acid (TFUC) at the 42oC for 17 hours. This supernatant (S3) was removed and pieces of membranes proektirovanii newly 1.0 ml of 80% acetonitrile, 0.1% tetracarboxylic acid (TFUC) at the 42oC for 1 hour. This supernatant (S4) was merged with the previously received supernatant (S1, S2, and S3) and dried in vacuum.

Dried CNBr fragments were then dissolved in 63 ál of 8 M urea, 0.4 M NH4THE HCO3. The fragments were recovered in 5 μl of 45 mm dithiothreitol (DDT) and subsequently preincubator at 50oC for 15 minutes. The solution was then cooled to room temperature and fragments proaccelerin by adding 5 ál of 100 mm iodoacetamide (Sigma, St. Louis, MO). After 15 minutes incubation at room temperature the sample was diluted 187 μl of Milli Q water to a final concentration of urea 2.0 M was Then added Trypsin (Worthington, Freehold, NJ) at a ratio of 1:25 (weight:weight) of enzyme to protein and protein is subjected to cleavage within 24 hours at 37oC. Cleavage was interrupted dobavlennaya (HPLC) system Waters 625 LC (Millipore, Milford, MA) using a 2.1 x 250 mm, 5 μm Vydac C-18 column (Vydac, Hespria, CA) equilibrated in 0.05% solution of TFWC and water for gel chromatography (buffer A). Proteins were suirable 80% acetonitrile with increasing concentration of 0.04% TFOC (buffer) gradient 38-75% buffer b for 65-95 minutes and 75-98% buffer b Within 95-105 minutes. Proteins were refractionary at a flow rate of 0.2 ml/min and detected at 210 nm.

After fractionation amino acid sequence of the proteins was analyzed by conducting automated Edman degradation on an Applied Biosystems Model 437A sequenator using standard cycles producer and program Model 610 A Data Analysis, version 1.2.1. All sequeiros reagents were supplied by Applied Biosystems. Amino acid sequences of seven of the eight internal fragments are given below, where "X" indicates that the amino acid identity was uncertain.

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Designing primer

One received internal amino acid sequence (SEQ ID NO:22) was then used to construct a fully reborn oligonucleotide primer, designated p4(R) in the form presented in SEQ ID NO:23.

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extracoporeal of detainee cDNA dog spleen using PCR.

A. Generating detainee cDNA dog spleen.

One gram of frozen material from the spleen of young individuals dog was crushed in liquid nitrogen in a dry ice and homogenized in 20 mm PHK-Stat buffer 60 (Tel - Nest, Inc. Friendswood, TX). Was added 4 ml of chloroform and the solution preextraction by centrifugation at 12000 g for 15 minutes. RNA was precipitated from the aqueous layer with 10 ml of ethanol. Then was selected poly AND+RNA on Dynal Oligo dT Dynabeads (Dynal, Oslo, Norwey). Five 100 μg aliquot of total RNA were combined and diluted with an equal volume of 2X binding buffer (20 mm Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA, 0.1% SDS), in accordance with the Protocol suggested by the manufacturer prior to elution of poly+mPHK 2 mm EDTA, pH 7.5. Then was generated dutina cDNA using elyuirovaniya poly AND+RNA and cDNA kit, Boehringer Mannheim for synthesis in accordance with the Protocol suggested by the manufacturer.

B. Allocation of partial dogTM1cDNA.

Oligonucleotide primers 5' Deg (SEQ ID NO:9) and p4(R) (SEQ ID NO:23) were used in standard PCR reactions using 150 ng detainee cDNA, 500 ng of each primer, 200 μm dNTPs, and 1.5 units of Taq polymerase (Boehringer Mannheim) in Taq buffer (Boehringer Mannheim) with magnesium. Received PR to increase product yield. This strip was subjected to elution from 1% agarose gel on Schleicher and Schuell (Keene.NH) NA 45 paper in buffer containing 10 mm Tris-HCl, pH 8, 1 mm EDTA, 1.5 M NaCl at 65oWith, besieged and Legerova in pCRtmII vector (Invitrogen, San Diego, CA) using the TA cloning kit (Invitrogen) and the Protocol suggested by the manufacturer. Legirovannye the mixture using electroporation was perencana in XL-1 Blue bacteria (Stratagene). Was identified one clone 2.7 containing sequences correspondingTM1peptide sequences that were not used in the design of primers.

Sequencing was performed with an Applied Biosystems 373A DNA sequestrum (Foster City, CA) with a set of Dye-deoxy termination cyclic sequence (ABI), which was introduced fluorescently-labeled dNTPs in asymmetric PCR reaction [McCabe, "Obtaining single-stranded DNA using asymmetric PCR reaction, PCR Protocol: A Guide to Methods and Applications. Innis. et.al.(eds), C. 76-83 Academic Press: New York (1990)] as follows. Samples kept at 96oC for 4 min and subjected to 25 cycles of sequential sequential processing: 96oC for 15 seconds, 50oC for 1 second; 60oC for 4 minutes. Serial data auto is of full inserts of clone 2.7 were presented in SEQ ID NO:24.

Attempt to select a dogTM1cDNA full length of the Stratagene library (as described in Example 2) was not successful. Were skanirovaniya approximately 1106phage plaques using hybridization in mild conditions using 30% formamide with clone 2.7 as samples, but was not obtained positive clones. Attempts extracapillary corresponding sequences of the lower thread from those presented in the clone 2.7 using specific oligonucleotides derived from clone 2.7, or degenerate primers based on the amino acid sequences of other peptide fragments, coupled with degenerate oligonucleotide based on the stored TM1subunit amino acid GFFKR motif [Tamura et. al., above], were also unsuccessful.

Example 5

The alleged cloning of the human homologue of canine cDNA.

In an attempt to highlight the human sequence homologous canineTM1as a sample, was used approximately 1 KB dogTM1fragment from clone 2.7. The sample was generated using a PCR reaction under conditions described in Example 2, using NT2 (presented in SEQ ID NO:25) primerahora, offered by the manufacturer. Purified DNA (200 ng) was machina 200 MK Ci32PdCTR using the Boehringer Mannheim Random Prime Labelling kit and the Protocol suggested by the manufacturer. Nevoshedshy isotope was removed by chromatography under the action of gravity with Sephadex G25 (fine-grained). Test to use was subjected to denaturing 0.2 N NaOH solution and neutralization of 0.4 M Tris-HCl, pH 8.0.

There were prepared a colony of cells (colony lifts) on Hybon filters (Amersham) with a cDNA library of the human spleen in pCDNA/Amp (Invitrogen, San Diego, CA). The filters were first are denatured and neutralized as described in Example 2, and subsequently are denatured in a solution of prehybridization (8 ml/filter) with 30% formamide at 50oWith careful stirring for 2 hours. To this solution was added to the labeled probe, as described above, and preincubation with filters for 14 hours at 42oC. the Filters were washed twice in 2X SSC/0.1% SDS at 37oAnd twice in 2X SSC/0.1% SDS at 50oC. Final thorough washing was conducted 1X SSC/0.1% SDS twice at 65o(1X SSC is 150 mm NaCl, 15 mm sodium citrate, pH 7.0). The filters were subjected to exposure on film Kodak X-Omat AR for 6 hours with intensifying (L)/carbenicillin and preincubator overnight at 37oC. Obtained sown stroke colonies were raised Hybond filters and these filters were processed as described above. Filters were prohibitionary at more stringent conditions with 1 KB breakdown of clone 2.7, the state, as described above, in 50% formamide hybridization solution at 50oC for 3 hours. The filters, which were introduced samples were washed with final care 0.1 X SSC/0.1% SDS at 65oWith and subjected to exposure on film Kodak X-Omat AR for 2.5 hours at -80oWith intensifying screen. Positive colonies were identified and subjected to cultivation in (L)/carbenicillin environment during the night. DNA from the cultures was prepared using minipreparation set Promega Wizard in accordance with the Protocol suggested by the manufacturer, and the resulting DNA was subjected to sequencing.

The initial screening resulted in 18 positive clones, while secondary screening at more stringent conditions of hybridization were given one positive clone, which was marked A. DNA and derived amino acid sequence of humandclone A presented in SEQ ID NO:1 and 2, respectively.

Characteristics Calouste for the Mature protein, plus 48 bases (16 amino acids) of the signal sequence 5 verkhnego flow and 214 bases 3' netransliruemoi sequence that is not broken in polyadenylation sequence. The molecular weight of the nucleus of the Mature protein is predicted equal to about 125 kD. Extracellular (interstitial) domain is predicted comprising amino acid residues from about 17 to 1108 SEQ ID NO:2. This extracellular region sequence with about 20 amino acids homologous to human CD11c transmembrane region (residues 1109 to 1128 SEQ ID NO:2). The cytoplasmic domain consists of approximately 30 amino acids (residues from 1129 until 1161 SEQ ID NO:2). The protein also contains a region (around 150-352 residues) of approximately 202 amino acids, homologous I (insertional) domain common to CD11a, CD11b and CD11c [Larson and Springer, above],E[Shaw, et. al. , J. Biol.Chem. 269:6016-6025 (1994)] and VLA-1 to VLA-2 [Tamura et. al. above]. It was shown that the I domain in other integrins involved in ICAM binding [Landis, et.al., J. Cell.Biol. 120:1519-1527 (1993)]; Diamond, et. al. , J. Cell.Biol. 120:1031-1043(1993)], confirming that thedcan also link the elements of the ICAM family of surface molecules. Has not been demonstrated that this area exists in any other is about 36% identity of amino acid sequence CD11a, approximately 60% identity CD11b and approximately 66% identity CD11c. The ordering of the amino acid sequences for (CD11b SEQ ID NO:3), CD11 (SEQ ID NO:4) andd(SEQ ID NO:2) is presented in Fig.1.

The cytoplasmic domains of the subunits in2the integrins are usually different from each other within the same species, while individual subunits show high degree of homology in specific microenvironments.

In accordance with these observations cytoplasmic region dmarkedly different from CD11a, CD11b, and CD11c except for the membrane proximal GFFKR amino acid sequence, which, as shown, is maintained among all integrins [Rojiani, et.al., Biochemistry 30: 9859-9866 (1991)] . Since the cytoplasmic tail region of integrins was involved in the "wrong" ("inside out") signalizovania and avidity regulation [Landis et. al., above], it is possible thatdinteracts with cytoplasmic molecules than the molecules interacting with CD11a, CD11b and CD11c, and as a result is involved in the ways of signalizovania other than ways, including other2integrins.

Extracellular domaindcontains saved DGSGS amino acid p is, is required for the interaction of the ligand [Michishita, et. al., 72:857-867 (1993)]. Three additional prospective customers, cation binding to CD11b and CD11c are stored indthe sequence in amino acids 465-474, 518-527 and 592-600 in clone A (SEQ ID NO: 1).dThe I-domain is 36%, 62% and 57% identity to the corresponding regions in CD11a, CD11b, and CD11c, respectively, and a relatively low serial homology in this region confirms thatdcan interact with a set of extracellular proteins, non-protein, interact with other famous2integrins. Or affinity dfor known2integranova ligands, for example, ICAM-1, ICAM-2 and/or ICAM-R may be different from that shown for other2integranova/ICAM interactions [see Example 12].

The allocation of additional humandcDNA clones for securitysage control.

To confirm the DNA sequence encoding the humandwere allocated additional human cDNA by hybridization of human splenic oligo-dt-premirovanii cDNA library (Invitrogen) in rdnk/AMR, (described in Example 5), the amount of which was selected from the 5' regiondas described below. The hybridization conditions were the same as described above for isolation of primary human dclone except that after hybridization the filters were washed twice in 2X SSC/0.1% SDS at room temperature and once with 2X SSC/0.1% SDS at 42oC. the Filters were subjected to exposure on film Kodak X-Omat AR during the night.

5'dprobe hybridization was generated by PCR reaction from A clone using primers CD11c 5' For (SEQ ID NO:94) and CD11c 5' for Rev(SEQ ID NO: 95) under the following conditions. Samples kept at 94oC for 4 min and subjected to 30 cycles of processing successive temperature stages (i) 94oC for 15 seconds; (ii)5oC for 30 seconds; (iii)72oC for 1 minute in the apparatus Perkin-Elmer 9600 thermoceycler.

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The amplification product was purified using the Bio Pad (Hercules, CA) Prep-A-Gene kit according to the Protocol suggested by the manufacturer. The resulting 5'dthe sample consisted of a length of about 720 base pairs corresponding to the region from nucleotide 1121 to nucleotide 1839 in SEQ ID NO:1. Purified DNA (approximately 50 ng) was mecena32P-dCTR using Boehringer Mannheim (Indianapolis, Indiana) Random Prime Labelling kit according to the Protocol,phia, NJ) according to the Protocol suggested by the manufacturer. Labeled probe was added to the filters in a solution of prehybridization containing 45% formamide and incubation was allowed to proceed over night at 50oC. After incubation, the filters were washed as described above.

Thirteen colonies gave signals on duplicate rises. Positive colonies were selected from the main cups diluted in LBM and corbellini (100 μg/ml) and cultivated at different dilutions on Hybon (Amersham) filters. Double filters were prohibitionary the same solution from the initial hybridization, and after hybridization the filters were washed with final care 2X SSC/0.1% SDS at 42oWith and subjected to exposure on the film.

Ten of the originally identified thirteen positive colonies were confirmed by secondary skanirovaniya. Of these ten two clones (designated A7.Q and A8.Q) were subjected to sequencing and determined to encode humand. It was found that the clone A7.Q represents a length of approximately 2.5 KB, including 5 Leeroy part, part, the coding region, and 60 additional bases 5' netransliruemoi sequence. Would the tee with a corresponding nucleotide 2152 SEQ ID NO:1. It was determined that clone A8.Q represents a length of approximately 4 KB, blocking alldthe coding region and including intron sequence at the corresponding base 305 of SEQ ID NO:1. Compared to the originally selecteddclone (SEQ ID NO:1) was observed one difference is that, as defined, both clone A7.Q and A8.Q have three insertions main CAG codon occurring at the base of 1495. Sequence for clone A7.Q and A8.Q are represented in SEQ ID NO:96 and 97, respectively, and the composite human sequence obtained from clone A7. Q and A8.Q and its corresponding deduced amino acid sequence represented in SEQ ID NO:98 and 99, respectively.

Example 6

Analysis Northern blot of humandexpression in tissues.

In order to determine the relative level of expression and tissue specificitydwas the analysis Northern blotting using fragments as samples from clone A. Approximately 10 μg of total RNA from each of several human tissues or cultured cell lines were loaded on a formaldehyde agarose gel in the presence of 1 µg acidini bromide. After electrophoresis at 100 B in accordance with the Membrane was annealed for 1.5 hours at 80oWith the vacuum. To block the membrane for 3 hours at 42oWas used a solution of prehybridization containing 50% formamide buffer 3-(N-morpholine)propane sulfonic acid (MOPS). Fragments of clone A was in the state of Boehringer Mannheim Random Prime Labelling kit according to the manufacturer's instructions, including the P32dCTR and a32PdTTR. Nevoshedshy label was removed on a column of Sephadex Q25 in THE buffer. The sample was introduced into the membrane with a score of 1.5106per ml buffer prehybridization. Then the blot was sequentially washed with 2X SSC/0.1% SDS at room temperature in 2X SSC/0.1% SDS at 42oWith 2X SSC/0.1% SDS at 50oWith 1X SSC/0.1% SDS at 50oWith 0.5 X SSC/0.1% SDS at 50oC and 0.1 X SSC/0.1% SDS at 50oC. Then the blot was subjected to exposure on the film for 19 hours.

Hybridization using BstX1 fragment from clone A (corresponding to nucleotides 2011-3388 in SEQ ID NO:1) led to the detection of a weak signal in the region of approximately 5 KB in the liver samples, planety, thymus and tonsils total RNA. The signal was not detected in samples of kidney, brain or heart. The amount of RNA present in renal lane, was minimal, as determined by staining of acidini bromide.

When used W is x different sizes in human multi-tissue Northern blot (MTN) using the poly+RNA (Clontech). Strip approximately 6.5 KB was observed in the spleen and skeletal muscle, while a band of 4.5 KB was detected in lung and peripheral blood leukocytes. Changes observed in the dimensions can be caused by specific adenalineage tissue cross-reactivity of the sample with other members of the family of integrins or by hybridization with mRNA subjected to alternative splicing.

Northern analysis using the third fragment from clone A, overlapping nucleotides 2000-3100 in SEQ ID NO:1, gave results that are consistent with the results using other fragments of clone A.

RNA from three liantian lines of cells of the spinal cord has also been tested using fragments corresponding to nucleotides 500-2100 and 2000-3100 in SEQ ID NO:1. TNR-1 cell line, previously stimulated PMA, gave a diffuse signal in the same area size (approximately 5.0 KB) with a slightly stronger intesively than in the case of tissue signals. RNA from unstimulated and DMSO-induced HL-60 cells hybridizeddthe breakdown at the same intensity as in the case of tissue samples, however, RMA processing, apparently, increased the intensity of the signal. As Rmueller, this result was confirmed by enhanceddthe expression in monocyte/mikrofalowe cell types. U937 cells were expressivelydtransfer, and this signal is not increased by PMA stimulation. There was no band in Molt, Daudi, H9, JY or cells Jukart.

Example 7

Unstable expression of human dstructures.

A. Generation of expression constructs.

Human clone A suffered from a lack of initiation methioninamide codon and possibly some 5' signal sequence. Therefore, in order to generate human expression plasmids containing A sequence, were used two different approaches. First, there were constructed two plasmids, in which the signal peptide sequences obtained from the genes encoding either CD11b or CD11c were subjected to splicing in clone A to generate chimericdsequence. The second approach was designed third plasmid, which was added adenosine base in position 0 in the clone A for encoding the initiating methionine.

Three plasmids contained different areas, which encode the 5 castdconsistently the Example 2) specific 3' primer Bam Rev (shown below in SEQ ID NO:26) and one of the three 5' primers. Three 5' primer contained the sequence: (1) Identical to nonspecific bases in positions 1-6, involving hydrolysis, EcoRI site of the provisions of 7-12 and consistent Kazak sequence of positions 13-18; (2)part CD11b (primer ERIB) or CD11c (primer ERIC) signal sequence, or adenosine (primer ERID); and (3)additional 15-17 grounds, specifically the overlapping 5' sequence from clone A in order to provide for primer annealing. Primers ERIB, ERIC and ERID presented in SEQ ID NO:27, 28 or 29, respectively, where meinenemy the initiation codon is 6 field is covered and EcoRI site is the field is covered twice.

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The resulting PCR product was hydrolyzed with EcoRI and BamHI.

All three plasmids contained a General seconddregion (which immediately incertitude in the bottom stream from the 5' region described in the previous paragraph), including the 3' enddthe clone. The seconddregion, which extends from nucleotide 625 in the Xbal site in the vector 3' polylinker region of clone A, was isolated by hydrolysis of the clone A BamHI and Xbal.

There have been three ligation reaction in which the 3' BamHI/Xbal fragment was Legerova in one of the three 5'dEcoRI/BamHI fragments using Ventouse number of vector rdnc. 3 (Invirogen), prohydrolized EcoRI and Xbal, was added to each reaction with an additional unit of ligase. Reactions were allowed to proceed for another 14 hours. Then one-tenth of the reaction mixture was transformed into XL-1 Blue competent cells. The resulting colonies were cultured, and the DNA is selected, as in Example 5. Hydrolysis EcoRI identified three clones that were positive for the indicated restriction site and, thus, designed signal sequence. The clones were designated RATM.B1 (CD11b/dfrom primer ER1B), RATM.C10 (CD11b/dfrom primer ER1C) pATM.D12 (adenosine/dfrom primer ER1D). The presence of the respective signal sequences in each clone was confirmed by sequencing the nucleic acid.

B. Transfection of COS cells.

Expression ofdplasmids discussed above, was carried out using cotransfected COS cells with individual plasmids and CD18 expression by plasmid, pRC. CD18. As a positive control, COS cells were subjected to co-transfection with the plasmid pRC.CD18 and CD11 expression by plasmid pDC.CD11A.

Cells were passaged in culture medium (DMEM/10%FBS/penstrep) in 10 cm Petri dishes treated with Sulphate in PBS) without trypsin for all procedures. Before transfection cups washed once free from serum DMEM. Fifteen micrograms of each plasmid were added to 5 ml of transfection buffer (DMEM with 20 μg/ml DEAE-dextran and 0.5 mm of chloroquine) in each Cup. After 1.5 hours incubation at 37oWith cells were subjected to shock within 1 minute 5 ml DMEM/ml DMSO. This DMSO solution was then replaced with 10 ml/Cup cultural environment.

The resulting transfectants were analyzed using ELISA, FACS and thus, as described in Examples 8, 9 and 10.

Example 8

ELISA analysis of COS transfectants.

In order to determine whether COS cells co-transfectional with the CD18 expression plasmid pRC.CD18 anddthe plasmid expressed don the cell surface together with CD18, the analysis was performed by ELISA using primary antibodies arising against CD18 (e.g., TS1/18 purged from ATS NV). As a positive control were also performed the ELISA assays on cells, co-transfection with the CD18 expression plasmid and the CD11a expression plasmid pDC.CD11A. The primary antibodies in this control induced CD18 antibodies and anti-CD11a antibodies (e.g., TS1/22, purified from ATS NV).

For ELISA kladivo culture Corning. The cells were allowed to incubated in culture medium for 2 days before analysis. Then the cups are washed twice with 150 μl/well of D-PBS/0.5% solution of gelatin skin teleost (Sigma). This buffer was used in all stages except stage of growth. All washing and incubation were carried out at room temperature. The wells were blocked with a solution of gelatin for 1 hour. Primary antibodies were diluted to 10 μg/ml solution of gelatin and then 50 μl were added to each well. For each primary antibodies were used triple wells. After 1 hour incubation cups were washed 3X a gelatin solution with a concentration of 150 μl/cell. Secondary antibody (specific sheep antimachine Ig/HRP-Fc [Jackson, West Grove, PA]) at a dilution of 1:3500 was added at a concentration of 50 μl/well, and cups were preincubator for 1 hour. After three washings of cups were manifested within 20 minutes, 100 μl/well of a solution of o-phenyldiamine (OPD)(Slgma) (concentration 1 mg/ml OPD in citrate buffer) before adding 50 µl/well of 15% sulfuric acid.

Analysis of transfectants in the format of ELISA with specific antibodies anti-D18 showed little expression above background in the cells, transfection plasmid only, to whom, and against the background when analyzed with specific antibodies to CD18 or reagents specific for CD11a. Further analysis of cells co-transfection with plasmids encoding CD18, and one of the designsdexpression (RATM.C10 or RATM.D12) showed that the cell surface expression of CD18 excluded concomitant expressiond. A detectable increase in CD18 expression in COS cells, transfection RATM.C10 or RATM. D12 was comparable to the expression observed in the co-transfection CD11a/CD18 positive control cells.

Example 9

FACS analysis of COS transfectants.

For FACS analysis cells in Petri dishes were loaded with fresh culture medium after day after transfection, and they were allowed to incubated for 2 days before analysis. Transfectant cells were removed from the cups 3 ml Versene, washed once with 5 ml of FACS buffer (DMEM/2% FBS/0.2% sodium azide) and diluted to 500,000 cells/sample in 0.1 ml of FACS buffer. Ten microlitres or 1 mg/ml FITC-conjugated CD18, CD11a or CD11b (Becton Dickinson) specific antibodies or 800 μg/ml CFSE-conjugated mouse 23F2G (anti-CD18)(ATCC HB11081) were added to each sample. Then the samples were preincubator in ice for 45 minutes, washed, and data were analyzed using Lysys II software (Becton Dickinson).

COS cells, transfection only CD18 sequences, not stained in CD18, CD11a or CD11b. If he transfectional with CD11a/CD18, about 15% of cells were stained by antibodies in CD11 or CD18. All cells, transfection with CD18 and any designddid not cause detectable staining in CD11a and CD11b. RATM.B1, RATM.C10 and RATM.D12 group were stained 4%, 13% and 8% positive in CD18, respectively. Fluorescence in positive population in CD11a/CD18 group was 4 times higher than the background. Compared with transfectional ddesigns CD18 design was produced positive population, which showed a 4-7-fold increase in fluorescence intensity compared to the background.

Example 10

Biotin-labeled immunoprecipitate humand/D18 complexes from co-transfection COS cells.

An attempt was made thus to the cells, co-transfection with CD18 and each of the plasmidsdexpression, separately described in Example 7 to determine whether or not to bedselected as part of a heterodimeric complex parameter integrins.

Transfection cells (1-3108cells/group) were removed from the cups Pechenie 15 minutes at room temperature. The reaction was suppressed by washing 3 times with 50 ml/group cold D-PBS. The washed cells resuspendable in 1 ml lisanova buffer (1% NP40, 50 mm Tris-HCl, pH 8.0, 0.2 M NaCl, 2 mm CA++, 2 mm MD++, protease inhibitors) and preincubator for 15 minutes in ice. The insoluble substance was separated by centrifugation at 10000 g for 5 minutes, and the supernatant removed to a fresh tube. In order to remove non-specific reactive substance with mouse immunoglobulin was conducted preliminary stage of enlightenment. Twenty-five grams of mouse immunoglobulin (Cappel, West Chester, PA) were preincubator supernatants at 4oC. After 2.5 hours, 100 μl (25 μg) rabbit antimisting Ig conjugated to separate (prepared from Protein A Sepharose 4B and rabbit antimisting IgG, both from Zymed, San Francisco, CA), were added to each sample; the incubation was continued at 4oWith shaking for 16 hours. Sepharose granules were removed from supernatant by centrifugation. After pre-enlightenment supernatant were then processed 20 μg antibody anti-D18 (TS1.18) for 2 hours at 4oC. the Antibody/antigen complexes were isolated from supernatants by incubation of 100 μl/ES, 0.2 M NaCl and 1% Triton-X 100. The washed pellets were collected and subjected to boiling for 10 minutes in 20 μl of 2X Laemmli sample buffer with 2% - mercaptoethanol. The samples were centrifuged and loaded on 8% pre-poured Novex polyacrylamide gel (Novex) at 100 B within 30 minutes. Protein was transferred to nitrocellulose membrane (Schleicher and Schuell) in TBS-T buffer at 200 mA for 1 hour. Membranes were blocked for 2 hours with 3% BSA in TBS-t, Membranes were treated with a 1:6000 dilution steppedinto horseradish peroxidase (POD) (Boehringer Mannheim) for 1 hour followed by three washes in TBS-T. Then for the manifestation of the blot was used to set Amersham Enhanced Chemiluminescence according to the manufacturer's instructions. The membrane was subjected to exposure to Hyperfilm MP (Amersham) for 0.5-2 minutes.

Immunoprecipitate CD18 complexes from cells transfection PRC. CD18 and or RATM.B1, RATM.C10, or pATM.D12 that detect surface expression of heterodimeric samples, consisting of approximately 100 kD chains, consistent with the predicted size of CD18 and chain approximately 150 kD, corresponding tod.

Example 11

Stable transfection of humandin the ovary cells Chinese hamster.

< cDNA-you encoding each chain were both transient and steady transfection in a cell line that lacked both componentsdand CD18.

For these experimentsdcDNA was subjected to additional stimulation of the leader sequence and Kozak agreed sequence, as described in Example 7, and subcloned in the expression vector rdnc. The final design, marked pATM.D12, was co-transfection with modified commercial vector, pDCl.CD18 coding for human CD18 in dihydrofolate reductase (DHFR) cells of the ovary (Cho) Chinese hamster. Plasmid pDC1.CD18 encodes DHFR+marker and transfectant could be selected using the corresponding nucleoside-deficient medium. Modifications, which gave pDC1.CD18, are as follows.

Plasmid pRC/CMV (Invitrogen) is a vector expression of the mammal with a CMV promoter and with the resistance gene marker ampicillin. DHFR gene from plasmid pSC1190-DHFR was insertion in pRC/CMV 5' SV40 replication source. In addition, polylinker of the 5' region of plasmid pHF2G-DHF was Legerova in pRC/CMV/DHFR construction, 3' in the DHFR gene. CD18 coding sequence was subsequently cloned in CLASS="ptx2">

Surface expression of CD18 was analyzed by cytometry in flow using monoclonal antibodies TS1/18. Education heterodimer found between dand CD18, in this cell line is consistent with immunoprecipitates described in Example 10, with transient expression in COS cells.

Example 12

Linking humandwith ICAM-R in D18-dependent hybrid.

In the light of what was reported, demonstrates the interaction between leikotitami by integrins and molecules intercellular adhesion (ICAMs), which mediate the contact cell-cell [Hynes, Cell 69:11-25 (1992)], the ability of Cho cells expressingd/CD18, to bind SAM-1, ICAM-R or VCAM-1, defined in two ways.

In replicating the analyses of soluble SAM-1, ICAM-R or VCAM-1, IgG1 integral proteins were immobilized in plastic and was defined as the abilityd/CD18 SNO transfection cells for binding to immobilized ligands. Transfection cells were subjected to RET callainos, washed in binding buffer (PRMI with 1% BSA), and preincubator or only in the buffer (with or without 10 ng/ml PMA), or in buffer with anti-CD18 monoclonal antibodies with a concentration of less than 10 soluble ICAM-1/IgGt, ICAM-R/IgGI, VCAM-1/IgG1 composite protein or bovine serum albumin (BSA) as a negative control. The design of the soluble forms of these adhesion molecules is described and fully disclosed in the joint application U.S. No. 08/102852, filed August 5, 1993, the Wells were blocked with 1% BSA in PBS before the addition of labeled cells. After washing tablets immersion in PBS with 1% BSA for 20 minutes was measured fluorescence remaining in each well, using a Cytofluor 2300 (Millipore, Milford, MA).

In experiments with immobilized ICAMs,d/CD18 co-transfectants, consistently shows a 3-5-fold increase in binding to ICAM-R/IgG1-hole compared to wells coated with BSA. Was demonstrated specificity D18-dependence of the binding with inhibitory effects of anti-D18 antibodies TS1/18. Linking cells, transfection CD11a/CD18 with ICAM-1/IgG1-hole, was comparable to the binding observed with the holes filled BSA. CD11a/CD18 transfection cells showed a 2-3 fold increase in binding to ICAM-1/IgG1-hole, only after pre-processing the RMA. PMA treatmentd/CD18 of transfectants did not affect binding to ICAM-1/IgG1 or ICAM-R/IgG1 holes. Not watched Oberfrohna cells with soluble ICAM-1/IgG1, ICAM-R/IgG1 or VCAM-1/IgG1 integral proteins was determined using flow cytometry. Approximately one milliond/CD18-transfection Cho cells (grown in centrifuge tubes to a higher expression) measurement was suspended in 100 μl of binding buffer (PRMI and 1% BSA) with or without addition of 10 μg/ml anti-CD18 antibody. After 20 minutes incubation at room temperature, the cells were washed in binding buffer and was added to soluble ICAM-1/IgG1 or ICAM-R/IgG1 protein compound with a final concentration of 5 μg/ml of Binding was allowed to proceed for 30 minutes at 37oC, after which cells were washed three times and resuspendable in 100 μl of binding buffer containing FITC-conjugated sheep anti-human IgG1 at razbavlenii 1:100. After 30 minutes incubation the samples were washed three times and suspended in 200 µl of binding buffer for analysis Becton Dickinson FaCScan.

Approximately 40-50%d/CD18 of transfectants indicated binding to ICAM-R/IgG1, but not binding to ICAM-1/IgG1 or VCAM-1/IgG1 protein.

Pre-treatment transfection cells PMA had no effect ond/CD18 binding to either ICAM-1/IgG1, ICAM-R/IgG1 or VCAM-1/IgG1, which soglasovat is her background after processingd/CD18 of transfectants anti-D18 antibody TS1/18.

Overall, the data from these two analyses on binding illustrate thatd/CD18 binds to ICAM-R and binds preferentially compared to SAM-1 and VCAM-1.d/CD18 binding preference for ICAM-R, compared to SAM-1 is opposite to that observed with CD11a/CD18 and CD11b/CD18. Thus, modulation ofd/CD18 binding can be selectively influence normal and pathological immune function, where ICAM-R plays a prominent role. In addition, the results of similar analyses, which were tested antibodies, immunospecificity for different extracellular domains of ICAM-R on their ability to inhibition of binding of ICAM-Rd/The CD18 transfectants, indicate thatd/CD18 and CD11a/CD18 interact with different domains of ICAM-R.

The inability of CD11a/CD18 binding of ICAM-1/IgG1 in solution confirms that the ability of binding between CD11a/CD18 and SAM-1 or ICAM-R is too low to allow binding in solution. Detectiond/CD18 binding to ICAM-R/IgG1, however, acknowledges the unusually high binding capacity.

Was used adhesive FACS analysis, as described above, to explore St. the existence in LFA-1/Ig the Chimera [Sadhu, et. al. Cell Adgesion and Communication 2:429-440 (1994)]. The mutant protein was expressed in soluble form from sustainably transfectional SNO cell lines and purified on a column (Prosep A, as described Sadhu, et.al.,above.

E37A/Ig bindingd/The CD18 transfectants was not detected in repeated analyses. The value of fluorescence intensity (MFI) E37A/Ig chimeras detected using FITC-conjugated anti-human antibodies was identical to the one MFI detectable antibodies, indicating that no detectable signal above background in the analysis E37A/Ig mutant protein. Similarly, in ELISA performed as described in Example 14, E37A/Ig mutant does not cause binding of immobilizedd/CD18.

dBinding to iC3b.

Complementry component C3 can be proteoliticeski split with the formation of the complex iC3b, which initiates an alternative path complemental activation and leads simultaneously to the cell-mediated destruction of the target. Both subunit CD11b and CD11c were involved in iC3b binding and subsequent phagocytosis of iC3b-coated particles. Protein fragment in the CD11b I domain has recently been identified as a site iC3b interaction [Ueda, et.al., Proc.Natl.Acad.Sci., (USA) 91:10680-10684 (19 binding interaction.

Linkdwith iC3b was conducted using transfectants or cell lines naturally expressing d(for example, PMA-stimulated HL60 cells) and iC3b-coated sheep red blood cells (sRBC) in the analysis rosethorne patterns [Dana, et. al., J. CIin. Invest. 73: 153 to 159 (1984)] . Abilityd/CD18 of CHO transfectants, VLA4-of CHO transfectants (negative control) and PMA-stimulated HL60 cells (positive control) education rosethorne structures are compared in the presence and absence of anti-CD18 monoclonal antibodies (e.g., TS1/18).

Example 13

Screening analysis, close to scintillation.

Specific inhibitors of binding betweendligands of the present invention and their binding partners (pairdligand/inteligent) can be determined using various methods, such as analysis technique that is close to scintillation, as described in U.S. patent 4271139, Hart and Greenwald, Mol.Immunol. 12:265-267 (1979) and Hart and Greenwald, J. of Nuc.Med. 20:1062-1065 (1979), each of which is presented here as a reference.

One element of a pairdligand/inteligent associated with the solid substrate, either directly or not directly the AET specific epitope at the C-Terminus of the soluble integranova chain of the protein. This epitope is either hemagglutinin protein or mycobacterial IIIE9 epitope [Anderson, et.al., J. Immunol. 141:607-613 (1988)]. A fluorescing agent is also associated with the substrate. Or fluorescent agent can be integrated into the solid substrate, as described in U.S. patent 4568649 presented here in the form of links. Not associated with substrate element pairdligand/inteligant is labeled with a radioactive compound in such a way that emits radiation capable of exciting the fluorescent agent. When the ligand binds radioactivedecay intiligent, the label is entered close enough to the fluorescent agent that is associated with the substrate for excitation of the fluorescent agent and for light emission. When the ligand is not bound, the label is usually too remote from the solid substrate to excite the fluorescent agent, and light emission is too weak. Emitted light is measured and correlated with the binding between the ligand and inteligenta. Addition of an inhibitor binding to the sample will reduce the emission of fluorescence due to the protection of radioactive label from being captured near solid surfaces. Therefore, inhibitors of the binding can be identified C is identified using similar methods.

Soluble recombinantd/CD18 latinboy constructions "zip" (see Example 14) is used in the analysis close to the scintillation in skanirovaniya in the modulators, the binding using the following method. Recombinant integrin immobilized nonblocking anti - subunit or anti - subunit antibodies, previously deposited on the die with the entered scintillator. Chemical library of compounds and specific biotinylation CAM/Ig Chimera simultaneously added to the Cup. The binding of CAM/Ig chimeras detected using labeled streptavidin. In the analysis of ICAM-1/Ig and ICAM-3/Ig biotinylated NHS-sulfobutyl-LC (long, Pierce) according to the Protocol suggested by the manufacturer. Labeled proteins are more reactive with ITSELF a specific antibody and can be shown in the reaction with immobilized LFA-1 using ELISA, with detection using Streptavidin-HRP and the subsequent manifestation of OPD.

Or recombinant Lazenby protein type "zip" cleared or partially cleared and zapolnyaetsya directly into the Cup with embedded scintillant. Unlabeled CAM/Ig Chimera and chemical library compounds are added to ignoreme the aqueous integrin is detected labeled non-blocking or subunit antibodies.

Example 14

Soluble humandexpression design.

The expression of soluble humand/CD18 heterodimeric protein of full length provides easy-to-clean material for immunization and analysis of binding. The advantage of generating soluble protein is that it can be cleaned rather from supernatants than from cell lysates (as with membrane-boundd/CD18 full length); therefore, enhanced regeneration and reduced amount of impurities.

Solubledthe expression plasmid was constructed as follows. Nucleotide fragment corresponding to a region of the grounds from 0 to 3161 in SEQ ID NO:1, cloned in plasmid pATM.D12, was isolated by hydrolysis of Hind III and AatII. PCR fragment corresponding to bases 3130-3390 in SEQ ID NO: 1, overlapping Hind III/AatII fragment containing additional MIuI restrictly site at the 3' Terminus, was amplified from pATM. D12 with primers sHAD.5 sHAD.3, is presented in SEQ ID NO:30 and 31, respectively.

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The product of PCR amplification was hydrolyzed with AatII and MIuI and Legerova in Hind III/AatII fragment. The resulting product was Legerova in Hind III/MIuI gidralizovanny plasmid pDC1.s.

This Cassia was detected using autoradiographically visualization immunoprecipitating CD18 complexes, retrieved from35S-labeled methionine cells. The design was also coexpression with soluble CD18 in 293 cells [Berman, et.al., J. Cell Biochem. 52:183-195 (1993)].

Solubleddesign full length.

The invention also considered alternative dexpression design. To facilitate the expression and purification of intactd/CD18 of heterodimer will be constructed solubledand CD18 expression plasmids for inclusion in the composite sequence of type 'zip', which should stabilize heterodimer in the cleaning process [Chang, et. al. , Proc.Natl.Acad.Sci (USA), 91:11408-11412 (1994)]. DNA encoding acidic and basic amino acid chain of type 'zip', was generated using primer annealing using oligonucleotides described by Chang, et. al. DNA sequences were further modified to include additional MIuI and b1 restricted sites at the 3' and 5' ends, respectively, of the DNA to facilitate sublimirovannydor CD18 expression constructs described earlier. The addition of sequences representing either hemaglutinin protein or polyhistidine sequence, the tis are introduced to facilitate affinity purification of expressed protein. The sequence encoding the main chain type "zip", introduced into the plasmid vector expressing CD18; acid chain is introduced into the circuit design. When the modified expression dand CD18 proteins in the cells of the owners, probably, the interaction between the acid and the basic circuit structure of type ' zip ' will stabilize heterodimer and will allow the selection intactd/CD18 molecules due to affinity purification as described above.

For the expression of solubledand CD18 sequences with acidic and basic elements of type ' zip ' were constructed plasmids and transfection in COS cells using the method of DEAE/Dextran as described in Example 7. The obtained protein was referred to asd/CD18LZ. Hemagglutinine and polyhistidine target was not introduced ind/CD18LZ. Transfection cells were grown for 14 days under conditions containing a reduced amount of serum (2%). Supernatant of transfection cells collected every five days, were analyzed for protein production using ELISA as described in Example 8. In short,d/CD18LZ heterodimer was immobilized in a Cup filled with anti-dthe monoclinic crystal is th anti-CD18 monoclonal antibodies TS1/18.1 (see Example 8), with the subsequent addition of the conjugate streptavidin/horseradish peroxidase (HRP) and o-phenyldiamine (OPD). Protein was clearly detectable in supernatant.

Analyses on binding with solubledproducts of expression of full length.

Functional tests for linkage using heterodimerd/CD18LZ full length, described above, were carried out due to the immobilization of heterodimer on tablets, filled with a monoclonal antibody 169C or non-blocking anti-CD18 monoclonal antibody (see Example 15). The wells were blocked with gelatin from fish skin to prevent nonspecific binding to add a CAM/Ig chimeras (see Example 12) at initial concentrations of 10 µg/ml the Binding of the chimeras withd/CD18 was detected with sheep anti-human Ig-HRP conjugate (Jackson Labs) and the subsequent manifestation of OPD.

VCAM-1/Ig was observed in binding to capturedd/CD18LZ at levels 3-5 times higher than the level from the captured CD11a/CD18. ICAM-1/Ig and ICAM-2/Ig bound soluble heterodimer CD11a/CD18 around level 15 and 10-fold greater than the background, respectively, but did not d/CD18. VCAM-1 binding was reduced by approximately 50% in the Yu was also conducted with ICAM-1/Ig protein, immobilized in 96-lanoche tablets followed by the addition of recombinant soluble integrin in the cell supernatant. Binding of soluble integrins was detected with unlabeled non-blocking and subunit-specific mouse antibodies followed by incubation with HPR-conjugated sheep artemisinin antibody and expression of OPD.

The results indicate that non-blocking antibody was detected d/CD18LZ binding to ICAM-R/Ig, 10-fold greater than the binding detected in control wells containing no antibody. Binding of solubled/CD18 was not found with immobilized ICAM-1/Ig, however, the binding was detected betweend/CD18 and immobilized CD11b/CD18 and CD11a/CD18 15 and 5 times, respectively, above the background binding.

As previous studies have demonstrated that CD11b and CD11c bind lipopolysaccharide (LPS) [Wright, Curr. Opin.lmmunol. 3:83-90 (1991); Inhalls and Golenbock, J. Exp. Med. 181:1473-1479 (1995)], the binding of LPS was also analyzed using flow cytometry and analysis on the dice. The results indicate that FI-labeled LPS isolated from S. Minnesota and S. typhosa (both obtained from Sigma) at a concentration of 20 μg/ml were able to weak with the elegance Cho cells. In the format of ELISA assays biotinylated LPS [Luk, et.al., Alan. Biochem. 232:217-224 (1995)] at a concentration of 0.5-3.0 µg linked immobilizedd/CD18LZ with the signal at four times the signal captured antibody and the blocking antibodies. The apparent binding of LPS with CD11a/CD18 was fixed on the excess of extracting from each experimental values of background binding with anti-CD11 and antibody TS2/4.

In order to identify other ligands ford/CD18, recombinant d/CD18LZ protein was used in drugabuse study. The binding of different cell types with immobilized protein was used to determine which cells Expressdthe ligands on the cell surface. Then used the inhibition antibodies to determine whether the observed cell binding to occur from interactions with known molecules with surface adhesion. If the result is not a inhibition, co-immunoprecipitated/CD18LZ related proteins from lysates of cells that will binddused in the attempt to identify the ligand.

Expression constructs soluble humandI domain.

dI domain and a human lgG4,dI domain was amplificatoare using PCR reactions using primers designed to attach flanking BamHI and XhoI restricted sites to facilitate sublimirovanny. These primers are presented in SEQ ID NO:32 and 33 with restrictee Sagami below.

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With the nucleotide immediately 3' to the BamHI site in SEQ ID NO:32 corresponds to the nucleotide 435 in SEQ ID NO:1; G nucleotide 3' XhoI site in SEQ ID NO: 33 is complementary to the nucleotide 1067 in SEQ ID NO:1. Amplificatory I domain hydrolyzed relevant enzymes, purified fragment legasuite in the expression vector of pDCs and the prokaryotic expression vector pGEX-4T-3 (Pharamacia) and a fragment of the I domain sequanorum. Composite protein then expressively in COS, CHO or E. coli cells, transfection or transformed with the appropriate expression construct.

This affinitydfor ICAM-R expressiondI domain may have a sufficient affinity to be a useful inhibitor of cell adhesion involvingd.

It was determined that the detected protein migrates at about 120 D under reducing conditions. Small bands were also detected in non-gels at approximately 40-45 D that were reactive with anti-human, but not antibiotiki antibodies. It was determined by analysis of the Western blot that 200 kD small band is bullish Ig.

Tests for linkage using the expression products of the I domain.

The ability of the I domain to a specific recognition of ICAM-R/IgG chimeric protein was tested in the ELISA format. Serial dilutiondI domain IgG4 protein compound (Id/IgG4) in TBS were preincubator ICAM-1/IgG, ICAM-R/IgG or neuroaxis to IgG1 myeloma protein immobilized on Immulon IV RIA/EIA die. CD11a I domain/IgG chimeric protein and a human IgG4/Kappa myeloma protein were used as negative controls. The binding of IgG4 was detected with biotinylated anti-IgG4 monoclonal antibody NR with posleduyuschimi analyses found no binding of CD11a/IgG4 protein or IgG4 myeloma protein with any of immobilized proteins. Id/IgG4 protein was not associated with fish skin gelatin or blocking agents of bovine serum albumin, human IgG1 or ICAM-1/IgG. Two-three-fold increase in binding signal compared to background was detected in the wells filled with ICAM-R/IgG protein using a concentration of 1-5 µg/ml of Id/IgG4 protein. The signal holes filled VCAM-1/IgG protein was 7-10 times higher than the background level. In a previous analysis d/CD18 transfection Cho cells does not bind VCAM-1/IgG protein, confirming that the VCAM-1 binding may be characteristic of the selected I domain amino acid sequence.

Additional designdI domain.

Additional designdI domain were generated in the same way as the previous design, but with the introduction of a larger number of amino acids arounddI domain. Specific design included: (i) Sequence from exon 5 (amino acids 127-353 in SEQ ID NO:2) preceding the current design, (ii) related to EF replays (EF-hand repeats) (amino acids 17-603 in SEQ ID NO:2), after the I domain and iii)alpha chain, a truncated transmembrane region (amino acids 17-1029 in SEQ ID NO:2) with IgG4 tail for the purposes of purification and detection. These Cassie pGEX-4T-3 (Pharmacia) and the I domain was sequentials. Then integral proteins expressibility in COS, Cho, or E. coli cells, transformed or transfection corresponding expression design. Protein was purified on ProSepA column (Bioprocessing Limited, Durham, England), was tested for reactivity with anti-IgG4 monoclonal antibody NR and were visualized on polyacrylamide gels kulasingam staining.

In order to construct the expression plasmid for full dpolypetide, pATM. D12, described above, was modified for expressiond-IgG4 protein compound as follows. IgG4 encoding DNA was extracted from vector pDCS1 due to PCR using primers that were individually introduced 5' AatII restrictly site (SEQ ID NO:89) and 3' XbaI restrictly site (SEQ ID NO:90).

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Plasmid pATM. D12, hydrolysates AatII and XbaI and respectively hydrolyzed and purified lgG4 PCR product was ligyrophobia in the linker vector.

Example 15

The production of humandspecific antibodies.

A. Production of monoclonal antibodies.

1. Unstable transfection cells from Example 7 were washed three times in Dulbecco phosphate buffered solution (D-PBS) and injected at a concentration of 5106cells/m same as with two-week intervals. Pre-bled and immunitary serum from mice was analyzed by FACS analysis, as outlined in Example 9, and the spleen of the mouse with the highest reactivity to cells, transfections d/CD18, was merged. The hybridoma supernatant cultures were then tested separately for the lack of reactivity against COS cells, transfections CD11a/CD18, and reactivity with cells, cotransfectiondexpression of the plasmid and CD18.

This method did not lead to a monoclonal antibody.

2. As an alternative method for producing monoclonal antibodies, soluble dI domain/IgG4 composite protein was affinity purified from the supernatant steadily transfection Cho cells and used to immunize Balb/c mice as described above. Hybridoma were confirmed and supernatant of these hybridomas were subjected to skanirovaniya using ELISA for reactivity againstdI integral domain protein. Then positive cultures were analyzed for reactivity withd/CD18 complexes expressed on Cho-transfectants.

Mouse 1908 received three initial immunization ddI domain/IgG4 protein compound. The hybrid was produced 270 IgG-producing wells. Supernatant from 45 wells showed using ELISA at least 7-fold greater binding to the Id/IgG4 composite protein than human IgG4. None of supernatant not interacted withd/CD18, transfectional Cho cells, as determined using FACS analysis.

To determine whether supernatant learn integranova alpha subunit protein in a different context, frozen splenic slices were stained with supernatants of 24 holes, out of a total number of 45 holes. It was determined that three of the supernatant are positive: one colored large cells in the red pulp, whereas the other two were stained with scattered cells in the red pulp, as well as in the crossbar.

These supernatant were further analyzed due to their ability to thus biotinylating CD18 complexes from eitherd/CD18, transfection Cho cells, or from PMA-stimulated HL60 cells. Hybrid wells with supernatants who learned the protein in detergent lysates (which should not be in the form of conformidade. Monoclonal antibodies that recognize the protein in detergent, may be more useful in thus heterodimeric complexes of transfectants, tissues and cell lines.

3. As another alternative for producing monoclonal antibody to CD18 complexes were immunoprecipitated from lysates of human seleski anti-CD18 monoclonal antibody 23F2G after enlightenment CD11a/CD18 (using monoclonal antibody TS2/4) and CD11b/CD18 (using monoclonal antibodies Mo-1). Five Balb/c mice at the age of ten to twelve weeks were immunitary subcutaneous injection approximately 30 μg of the obtained protein in complete Freund Freund on day 0, followed by two repeated injections 30 ung immunogen/mouse 28 and 43 days in incomplete Freund Freund. Selected ten sera on the tenth day after the last repeated injections and the reactivity was analyzed using a 1:500 dilution of each serum for the detection of 1 µg/track immunogen in the analysis of the Western blot. Sera from three mice were detected bands of approximately 95 and 150 kD; signal was not seen in the bands, treated with a 1:50 dilution pre-immunization. In addition, all pastamania syrocki immunoprecipitating protein from lysates biotinylated d/CD18 CHO cells, which migrate with the respective molecular weights on SDS-PAGE, was heterodimer. According to these results was selected mouse # 2212 and was further immunizaton via intraperitoneal injection on day 64 30 μg of immunogen in PBS. The mouse was put to death four days later, and spleen were removed under sterile conditions.

Was obtained single-cell suspension by grinding the spleen between the two ends of the frosted glass microscope slides submerged in free serum RPMI 1640, supplemented with 2 mm L-glutamine, 1 mm sodium pyruvate, 100 units/ml penicillin and 100 µg/ml streptomycin (RPMI) (Gibco, Canada). The cell suspension was filtered through sterile 70-mesh Nitex cell strainer (Becton Dickinson, Parsippany, New Jersey), and the filtrate is washed twice by centrifugation at 200 g for 5 minutes. Received tomecek was resuspendable in 20 ml of free serum RPMI. The thymocytes taken from three Balb/c mice, were prepared in the same way.

Before hybridization NS-1 myeloma cells, kept in log phase in RPMI with 10% serum Fetalclone (FBS) (Hyclone Laboratories, Inc., Logan, Utha) within 3 days the but in the above paragraph, and counted. Approximately 210ethe spleen cells were combined with 4107NS-1 cells, and the resulting mixture was concentrated by centrifugation at 200 g. The supernatant was discharged. Cellular bulb was removed by tapping the tube, and 2 ml of 50% PEG 1500 75 mm Hopes (pH 8.0, 37o(C) (Boehhnger Mannheim) was added in one minute under stirring. An additional 14 ml free from serum RPMI was incrementally added over the next seven minutes, followed by immediate addition of 16 ml of RPMI. The resulting mixture was centrifuged at 200 g for 10 minutes, and the supernatant was unloaded. The ball was resuspendable in 200 ml RPMI containing 16 mm FBS, 100 mm hydroxatone sodium, 0.4 mm of aminopterin, 16 mm thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5106thymocytes/ml and dispensed into 96-well flat-bottomed tablets tissue culture (Coming, United Kingdom) with a concentration of 200 μl/well. Cells were loaded with 2, 4 and 6 days after hybridization the absorption of approximately 100 ál from each well of the 18G needle (Becton Dickinson) and add 100 ál/well of microbiological environment described above, except the content of 10 units/well of IL-6 and lacking thymocytes.

At 7-10 days after hybridization of the supernatant from the ynatech, Cambridge, Massachusetts) were filled with 50 µl/well of sheep antimisting IgA, IgG or IgM (Organon Teknicka), diluted 1: 5000 in 50 mm carbonate buffer, pH 9.6, at 4oC. Plates were washed 3X PBS containing 0.5% tween-20 (PBST) was added 50 μl of culture supernatant from each well. After incubation at 37oC for 30 minutes, the wells were washed with PBST as above, and 50 μl of horseradish peroxidase conjugated with sheep artemisinin IgG (fc) (Jackson ImmunoRasearch, West Grove, Prnnsylvaia), diluted 1:3500 in PBST was added to each well. The tablets were proskurovsky, as described above, washed 4 PBST, was added 100 μl of substrate containing 1 mg/ml o-phenylenediamine (Sigma) and 0.1 μl/ml 30% hydrogen peroxide in 100 mm Citrate, pH 4.5. The staining reaction mixture was stopped after 5 minutes by adding 50 μl of 15% sulfuric acid. The absorbance at 490 nm was recorded for each well using the apparatus for reading tablet (Dynatech).

Hybridoma were further characterized as follows. Supernatant of IgG-producing cultures were analyzed by flow cytometry for reactivity tod/CD18 transformed Cho cells, but not the JY cells (b-cell line positive for LFA-1, but not otherd/CD18-JY cells were suspended in 50 μl of RPMI containing 2% PBS and 10 mm NaN3(FACS buffer). Individual cell suspension were added to 50 μl IgG supernatant positive hybridoma cultures in wells of 96-well round-bottom of the plates (Corning). After 30 minutes incubation in ice, the cells were twice washed with a concentration in clinical centrifuge, supernatant from each well was unloaded, and lumps resuspendable in 200-300 μl of FACS buffer. The last wash was replaced with 50 μl/well dilution 1:100 F(ab')2 fragment of sheep antimisting IgG (H+L)-FITC conjugate (Sigma, St.Louis, Mussouri), prepared in FACS buffer. After incubation, as described above, the cells were twice washed Dulbecco''s PBS (D-PBS) with addition of 10 mm NaN3and, finally, was resuspendable in D-PBS containing 1% paraformaldehyde. Then the samples were transferred to polystyrene tubes for analysis of flow cytometry (FACS) analyzer Becton Dickinson FACsan.

Hybridization was given four cultures that are considered positive by both criteria. When the secondary screening was repeated on the increase in volume of supernatant after approximately four days, three of the four cultures remained positive. Three holes marked A, 169 the rija, 16 mm thymidine and 10 units/ml IL-6. Wells tablets clones were counted visually after four days, and was zaregistrirovan the number of colonies in the hole with the lowest density. Selected wells of each clone were analyzed by FACS after 7-10 days. Activity was detected in two cultures A and 169C. In the final cloning of positive wells containing single colonies were increased in volume in RPMI with 11% FBS. Antibodies of clonal supernatants A and 169C were ittipiboon using IsoStrip kit (Boehringer Mannheim) according to the method proposed by the manufacturer, and found that they are of the IgG1 isotype.

Immunoprecipitated/CD18 complexes from SNO of transfectants and PMA-stimulated HL-60 cells was used as the tertiary screening for specificity. Hybridoma A and 169C besieged corresponding strips of SNO lines and samples of single chain 150-160 kD of HL60 cells, as determined by SDS-PAGE. Hybridoma A and 169C were deposited on may 31, 1995 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Meryland 20852 and designated Accession Number HB11907 and HB 11906, respectively.

In order to more fully characterize the binding properties A and 169C, has been studied is th d/CD18. Solubled/CD18 full length was immobilized each unlabeled antibody separately in the format of a 96-hole tablet, and have been used biotinylated antibodies to detect the protein associated with the same or other unlabeled antibodies. Binding was detected using sheep antimisting Ig/HRP conjugate followed by the addition of OPD substrate. The results indicate that the antibody A was capable of blocking the binding of the biotinylated A and TS1/18, while the antibody 169C blocked the binding of only herself.

4. Was selected another mouse (#2214), immunitary with the same Protocol as the mouse #2212, and then immunizaton using pre hybridizing re-immunization for 70 days 30 μg of purifieddfrom splenic lysates in PBS. The mouse was put to death four days later. and spleen were removed under sterile conditions.

Hybridization and cloning of positive cells were carried out as described above. Hybridization has produced five anti-dmonoclonal hybridomas, designated 170D, 170F, T, H and N that were ittipiboon in the form of IgG1using IsoStrip kit (Boehringer Mannheim) according to the methods, the initial Protocol, as mouse #2212 and mouse #2214, and then immunizaton 88 day 30 μg of immunogen and pre hybridizing repeated immunization with 30 μg of immunogen for 203 days. The mouse was put to death four days later, and spleen were removed and held hybridization as described above. Hybridoma supernatant was subjected to skanirovaniya using ELISA capture antibody and using flow cytometry, as described in the above paragraph.

Were identified fifteen positive hybridomas destined A, V, S, E, 188F, 188G, 188I, 188J, K, 188L, M, 188N R, 188R and T and ittipiboon in the ELISA. In short, Immunolon 4 tablet (Dynatech, Cambridge, Massachusetts) were completed at 4oWith 50 µl/well of sheep artemisinin IgA, G, M (Organon Teknicka), diluted 1:5000 in 50 mm carbonate buffer, pH 9.6. The tablets were subjected to blocking for 30 minutes at 37oWith 1% BSA in PBS, washed 3 times with PBS containing 0.05% tween-20 (PBST) was added 50 μl of culture supernatant (diluted 1: 10 in PBST). After incubation and washing, as described above, was added 50 μl of horseradish peroxidase conjugated rabbit artemisinin IgG1, G2aor G3(Zymed, San Francisco, California) diluted 1:1000 in PBST with 1% normal Ovech the CL substrate, containing 1 mg/ml o-phenylenediamine (Sigma) and 0.1 μl/ml 30% hydrogen peroxide in 100 mm citrate, pH 4.5. The staining reaction mixture was stopped after 5 minutes by adding 50 μl of 15% sulfuric acid. AND490was registered by the use of apparatus for the reading of the tablet (Dynatech) for all fifteen of antibodies, and antibodies were determined, which is IgG1.

The excess of the splenic cells from mouse #2211 was frozen in the cryostat and stored in liquid nitrogen. The cryostat was subjected to rapid defrosting by placing in a water bath at 37oWith, and was stirring his it in a circular motion until, until the contents are thawed. Cells were transferred to 15 ml centrifuge tubes, which were slowly added 1 ml of warm RPMI containing 11% FBS, with intervals between the additions of three to five minutes. Was added 5 ml of warm RPMI and after five minutes of waiting the tubes were centrifuged at 200 g for five minutes, and the supernatant was aspirated. Cells were resuspendable in RPMI and was held hybridization as described above. Hybridoma supernatant was characterized using capture antibodies and flow cytometry, as described above.

Hybridization was given five clones, designated A, S, 195D, E and N. Clones were Elena as being IgG2a.

6. In order to identify the ability of antibodies to inhibit functionaldlinking for immunization was used solubled/CD18LZ (see Example 14). Protein was separated on a resin used in affines chromatography of the supernatant unstable transfection COS cells anddassociated with the resin, was used as immunogen. Selected mouse was immunotherapies, as described above, and repeated the final immunization was performed two weeks after the initial immunization. Immunization using this technique prevents the possible changes in the conformation of the protein, often associated with detergent lysis of the cells. Additional mouse immunities recombinant protein is also associated with the resin, but was not originally immunizaton recombinant protein, purified from the cell lysate.

Hybridoma, prepared as described above, which arose after immunization, skanirovaniya using ELISA on recombinant protein immobilized from the cell supernatant using Fab fragment not blocking antibodies. Or used flow cytometry to analyze the reactivity to the JY cell of the monoclonal antibodies were generated as follows. Affinity purifiedd/CD18 heterodimeric protein from detergent lysates of stably respectively Cho cells were used with 50 µg/ml muramyl dipeptides to immunize Balb/c mice as described above. Mice received three immunizations to reactivity sivertone towardsd/CD18-defined immunoprecipitate biotinylated complexes in Cho-transfectants. Hybridoma from positive animals was defined according to standard protocols, and then selected hybridoma culture using flow cytometry usingd/CD18 of transfectants. CD11a/CD18, the transfectants were used in the control of reactivity only for CD18.

8. As another alternative for producing monoclonal antibodies Balb/c mice were subjected to the Protocol of immunization/immunosuppressive designed to reduce reactivity SNO cell determinants on transfectant used for immunization. This Protocol included immunization with retrospektivnyi Cho cells and subsequent destruction SNO-reactive b-cell blasts cyclophosphamide processing. After three laps immunization was carried out and ciclofosfamida processing, and the mouse is about alternative CD18 complexes from detergent lysates of PMA-stimulated HL60 cells were enriched by pre-enlightenment as explained above. Other2integrins shone on the same columns. Immunization received by the complexes, hybridoma production and protocols for screening was performed as described above.

C. Production of polyclonal serum.

PurifieddI domain/IgG4 Chimera (Example 14) was used to generate polyclonal antisera in rabbits. Was held injectiondI domain/IgG4 antigen concentration of 100 μg/rabbit at first in complete Freund Freund, followed by three repeated immunizations with the same amount of protein in incomplete Freund Freund. Feel slopes blood were analyzed after the third and fourth injections. Rabbit immunoglobulin (Ig) was purified from serum by protein a-sepharose column, and the pre-enlightened anti-human IgG to human IgG/Affigel column. Reactivity determined by ELISA in the I domain of the Chimera, but not human IgG, was used to confirm complete pre-enlightenment.

Pre-coated polyclonal serum was used for thus protein from detergent lysates of surface-biotinylated SNO glue which can be found using the method previously described in Example 10. Pre-coated serum recognized a protein complex of the same molecular weight, molecular weight, precipitiously using anti-CDIS monoclinal antibodies TS1.18. In addition, serum recognized a single band of the appropriate size in the analysis of the Western blot, CD18 complexes fromd/CD18 transfection Cho cells. Affinity purified integrins CD11a/CD18, CD11d/CD18 and VLA4 from human spleen were not detected using rabbit polyclonal serum. Serum was not able to interact withd-transfectional SNO cells in solution, as determined using flow cytometry. Therefore, it was concluded that the polyclonal rabbit serum are able to identify only denaturateddI domain/IgG4 proteins.

When attempting the production of polyclonal antisera againstd/CD18, the mouse was immunizaton 3 timesd- transfectional Cho cells (D6.CHO,d/CD18) with adjuvant protein and once cleanedd/CD18-heterodimers. The final re-immunization included onlyd/CD18 heterodimer. Approximately 100 µl impunitivnoj savored Uchenie 2 hours at 4oC. the Obtained serum was analyzed fordreactivity at dilutions 1/5000, 1/10000, 1/20000 and 1/40000 on normal human spleen. Polyclonal antibody was reactive at 1/20000 dilution, while the dilution 1/40000 gave very weak staining.

Example 16


Tissue distributiond/CD18 was determined using antisera generated as described in Example 15.

Purified rabbit polyclonal antibody was used at concentrations between 120 ng/ml and 60 ng/ml for immunohistochemical analysis of frozen sections of human spleen. Sections of 6 μm thickness were distributed in the form of layers in Superfrost Plus Slides (VWR) and stored at -70oC. Before use, the slides were removed from the freezer at -70oWith and placed at 55oWith in 5 minutes. Then the sections were fixed in cold acetone for 2 minutes and dried in the air. Slices were blocked in a solution containing 1% BSA, 30% normal human serum and 5% normal rabbit serum for 30 minutes at room temperature. Primary antibody was added to each slice within 1 hour when contpromise. Then for each slice in the same TBS buffer was added rabbit antimurine associated IgG antibody. Antibody mouse alkaline phosphatase antimelanoma phosphatase (ARAR), proinsurance for 30 minutes at room temperature, was used to detect the secondary antibody. Then the sections were washed 3 times in TBS buffer. Was added as substrate and Fast Blue (Vector Labs) and the color development was stopped by immersion. The slices were subjected to protivorechivuyu in Nuclear Fast Red(Sigma) and washed before being placed on a glass slide Aqua Mount (Baxter). Staining was detected in the splenic red pulp with this reagent, but not with non-drug rabbit polyclonal Ig or untreated pre impunitivnoj serum from the same animals.

It was determined that one mouse serum has a specificdreactivity, it was used for dyeing various lymphoid and non-lymphoid tissues. Monoclonal antibodies recognizing CD18, CD11a, CD11b and CD11c were used in the same experiments as controls. Staining of normal splenic sectionsdpoklonilos serum and monoclonal antibodies to CD11a, CD11b, CD11c and CD18 showed the following results. CD11c and CD18. Was clear picture tagging some cells located in the marginal zone of the white pulp, and clear labelling of cells with peripheral marginal area. This pattern was not observed with other antibodies. Individual cells scattered throughout the red pulp that could be or could not be the same population or subnanogram, prominent with CD11a and CD18, have also been labeled.

Tagging CD11c were not detected in some cells, painted in the marginal zone, but the antibody did not show a clear ring patterns around the white pulp, when compared withdpolyclonal serum, only tagging in the red pulp gave the same pattern of staining, asdpolyclonal serum.

Therefore, the pattern of labeling observed withdpolyclonal serum was unique compared to that which was observed using antibodies with other2the integrins (CD11a, CD11b, CD11c and CD18), and confirmed that the distributiondin humans in vivo is different from the distribution of other2integrins.

Characterization of the humandthe expression of monoclonal antibodies.

Antibodies secreted by the hybridomas A and 169C, were used dleton of peripheral blood leukocytes using flow cytometry. The hybridoma supernatant used in both sets of experiments were undiluted.

Staining of tissue.

All staining procedures were performed as described above except liver slices, which were stained as follows. After fixation with acetone slices were blocked in 1% hydrogen peroxide solution and 1% sodium azide in TBS for 15 minutes at room temperature. After primary or antibody-based test coloring was added rabbit antimurine antibody directly conjugated with peroxidase for 30 minutes at room temperature. Sections three times washed in TBS buffer. Swine anti-rabbit antibody directly conjugated with peroxidase was preincubation for 30 minutes at room temperature to detect the secondary antibody. Then the sections were washed 3 times in TBS buffer and was added AEC substrate (Vector Labs), and color was allowed to occur. The sections were subjected to protivorechivuyu Hematoxylin Gill's No. 2 (Sigma) and then washed in water before dehydration and premises on a glass slide.

In splenic sections most of the expression was localized in the splenic red pulp cells was painted, while small subnavbar macrophages gave the signal. A small amount folicular dendritic cells in the white pulp were also poorly painteddthe antibodies. CD11a and CD18 staining was detected throughout the red and white pulp. CD11c staining was more noticeable in large cells, mainly macrophages in the splenic white pulp and marginal zone surrounding the white pulp; diffuse staining in the red pulp was also noticeable was that CD11b had distribution, overlapping, but not identicaldin the red pulp, without the inclusion of the white pulp.

A comparison was made between integranova expression in normal and (rheumatoid) arthritic synovial tissues. Minimal staining with all antiintegrin antibodies (including antibodies specifically immunoreactive with CD11a, CD11b, CD11c and CD18, as well as withd) was observed in normal tissues with a wide distribution in living cells, mainly macrophages. In the inflamed synovium expression of all integrins was more localized in the cells, clustered around the lymphatic vessels. While expression partnersdand CD11b were similar, CD11c did not show SEB is to, CD11b, but notdexpression was detected in liver macrophages, or Kuppfer cells. Staining of normal human liver slices (as previously described for the coloration of canine liver slice, above) confirms the preservation of this painting partner in humans. In addition, CD11c was found with low levels. In sections from a patient with hepatitis staining all Lagonegro was higher than observed in normal liver, whiledexpression in these samples was detected on macrophages and granulocytes.

Minimal staining of normal human kolorowych sections were observed with anti-dantibodies; observed weak staining of smooth muscle and staining of leukocytes. All LaMontagne were found with higher levels in slices from patients with the disease Crohb's.

Normal lung showed a limited number of poorlyd-positive cells were identified morphologically, these cells are macrophages and neutrophils. In lung tissue from patients with emphysemadstaining was observed on neutrophils and macrophages containing hemosiderin, iron-containing pigment, indicating absorption from patients with multiple sclerosis (MS) on integranova expression. In normal braindthe staining was less intense than the staining of CD11a, CD11b, CD11c, and restrictively in cells morphologically typically presented in the form of microglial cells and CD68 staining. CD11b positive cells were found around the blood vessels and throughout the fabric. CD11c+cells were placed inside blood vessels, whereas+dcells were surrounded by blood vessels. In MS tissue sectionsdexpression was found in microglial cells and necrophagous the leucocytes subnasale;+dcells were located inside damage in the form of plaques, as well as around the outer cover,dthe signal was equivalent in intensity CD11c, but less than the signal CD11b.

Both are slice of the thoracic aorta and abdominal aorta from PDAY (Pathological determinants of atherosclerosis in youth, LSU Medical Center) tissue samples were analyzed antileukotriene and anti-ITSELF antibodies. Investigated the damage was consistent with fatty streaks in the aorta (aortic fatty streaks), composed under the inner shell of the vessel aggregates large xantana cells (mostly macrophages with the absorbed lipids) and infiltrates smaller cells. The research is her (CD11a, CD11b, and CD11c), plus macrofamily marker (CD68), showed that a large part of macrophages loaded with lipids, expressed a moderate leveldand CD18, whereas the expression of CD11a and CD11c was on low or from low to moderate levels, respectively. CD11b was weakly expressed and besides only at the expense of subnebula macrophages.

Research on double tagging were performed to determine the relative localizationdand ICAM-R antigens in sections of the aorta. As Santanyi cells in these sections were stained with antibody Ham 56, specific to makrofagov marker, but not by antibodies actin smooth muscle, it was determined that Santanyi cells are not formed from under the inner shell of the vessel smooth muscle cells. CD68 positive macrophages expressing dwere surrounded and scattered small ICAM-R positive cells. It seems that this is a limitation of the small number of cells that were CD68 negative, but stained by both antibodiesdand ICAM-R.

It turns out that the distributiondin normal tissues were permanent leukocytes in the film overlapping CD11b and CD11c, but not being identical resistnace distribution of leukocytes. Cell morphology indicates thatdstaining mainly limited in macrophages and granulocytes with limited staining of lymphocytes. In General, tissue inflammation, it appears that increased the number and types of cells observed in specific tissues, together with increased staining lakeontario, includingd. As cellular and spatial distribution Lagonegro was not identical in pathological tissues, it was concluded that the distinct functions and ligands exist for each family member, includingdin specific contexts.

It turns out thatdexpression of early atherosclerotic lesions were more prominent than the expression of CD11a, CD11b and CD11c, confirming that thedmay play a Central role in the establishment of these injuries. The attached distributiondand ICAM-R positive cells is supported by data suggesting an interaction betweendand ICAM-R, assuming thatdmay be included in the recovery of white blood cells or activation at early stages in these lesions.

The line of cells and staining of peripheral blood leukocytes.

Antibodies A cladach negatively affected PMA stimulation, which, reportedly induces differentiation along the path of the macrophage, but not affected DMSO, which induces differentiation of granulocytes [Cjllins, et.al., Blood 70:1233-1244 1987)]. FACS profiles A and 169C were antithetical to PMA stimulation in relation to the profiles observed with anti-CD11b and CD11c monoclinal antibodies. Monolta cell line TNR-1 also showed weak staining A and 169C. In addition, Sobibor cells in the lymphocyte and monocytic ways of peripheral blood leukocytes, as it turns out, is weakly positive according to FACS. Subnavbar monocytes peripheral blood poorly painted A and 169C, while it was found that lymphocytes do not have surface expressiond. CD8+subnavbar T lymphocytes was+d. In addition, antibodies A and 169C were not able to detect the antigen In cell lines, JY, Ramos, basophilic line, KU812, and T cell lines, Jukart, SKW and Molt 16.

In the light of the results obtained with HL60 cells granulocytes were isolated from peripheral blood by centrifugation with ficol/giacomin ingredient and subsequent lysis of red blood cells. It was found that all the drugs are more than 90% of the PMA, through visualization of nuclear morfologiia (fMLP) for potential release of intracellular integranova pools. Estimulando populations showed low but significant expression A and 169C antigens compared with IgG1 control with detectable increase in stimulation. In PMNs, levelsdand CD11c surface expression were more similar than the levels that were observed in HL60 cells. Antibody 169C was subsequently used for deposition of heterodimeric molecules of the detergent lysate biotinylated PMNs subunit with approximately 150 and 95 kD correspondingdand CD18, respectively.

The presence of a din PMNs cannot be assumed from the information known about Sabazia dexpression. Canine neutrophils, unlike their human contrasty, Express T helper cell marker CD4 and integrity VLA-4, and therefore may have other ligands and function in dogs than in humans.

Staining of PBL sub-groups.

The present study attempted to determine the distribution of this2integrin in human peripheral blood leukocytes. In addition, we compared the density of cell surface drelative to other2integrins. Finally, strong regulation dexpression in purified by chelovekoorientirovannoe with density gradient in maneadero cell fraction (containing monocytes, lymphocytes and basophils) and granulocytes (neutrophils and eosinophils) [Warner et.al., J. Immunol.Meth. 105:107-110 1987)]. For some experiments, eosinophils were purified using the CD16 immunomagnetic breeding with admixtures of more than 95% [Hansel, et.al., J. lmmunol.Meth., 122:97-103 (1989)] . The skin cells of the breast (skin mast cells) were enzymatic dispergirovannykh leather and enriched, as previously described [Lawrence, et.al., J. lmmunol. 139:3062-3069 (1987)].

Cells were outline of appropriate dilutions of monoclonal antibodies specific for either CD11a(MHM24), Cd11b (NA), CD11c (BU-15) or d(169A). Was also used mouse control IgG1. Cells were washed and then preincubator excess mouse IgG and FITC-labeled mouse monoclonal antibody or sheep polyclonal antibody, specific for a particular cell (e.g., CD3, CD4, or CD8 for T cells; CD16+ lymphocytes for NK cells; anti-IgE for basophils [Bochner, et.al. , J. lmmunol.Meth. 125:265-271 (1989)]. Then the samples were investigated using flow cytometry (Coulter EPICS Profile) using the opening of membrane channels for the identification of cell subnanogram.

For the study of human eosinophils, which was investigated strong sverrehelenadexpression, the cell is 991)], or IL-5 (10 ng/ml) before tagging different monoclonal antibodies, as described above.

The results showed thatdattended all eosinophils, basophils, neutrophils, peripheral blood monocytes and NK cells. Small Sobibor (approximately 30%) CD8+lymphocytes was also found in the expressiond. The skin cells of the mammary gland and CD4+lymphocytes did not expressivelyd. In General, CD11a and CD11b are present at higher densities on leukocytes thandthe latter is expressed at relatively low levels like CD11c. Among leukocytes, monocytes and CD8+cells had the highest densityd, while the eosinophils had the lowest leveldexpression. The expression on neutrophils, basophils and NK cells was intermediate.

Stimulation of peripheral eosinophils CC-chemokine RANTES did not cause changes in the expression of any2integrins. Processing turbolover ether, however, has produced a two-threefold increase in the expression and CD11b andd, but had no effect on the expression of CD11a or CD11c. IL-5 treatment resulted in selective sverhnegativny CD11b expression without affecting the levels of other Inskoy blood dusually expressed with a level comparable to D11. The highest levels were found in monocytes and subnasale CD8+the lymphocytes. Human skin cells of the mammary gland is not expressedd. Purified eosinophils, apparently, had previously formed vnutritelostnoe storage pools CD11c andd. However, differential sverhnegativny shown IL-5 in comparison with HVF, confirms that these storage pools are separated from each other.

Painting staining for sub-groups of peripheral blood leukocytes (PBL) were determined using flow cytometry with a combination of open membrane channel and surface markers, as described above, in an attempt to more accurately determine A/negative limfocitna group. PBL were isolated on Ficoll as described previously, and painted separately A, 169C, and monoclonal antibodies to CD14 (monocyte/macrofamily marker), CD20 (cell), CD56 (NK cell), T cell receptor / (T cell), CD16 (neutrophils, Nks), and 4 (negative marker for neutrophils). The paths were determined by the size and distribution of the marker.

The results indicate that cells in CD14+ monolitom channel show low levels A and 169C OCD is presennol due to the increase in forward scattering. Mixed TCR/CD20+population, apparently, had low, but homogeneous levels A/expression, whereas the population, mapped at a slightly higher side scatter (cell complexest), which were stained with 50% positive populations for CD56, apparently, had a clear A/negative population. Negative population was not recognizable TCP, CD20, CD14 or CD16 antibodies.

Synovial distributiond.

To determine the cellular distribution dother2integrins and their contraception in inflammatory and noninflammatory synovium were used monoclonal antibodies in various2integranova and immunoglobulin supergene families in immunohistological studies. Protein expression was determined in samples of normal, osteoarthritic and rheumatoid synovial tissues.

The results indicate that the layer of synovial lining cells were expressional high levels of VCAM-1, CD11b/CD18 andd/CD18. In these cells CD11c/CD18 expression was limited, and CD11a/CD18 usually were not found. In rheumatoid arthritic synovitis expression2integrins in the synovial cell layer uvelichivayutsa, approaching the ratio of CD11b anddbut there was no increase in CD11a expressii.

In subjectivism space fabric, aggregates and diffuse infiltrates of CD3/CD11a/ICAM-R+lymphocytes were redistributed among CD68/CD11b/dmacrophages. A significant number of units showed intensedstaining, especially in areas enriched in T cells.

Synovial endothelium variable expressed SEM-1 and ICAM-2 with minimal presence of ICAM-R expression.

Taken together, these results indicate that synovial macrophages and macrophagecolony synovial cells Express high levels2integrins CD11b andd. In synovitis is an extension of this subnebula cells in both areas in lined and subsistence together with the apparent increase in the expression of CD11c. Specific populations rheumatoid synovial T lymphocytes in addition to the expression of CD11a and ICAM-R is also expressed high levelsdlast molecule that has been shown above, was expressed at low levels in the peripheral blood lymphocytes.

Example 17.

The allocation of the rat cDNA clones.

In light of the similar genes in other species including rats (this example) and mice (Example 17, below).

Partial sequence of rat cDNA showing homology with humandgene was obtained from rat spleen gt10 library (Clontech). The library was seeded in the plate at 2104pfu/Cup 150 mm LBM/agar Cup. The library was raised on Hybon membranes (Amersham), are denatured for 3 minutes, neutralized for 3 minutes and washed for 5 minutes in buffer, as described in standard protocols [Sambrook, et. al. , Molecular Cloning: a laboratory manual. 0.2.110]. Membranes were immediately placed in a Stratalinker (Stratagene), and DNA stitched using autorevue set. Membranes were prehybridization and hybridized 30% or 50% formamide, laboristic and high rigid conditions, respectively. Membranes were initially skanirovaniya P-labeled sample, generated from humandcDNA corresponding to the bases of 500 to 2100 in the clone A (SEQ ID NO:1). Samples were outline using Boehringer Mannheim's Random Prime kit according to the Protocol suggested by the manufacturer. The filters were washed with 2X SSC at 55oC.

Identified two clones, designated 648.3 and 705,1, which showed sequence homology to people who/SUB> gene in 3 oblasti gene, starting from the base of 1871 and extending to the base 3012 for clone 648.3 and from the Foundation 1551 to 3367 for clone 705.1.

In order to allocate more complete rat sequence, which included the 5' region, the same library was re-skanirovana using the same Protocol that was used for initial screening, but using a mouse sample generated from clone EN 60 (see Example 17 below). Were selected single dedicated plaques from the second screening and supported in the form of single clones in LBM/agar plates. Sequenced primers 434FL and 434FR (SEQ ID NO: 34 and 35, respectively) were used in the standard Protocol to generate DNA for sequencing.

< / BR>
DNA from PCR was purified using Qick Spin column (Quagen) in accordance with the Protocol suggested by the manufacturer.

Identified two clones, designated 741.4 and 741.11 overlapping clones 648.3 and 705.1; in areas of overlapping clones 741.1 and 741.11 were 100% homologous clones 648.3 and 705.1. The composition of the rat cDNA with homology to humandthe gene represented in SEQ ID NO: 36; the predicted amino acid sequence represented >gene using clone set Clonetech rat spleen RAGE in accordance with the Protocol suggested by the manufacturer. Used gene-specific oligonucleotides were labeled 741.11#2R and 741.2#1R (SEQ ID NO:58 and 59, respectively).

< / BR>
Oligo 741.11# 2R includes a pair of bases 131-152 in SEQ ID NO:36, in the reverse orientation and 741.2#1R includes a pair of bases 696-715 in SEQ ID NO:36, also in the reverse orientation. Primary PCR was performed using the big 3' oligo, 741.2# 1R. The second PCR was followed, using oligo 741.11# 2R and DNA generated in the primary reaction. Strip approximately 300 base pairs was detected on 1% agarose gel.

The secondary PCR product was Legerova in plasmid pCRTAII (Invirogen) in accordance with the Protocol suggested by the manufacturer. Were selected white positive colonies and added to 100 μl of LBM, containing 1 ál of 50 mg/ml solution carbenicillin and 1 ál MC ragovoy culture in individual holes in round-bottom 96-well tablets tissue culture. The mixture was preincubation at 37oC for from 30 minutes to 1 hour. After the initial incubation period were added 100 μl of LBM (containing 1 ál of 50 mg/ml carbenicillin and 1:250 dilution of 10 mg/ml of races is inuu 96-well metal comb for migration the supernatant from the 96-well plate was transferred to four nylon filter Amerscham Hybond. Filters were are denatured, neutralized and cross-linked using a standard Protocol. Filters were prehybridization in 20 ml of buffer prehybridization (5X SSPE; 5X Denhardts; 1% SDS; 50 ung/ml DNA denatured salmon sperm) at the 50oWith in a few hours with shaking.

Oligopoly 741.11# 1 and 741.111 R (SEQ ID NO:56 and 57, respectively), including base pair 86-105 (SEQ ID NO:36) in forward and reverse orientation, respectively, was in the state as follows.

< / BR>
Approximately 65 ng oligo DNA in 12 ál dH2O was heated to 65oC for 2 minutes. 3 μl of 10 MCI/ml32P-ATP were added to a test tube with 4 ál of 5x kinase buffer (Gibco) and 1 μl of T4 DNA Kinase (Gibco). The mixture was preincubation at 37oC for 30 minutes. After incubation were added to 16 μl of each labeled oligoprobes to prehybridization buffer and filters, and hybridization was continued overnight at 42oC. the Filters were washed three times in 5x SSPE; 0.1% SDS for 5 minutes per wash at room temperature, autoradiographically within 6 hours. Positive clones were multiplied, and the DNA purified using the selected clone 2F7, showing 100% homology to clone 741.11 in the overlapping region. Full rat sequencedthe nucleic acid represented in SEQ ID NO:54; amino acid sequence presented in SEQ ID NO:55.

Characteristics of rat cDNA and amino acid sequences.

Any nucleic acid or amino acid sequence has not been previously reported for the rat subunits in2the integrins. However, comparison of the sequences with reported human2integranova subunit confirmed that the selected rat clone and its predicted amino acid sequence is most closely related todthe nucleotide and amino acid sequences.

On nukleinovokisly level, the allocation of the rat cDNA clone showed 80% identity, compared with humandcDNA; 68% identity in comparison to human CD11b; 70% identity compared to human CD11c; and 65% identity compared to the mouse CD11b. Significant identity is not observed in comparison with human CD11 and mouse CD11.

At the amino acid level predicted rat polypeptide encoded to vydelennaya with human CD11a; 58% identity in comparison to human CD11b; 61% identity in comparison to human CD11c; 28% identity compared to the mouse CD11a and 55% identity compared to the mouse CD11.

Example 18.

Production and characterizationdspecific antibodies rodents.

A. Antibodies against rat I domain/uIgG4 integral proteins.

In light of the fact that the I domain of human 2integrins, as has been demonstrated, was involved in ligand binding, it has been suggested that the same will occur for ratdthe protein. Monoclonal antibodies immunospecificity for ratdI domain can be so useful in the rat models of human States of sablania, where implicitdthe binding.

Oligopsony of rat alpha-D15 (SEQ ID NO:87) and "rat alpha-D13" (SEQ ID NO:88) were generated from ratdsequence corresponding to base pairs 469-493 and base pairs 1101-1125 (in reverse orientation), respectively, in SEQ ID NO:54. Oligopsony were used in standard PCR reactions to generate rat dThe DNA fragment containing the I domain, which includes the range of par espressomachines. Was selected positive colony and reproduced for DNA purification using Qiagen (Chatswoth, GA) Midi Prep kit according to the Protocol suggested by the manufacturer. DNA was hydrolyzed with XhoI and UD standard restriction the enzyme hydrolysis and strip 600 base pairs was purified on a gel, which was subsequently Legerova in pDCS1/HuIgG4 the expression vector. Was selected positive colony, multiplied, and DNA purified using Qiagen Mahu Prep set.

COS cells were seeded at half the confluence in 100 mm plates with culture and grown overnight at 37oWith 7% CO2. Cells were washed once with 5 ml of DMEM. To 5 ml of DMEM was added 50 μl of DEAE-dextran, 2 ál of chloroquine and 15 µg of ratdI domain/uIgG4 DNA described above. The mixture was added to COS cells and preincubation at 37oC for 3 hours. Then the medium was removed and was added 5 ml of 10% DMSO in CMF-PBS accurately within 1 minute. Cells were gently washed once with DMEM. Ten ml of DMEM containing 10% FBS was added to the cells and incubation continued overnight at 37oWith 7% CO2. The next day, the medium was replaced with fresh medium and incubation continued for three additional days. Wednesday was SOBR the fool was repeated until until it was collected 2 l of supernatant culture.

Supernatant collected as described above was loaded in a Prosep-A column (Bioprocessing Limited), and the protein purified as described below.

The column was initially washed proryvnym buffer in a volume equal to 15 column volumes containing 35 mm Tris and 150 mm NaCl, pH 7.5. The supernatant was loaded on a low speed with a volume less than a volume of approximately 60 column volumes per hour. After loading the column was washed proryvnym buffer in a volume equal to 15 column volumes, 0.55 M diethanolamine, pH 8.5, in the amount equal to 15 column volumes of 50 mm citric acid, pH 5.0, in the amount equal to 15 column volumes. Protein was blueraven 50 mm citric acid, pH 3.0. The protein was neutralized 1.0 M Tris, pH 8.0 and Valitova in sterile PBS.

RatdI domanovic protein was analyzed as described in Example 14. Detected protein migrated in the same manner as described with human I domenovym protein.

C. Production of monoclonal antibodies with ratdI domenovym/uIgG4 integral proteins.

Mice were individually immunitary 50 µg purified ratdI Domanovo/uIgG4 integral protein, pre-emulsified injected in 4 points of the back and flanks of each mouse. Four weeks later, mice were re-immunitary injection of 100 ál of ratdI Domanovo/uIgG antigen (50 μg/mouse), a pre-emulsified in an equal volume of complete Freunds adjuvant (FCA). Additionally, two weeks later, mice were re-immunitary 100 μg of antigen in 200 μl of PBS in the form of intravenous injections.

To assess serum titers at immunitary mice was conducted by retro-orbital phlebotomy animals ten days after the third immunization. Blood was allowed to clot and serum allocated by centrifugation. Serum was used thus on biotinylated (BIP) rat splenocytes. Serum from each mouse was immunoprecipitated protein bands of the expected molecular weight for the ratdand rat CD18. To obtain hybrid was chosen as one mouse and subjected to repeated immunization fourth time, as described above for the third re-immunization.

The hybridoma supernatant were subjected to skanirovaniya using capture antibodies, described as follows. Immunolon 4 plates (Dynatech, Cambridge, Massachusetts) were loaded at 4oWith 50 µl/well of sheep artemisinin IgA, IgG or IgM (Organon Teknika), rubbable is 0 μl of the supernatant of the culture. After incubation at 37oC for 30 minutes and washing, as described above, was added 50 μl of horseradish peroxidase conjugated with sheep artemisinin IgG9(Fc) (Jackson ImmunoReserch, West Grove, Pensylvania), diluted 1: 3500 in PBST. Immediately after this was added 100 μl of substrate containing 1 mg/ml o-phenylenediamine (Sigma) and 0.1 μl/ml 30% H2O2in 100 mm citrate, pH 4.5. The staining reaction products was stopped after 5 minutes by adding 50 μl of 15% H2SO4. The absorbance at 490 nm was recorded on a Dynatech plate reader.

The supernatant from the wells containing antibody was analyzed using ELISA with immunogenum mouse dI domain/uIgG4 composite protein. In the ELISA plates, loaded HuIgG4 antibody served as a control for reactivity against IgG hybrid partner. Positive wells were selected for further validation IN rat splenocytes the lysate using the techniques described below.

C. Production of polyclonal serum in the ratdI Dominova/uIgG4 composite protein.

Two rabbits were previously performed bloodletting before immunization with 100 µg of purified ratdI domenovym Freund Freund''s (IFA). After three injections of rabbits was performed phlebotomy and stored serum used in the standard thus in the rat splenocyte lysates. It was determined that sera from both rabbits were immunoreactive with moused. The rabbits were again re-immunitary 100 ung antigen in IFA, and collected serum was analyzed for increased immunoreactivity with mousedusing thus. The animal was held the final re-immunization, and after 10 days was performed phlebotomy and collected serum.


Rabbit polyclonal serum generated against ratd"I" domain, was used for immunohistochemical staining of sections of rat tissues using the techniques described in Example 16. Painting staining detected on frozen and embedded in paraffin rat splenic sections, was essentially identical to that observed with antibodies against humand, with staining of individual cells throughout the red pulp. Painting staining differed from the pattern observed with monoclonal antibodies against rat CD11a, C is the cortex. None of these tissues did not give any signal when stained rabbit preimmune serum.

D. analysis of the specificity of the antibody.

Rats were euthanized with asphyxia CO2, and spleen were removed using standard surgical techniques. Splenocytes were collected by gentle squeezing of spleen through a wire sieve 3 cm3syringe 20 ml RPMI. Cells were collected in 50 ml conical tubes and washed in a suitable buffer.

Cells were washed three times in cold D-PBS and resuspendable at a density of 108-109cells in 40 ml PBS. Four mg of NHS-Biotin (Pierce) were added to the cell suspension, and the reaction was allowed to proceed for exactly 15 minutes at room temperature. Cells were collected in lumps and washed three times in cold D-PBS.

Cells were resuspendable at a density of 108cells/ml in cold lisinop buffer (1% NP40, 50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 2 mM CaCl; 2 mM MgCl; 1: 100 solution of pepstatin, leupeptin and Aprotinin added before adding to the cells; and 0.0001 g of PMSF crystals added before adding to the cells). Lysates were mixed with pokracovanim for about 30 seconds, Princeville subjected to centrifugation for 10 minutes at 10,000 g to the formation of insoluble material in the form of lumps. The supernatant was collected into new tubes and stored at temperatures between 4oAnd -20oC.

One ml of cell lysate was pre-enlightened by incubating 200 µl And sepharose suspension containing protein (Zymed) overnight at 4oC. Aliquots of pre-enlightened lysate were placed in appendrows tubes at 50 ál/tube for each of the antibodies that were tested. Twenty-five μl of polyclonal serum or from 100 to 500 μl of the supernatant monoclonal antibodies were added to the pre-enlightened to the lysate and the mixture preincubation for 2 hours at 4oWith rotation. Then there was added one hundred μl of rabbit antimisting IgG (Jackson) associated with protein And sivaratnam granules in suspension PBS, and incubation continued for 30 minutes at room temperature with rotation. Pellets were collected by gentle centrifugation and washed three times with cold proryvnym buffer (10 mm HEPES, and 0.2 M NaCl; 1% Triton X-100). The supernatant was removed by suction, and was added 20 μl of 2X SDS buffer containing 10% -mercaptoethanol. The sample was subjected to boiling for 2 minutes in a water bath and the sample was loaded in 5% of the cellulose filters were blocked with 3% BSA in TBS-T for 1 hour at room temperature, and the blocking buffer was removed. Streptavidin-HRP conjugate (Jackson) in 0.1% BSA TBS-T was added, and incubation continued for 30 minutes at room temperature. Filters were washed three times for 15 minutes each TBS-T and autoradiographically using Amerscham's ECL kit according to the Protocol suggested by the manufacturer.

That is, the Production of monoclonal antibodies in rat dprotein full length.

Cleandthe rat protein.

Ratdwas purified from rat splenocytes for preparation of immunogen to generate anticriminedmonoclonal antibodies. Spleen respectively from 50 normal rat female Lewis 12-20 weeks of age were collected, and was prepared single-cell suspensions from tissue by forcing it through a fine wire sieve. Red blood cells were removed by lysis in buffer containing 150 mm NH4Cl, 10 mM knso3, 0.1 mm EDTA, pH 7.4, and the remaining leukocytes were washed twice with phosphate buffer solution (PBS). Splenocytes were transferred into lumps by centrifugation and lysed in buffer containing 50 mm Tris, 150 mM NaCl; 2 mM CaCl2; 2 mM MgCl2; 10 mm PMSF, leupeptin pepstatin, CD11b and CD11c were removed from the splenic lysate using thus as follows. 750 µl volume And sepharose suspension of protein was preincubation 2 mg rabbit antimisting immunoglobulin at 4oC for 30 minutes. Rabbit antimachine And separatly protein was washed three times Lisinym buffer and suspended in a final volume of 1.5 ml lisanova buffer. Approximately 200 μg of each rat 2integranova monoclonal antibodies, 515 F (specific for rat CD11a), OX-42 (specific for rat CD11b) and 100 g (specific for rat CD11c) were added to 50 ml of rat splenic lysate. After 30 minutes incubation at 4oTo it was added 500 μl of rabbit antimisting And Safronova protein to the splenic lysates and mixed with the rotation again and again for 30 minutes at 4oC. the Lysate was centrifuged at 2500 g for 10 minutes to form balls CD11a, CD11b and CD11c associated with rabbit artemisinin And separatim protein, and the supernatant transferred to a clean 50 ml centrifuge tubes. Immunoprecipitate antibodies 515 F, OX-42 and 100 g was repeated two additional times to ensure complete removal of CD11a, CD11b and CD11c.

2integrins remaining in the lysate, the high antibody SW conjugated to CNBr-separati was added to the lysate and mixed with the rotation again and again for 30 minutes atoC. Complexes of antibody/antigen were collected in lumps by centrifugation at 2500 g for 10 minutes, and lumps washed three times Lisinym buffer before storing at 4oC.

Immunization Armenian hamsters.

Armenian hamsters six to eight weeks of age were initially immunitary approximately 50 µg of recombinant protein consisting of the I domain of the ratdhybridizing with a human IgG4 heavy chain, emulsified in complete Freund Freund's. Primary immunization, followed by subsequent immunization rates I domain/uIgG4 was emulsiable in incomplete Freund Freund's 14, 33 and 95 days. It was subsequently made obtaining two separate hybrids identified 197 and 199.

Four days before the hybridization 197 (306 days) one hamster was introduced combination of rat protein, purified from splenocytes and Cho cells, transfection moused. Re hybrid immunization was carried out three days before hybridization (307 days) purified mousedprotein anddtransfectional Cho cells. Ratdtransfection Cho cells were prepared as described below.

EN by electroporation into Cho cells together with human CD18-pRC design. Transfection cells were grown in the presence of gipoksantina to hold a selection of cells, successfully transfection pCR design and in the presence of g418 in order to perform a selection of cells, transfection pDCl design. After three weeks, cells were stained rat dspecific rabbit polyclonal serum and subjected to sorting using FACS. A small number of cells that expressed the highest levels of surfaced(approximately 3% of the total population) were collected and further multiplied. FACS selection was repeated several times to ensure populations of cells with the highest levelsdsurface expression.

dtransfection cells were also characterized using flow cytometry using ratdspecific polyclonal serum and human CD18-specific monoclonal antibodies TS1.18.1. The results confirmed that transfection Cho cells expressed high levels and ratdand human CD18.

Finally,dand CD18 expression in cells was evaluated using thus. It was found that ratdd/human CD18 heterodimeric complex on the surface transfection Cho cells.

On the day of hybridization, the spleen was removed and the resulting single-cell suspension by grinding the tissue between the frosted ends of two glass microscopic slides submerged in serum free RPMI 1640, supplemented with 2 mm L-glutamine, 1 mm sodium pyruvate, 100 units/ml penicillin and 100 µg/ml streptomycin (RPMI)(Gibco, Canada). The cell suspension was filtered through a sterile sieve with cells 70 Nitex mesh (Becton Dickinson, Persyppany, New Jersey), washed twice by centrifugation at 200 g for 5 minutes, and balls resuspendable in 20 ml serum-free RPMI. The thymocytes taken from three native Balb/c mice, were prepared in a similar manner. NS-1 myeloma cells, kept in log phase in RPMI with 10% serum Fetaclone (FBS) (Hyclone Laboratories, Inc. Logan, Utha) for three days prior to hybridization, were centrifuged at 200 g for five minutes, the beads were washed twice as described previously.

Approximately 1.15108splenic cells were combined with 5.810o(50% in 75 ml Hopes, pH 8.0)(Boehringer Mannheim) was added over one minute, followed by adding 14 ml of serum free RPMI, for more than seven minutes. Was added a further eight ml RPMI, and the cells were centrifuged at 200 g for 10 minutes. The supernatant was removed, and the ball resuspendable in 200 ml RPMI containing 15% FBS, 100 mm gipoksantina sodium, 0.4 mm of aminopterin, 16 mm thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.106thymocytes/ml Suspension was distributed into ten 96-well flat-bottomed tablets with tissue culture (Corning, United Kingdom) at 200 μl/well, and cells were discharged on 4, 5, 6 and 7 days after hybridization suction of approximately 100 ál from each well of the 18G needle (Becton Dickinson) and add 100 ál of cultivated environment described above, except for the missing thymocytes.

On day 10 supernatant of hybridized holes were identified on reactivity using flow cytometry to moused/human CD18 transfections Cho cells. Approximately 5105ratdtransfection Cho cells were suspended in 50 μl of RPMI containing 2.0% FBS and 0.05% of sodium azide and added to pribliziteljny included rabbit anti-d- polyclonal serum and TS1/18 (anti-human CD18). Cells were preincubator for 30 minutes in ice, washed three times in FACS buffer (RPMI, 2.0% FBS, 0.05% Na-azide) and preincubator for 30 minutes in ice with FITC-conjugated sheep antimachus antibody (Jackson Immunol Reserch Labs) with a final dilution of 1:200 in FACS buffer. Cells were washed three times in FACS buffer and resuspendable in 200 ml of FACS buffer. The samples were analyzed on a Becton Dickinson FACscan analyzer. In order to ensure that the wells with a positive clones were specific for rat dthe determination was repeated with retrospektivnyi Cho cells, the Wells that met the criterion for reactivity with ratdCho-transfectants and did not meet the criterion of reactivity with retrospektivnyi Cho cells were cloned.

After the initial screening cells from the wells with a positive reaction were cloned initially double dilution and subsequently using limiting dilution in RPMI, 15% FBS, 100 mm gipoksantina sodium, 16 mm thymidine and 10 units/ml IL-6. In stage limiting dilution was determined quantity of holes, in which the observed growth, and predicted clonality with use 10-12 days. After the final clone positive wells were propagated in RPMI and 11% FBS. Cloning gave one culture is considered positive in terms of this criterion, which were replicated four separate subclone marked A-1, A-2, A-3 and A-4.

Before hybridization 199 second hamster was re-immunity 307 day 2.3106ratdtransfection Cho cells. Two final immunization was performed 4 days before hybridization (334 days) and again three days prior to hybridization (335 days). Re-immunization in 334 days consisted of 2106ratdtransfection Cho cells and 200 μl of purified ratdassociated with separate (previously described), introduced by intravenous infusion. Re-immunization 335 day consisted of 5106ratdtransfection Cho cells, also introduced by intravenous infusion. Hybrids and protocols for screening for hybridization 199 were identical hybridization 197, and were identified and cloned three hybridoma marked A, N and M, supernatant, reactive with moused. Hybridoma, destined M, was besieged March 1, 1996 merical Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and assigned to isolate anticriminedantibodies were prepared liver lysates labeled with Biotin as described in Example 18, section E, above. Lysates were pre-clarified prior to use in experiments on thus. Initially, 50 mg/ml normal mouse immunoglobulin was added to the lysate, and the resulting solution was mixed by rotation again and again for 30 minutes at 4oC. 75 μl of the suspension And Safronova protein loaded rabbit artemisinin immunoglobulin was added and mixed with a continuous rotation again and again within 30 minutes. Granules coated with rabbit artemisinin protein And were collected in lumps by centrifugation at 15,000 rpm in a tabletop microcentrifuge for five minutes at 4oC, and the supernatant collected. Material in the form of lumps was unloaded.

For each cloned hybridoma approximately 300 ál suspendant was placed in eppendorff centrifuge tube, to which was added 30 μl of 10% Triton X-100, 30 ál of 100X pre-prepared solution of pepstatin, leupeptin and Aprotinin, 100 μg PMSF crystals and 50 ál of pre-clarified biotinylated lysate rabbit spleen. The samples were carefully pokracovali and pomeshali mg/ml rabbit Anticriminaldspecific polyclonal antibody 50 ál of lysate of rat spleen.

After 30 minutes incubation was added 75 μl of the pellet And Safronova protein in suspension PBS to each sample and preincubation with the rotation again and again at 4oC for 30 minutes. A-coupled protein granules were collected in lumps by centrifugation at 15,000 rpm in a tabletop microcentrifuge for 5 minutes at 4oC, and the supernatant collected. Granules in the form of lumps were sequentially washed with series 1 ml detergent washes as follows. Buffer # 1, containing 10 mm Tris, 400 mm NaCl, 1.0% Triton X-100, pH 8.0; buffer #2, containing 10 mm Tris, 400 mm NaCl, 0.5 Triton X-100, pH 8.0; buffer #3, containing 10 mm Tris, 400 mm NaCl, 1.0% Triton X-100, 0.1% of deoxycholate, pH 8.0 and buffer #4, containing 10 mm Tris, 400 mm NaCl, 0.5 M LiCl2, pH 8.0. Final washing was carried out proryvnym buffer #1. The pellet was carefully pokracovali after each washing and collected in lumps using a desktop microcentrifuge. Supernatant was removed with a portable pipette, and after the last wash the rest of the buffer was removed from the pellet trap Hamilton. 50 µl aliquots of SDS sample buffer containing bromophenol blue and Proninoy Y dyes and Merce 1-2 minutes, and incubated at room temperature for 5-10 minutes. The samples were centrifuged for 5 minutes at 15,000 rpm in a tabletop microcentrifuge at 4oWith and released protein was collected and transferred to new microcentrifuge tubes. Aliquots from each sample was boiled for 4 minutes in a water bath before being loaded into 7.5% SDS-PAGE gels. After separation by PAGE proteins were transferred to nitrocellulose filters for 1 hour at 200 mA, and the filters were blocked in a solution of 3.0% BSA/TBS-T overnight at 4oC. a Solution of 0.1% BSA/TBS-T containing 1:6000 dilution of streptavidin-OPD was added to each filter, and the incubation continued for 1 hour at room temperature. The filters were washed five times for 5 minutes each in TBS-T, shown using Amerscham's ECL kit according to the Protocol suggested by the manufacturer.

It was found that the clone M immunoprecipitated heterodimeric protein. A large part of the protein subunit had an approximate molecular weight of 170-175 kD, which is consistent with the size of the protein, immunoprecipitating rabbit Anticriminal dpolyclonal control. The second protein was precipitated with a molecular weight of about 95 kD, consistent with the weight of CD18.

Example 19

Were cross-species hybridization using PCR-generated samples: 1.5 kb fragment corresponding to bases 522-2047 of human clone A (SEQ ID NO:1), and a 1.0 kb fragment of rat, which corresponded to bases 1900-2900 in human clone A (SEQ ID NO: 1). The human probe was generated via PCR using the primary primernih pairs, designated ATM-2 and 9-10.1, is presented in SEQ ID NO:38 and 39, respectively; rat probe was generated via PCR using the primary primernih pairs 434L and 434R presented in SEQ ID NO:34 and 35

respectively. The samples were preincubator at 94oC for 4 min and subjected to 30 cycles of the processing sequence of the temperature stages: 94oC; 50oC for 2 minutes; 72oC for 4 minutes.

< / BR>
PCR products were purified using Quagen Quick Spin of the dial in accordance with the Protocol suggested by the manufacturer, and approximately 180 ng of DNA was in the state 200 MX[32P]-dCTP using the Boehringer Mannheim Random Primer Labelling kit according to the Protocol suggested by the manufacturer. Nevoshedshy isotope was removed using message-sep Spin column (Princeton Separation, Adelphia, NJ) according to Protocol-HCl, pH 8.0,

Mouse limosna oligo dT-premirovany cDNA library in lambda ZAP II (Stratageen) was placed with a number of approximately 30,000 plaques 15 cm Cup. Blaskova lifts on nitrocellulose filters (Schleicher &Schuell, Keene, NH) were preincubator at 50oWith stirring for 1 hour in a solution of prehybridization (8 ml/rise) containing 30% formamide. Labeled human and rat samples were added to a solution of prehybridization, and incubation continued overnight at 50oC. the Filters were washed twice in 2X SSC/0.1% at room temperature, once in 2X SSC/0.1% SDS at 37oAnd once in 2X SSC/0.1% SDS at 42oC. the Filters were subjected to exposure on film Kodak X-Omat AR at -80oWith over 27 hours with intensifying screen.

Four plaques giving positive signals on duplicate rises, Persiani touch in LB medium with magnesium (L)/carbenicillin (100 mg/ml) in the cups and preincubator overnight at 37oC. Phage plaques were raised Hybon filters (Amerscham), explored in the initial screen and subjected to exposure on film Kodak X-Omat AR at -80oWith over 27 hours with intensifying screen.

Twelve plaques, giving the put the and 1 mm MgCl2. The size of the inserts was determined by PCR amplification using T3 and T7 primers (SEQ ID nos: 13 and 14, respectively) and the following reaction conditions. The samples were preincubator at 94oC for 4 min and subjected to 30 thermal cycles of the processing sequence of stages: 94oC for 15 seconds; 50oC for 30 seconds and 72oC for 1 minute.

Six samples were produced clear bands, which were in sizes from 300 bases up to 1 KB. Family were selected through co-infection with helper phage and recirculatory generate Bluescript SK-(Stratagen). The obtained colonies were cultured in LBM/carbenicillin (100 mg/ml) over night. DNA was isolated using the Promega Wizard minipreparation kit (Madison, WI) according to the Protocol suggested by the manufacturer. EcoRI restriction analysis of purified DNA confirmed the molecular weight, which were detected using PCR. Insertional DNA was sequenced with M13 and M13 reverse 1 primers presented in SEQ ID NO:40 and 41, respectively.

< / BR>
Sequencing was carried out as described in Example 4.

Of the six only two clones, designated 10.3-estalella 68% identity corresponding humandwith 40% identity to human CD11a, 58% identity to human CD11c and 54% identity murine CD11b. This fragment of 600 bp was then used to extract a more complete cDNA encoding mouse allegeddhomolog.

Murine splenic cDNA library (oligo dT and statistically premirovany) in lambda Zapll(Stratagen) was cultivated with a concentration 2.5104phage/15 cm LBM Cup. Plaques were lifted onto Hybond nylon portable membranes (Amerscham), are denatured with 0.5 M NaOH/1.5 M NaCl, neutralized in 0.5 M Tris base/1.5 M NaCl/11.6 HCl, and washed in 2X SSC. DNA was cross-linked to the filters due to UV exposure.

Approximately 500,000 plaques were tested using samples 10.3-1 and 10.5-2, pre-labeled, as described above. Samples were added to a solution of prehybridization and preincubator over night at 50oC. the Filters were washed twice in 2X SSC/0.1% SDS at room temperature, once in 2X SSC/0.1% SDS at 37oAnd once in 2X SSC/0.1% SDS at 42oC, the Filters were subjected to exposure on film Kodak X-Omat AR at -80oWith over 13 hours with an intensifying screen.

Eighteen positive plaques were transferred to phage above. Seven samples gave the signal band, which have a size in the range from 600 bp to 4 kb. EcoRI restriction analysis of purified DNA confirmed the sizes observed from PCR, and DNA was sequenced with primers M13 and M13 reverse 1 (SEQ ID NO:40 and 41, respectively).

One clone, designated V, contained a 4 kb insert, which corresponded to the region of 200 bases of the lower thread 5' end of the humandA clone and included 533 grounds 3' netransliruemoi area. Clone V showed 77% identity corresponding humand, 44% identity corresponding to human CD11a, 59% identity corresponding human CD11c and 51% identity corresponding region of mouse CD11b. The second clone A was 1.2 kb insert, which was added to 5 concu coding region of the humand, 12 nucleic acids of the lower stream of the initiating methionine. Clone A showed 75% identity corresponding humand, 46% identity corresponding to human CD11a, 62% identity corresponding human CD11 and 66% identity corresponding region of mouse CD11b.

Fragment of clone A closer to the 5' end of the human qlprazolam to the base 1134. Clone A contained insertions 110 grounds (grounds 704-814 clone A), not present in the region overlapping clone W. This insertion had taken place, probably at the exon-intron binding [Fleming, et.al., J. Immunol. 150:480-490 (1993)] and was removed prior to subsequent ligation of clones A and B.

Quick ekstragirovanie 5' end of the cDNA alleged mouse dthe clone.

RACE PCR [Frohman, "RACE: Rapid amplification of cDNA ends," in PCR protocols: A Guide Methoda and Applications. Innis, et.al. (eds) pp.28-38, Academic Press: New York (1990)] was used to retrieve lost 5' sequences of the alleged mousedthe clone containing the 5' netransliruemuyu sequence and initiating methionine. Murine splenic RACE-Ready kit (Clonech, Palo Alto, CA) was used according to the Protocol suggested by the manufacturer. Two anticholesterolemic gene-specific primer, A RACE1-primary and A RACE-vnutrenniy (SEQ ID NO: 42 and 43) were designed to perform primary and vnutrivennoi PCR reactions.

< / BR>
Primers SEQ ID NO:42 and 43 correspond to areas beginning 247 302 and bases from the 5 ' end, respectively. PCR was carried out as described above using a 5' anchor primer (SEQ ID NO:44) and murine sales what Merom 280 grounds which was subcloned using the TA cloning kit (Invitrogen) according to the Protocol suggested by the manufacturer. Ten of the resulting colonies were cultured and the DNA isolated and sekvenirovano. Additional 60 bases 5' sequence were identified by this method, which corresponded to the grounds 1-60 in SEQ ID NO: 45.

Characteristics mouse cDNA and predicted amino acid sequence.

The composite sequence of the mouse cDNA encoding the presumed homolog of the humandpresented in SEQ ID NO:45. Although the homology between the external domains of human and mouse clones is high homology between the cytoplasmic domains is only 30%. The observed change may indicate the C-terminal functional difference between human and mouse proteins. Or change in the cytoplasmic domains may arise from the splice variations, or may indicate the existence of additional integranova gene(new).

At the amino acid level murine cDNA predicts a protein (SEQ ID NO:46) with 28% identity murine CD11a, 53% identity to mouse CD11b, 28% identity to human CD11a, Comparison of amino acid sequences of the cytoplasmic domains of humandand predpolagaemogo murine homologue indicates the scope of the same length, but with divergent primary structure. Similar to the length of the sequence in these areas rather confirms the change of the sample than splice variant forms. If to compare with the predicted mouse-polypetides of Example 16, above, mouse and rat cytoplasmic domains show more than 60% identity.

Example 20

The allocation of additional murine cDNA clones for further confirmation.

In order to confirm the nucleic and amino acid sequences described in Example 19 for mousedwere allocated additional mouse sequence to confirm.

Selection of mouse cDNA hybridisation of two homologousdsamples (3' and 5') was performed using both libraries and murine splenic statistical premirovanii library and oligo dT-premirovanii cDNA library in labda ZAPll(Stratagen). Biblioteca was cultivated at 5105phages 15 cm LBM Cup. Plaques were lifted onto Hybond nylon membrane (Amerscham) and membranes were are denatured (0.5 M NaOH/1.5 M NaCl), neutralized (0.5 Tris base/1.5 M NaCl/11.6 M HCl) and projoba were generated using primers described below in a PCR reaction under the following conditions. Samples kept at 94oC for 4 minutes and then was passed through 30 temperature cycles with a sequence of stages (94oC for 15 seconds, 50oC for 30 seconds; 72oC for 1 minute in the apparatus Perkin-Elmer 9600 thermocycler).

3' sample had a length of approximately 900 bases and stretched in the region from nucleotides 2752 up 3651 (SEQ ID NO:1)(5'-3') and was produced by primers 11. b-1/2FOR11 and 11.b-1/2REV2 as shown in SEQ ID NO:69 and 74, respectively. This sample was used in the first set of climbs.

5' sample had a length of approximately 800 bases and stretched in the region from nucleotides 149 to 946 (SEQ ID NO:1)(5'-3') and was produced by primers 11.b-1/2FOR1 and 11.a-1/1REV1 as shown in SEQ ID NO:50 and 85, respectively. This sample was used in the second set UPS.

In the third set UPS both samples described above were used together on the same cups.

Skanirovaniya were subjected to approximately 500,000 plaques using two of the above samples, which was in the state in the same manner as described in Example 17. Labeled samples were added to a solution of prehybridization containing 45% forms is the temperature (22oC). Final washing was performed in 2X SSC/0.1% SDS at 50oC. Autoradiography was carried out for 19 hours at -80oWith film Kodak X-Omat AR with intensifying screen.

Thirteen plaques giving positive signals on at least duplicate rises were subjected to secondary skanirovaniya performed as described for the initial screening except that both 3'and 5' - labeled samples were used for hybridization and introduced an additional final wash using 2X SSC/0.1% SDS at 65oC. Autoradiography was performed as described above for 2.5 hours.

Thirteen plaques (marked from MS2P1 to MS2P13), giving positive signals were transferred into phage diluent with a low content MD++. The size of the inserts was determined by PCR amplification (Perkin-Elmer 9600 thermocycler using primers T3 and T7, which was annealed in Bluescript fahmida in ZAP II (sequence described earlier) under the same conditions as shown above. The sizes of the bands were in the region from 500 bases up to 4 kb. T

1. Hybridoma M, at SS NWO, secreting a monoclonal antibody reactive with a-subunit of rat multi is terisolasi the property it is secreted by hybridomas on p. 1.


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