Method for immunoenzyme analysis for assay of von willebrand factor, monoclonal antibody to von willebrand factor (variants) and strain of hybrid cultured mammal cells mus musculus l - producer of monoclonal antibodies to von willebrand factor (variants)

FIELD: immunology.

SUBSTANCE: invention relates to immunoenzyme analysis and can be used for assay of von Willebrand factor. Method involves immunoenzyme analysis wherein monoclonal antibody 5C3 is used as an immobilizing antibody, and a mixture of biotin-labeled monoclonal antibodies 2H2 and 7D12 is used as a detecting antibody. Also, invention relates to monoclonal antibodies produced by the strain of hybridoma cultured cells Mus musculus L. and directed against von Willebrand factor, and to strains of hybrid cultured cells Mus musculus L. producing indicated monoclonal antibodies. Invention provides the development of highly sensitive method for assay of von Willebrand factor.

EFFECT: improved method for analysis.

9 cl, 1 tbl, 2 dwg, 3 ex

 

The invention relates to the field of biotechnology, and in particular to methods enzyme-linked immunosorbent assay (ELISA), and monoclonal antibodies (monot) for their implementation, in particular to systems for the analysis of von Willebrand factor (PHI), and technologies of their production.

The von Willebrand factor (EF) is a multimeric glycoprotein (GP) of the blood plasma (0.5 to 20×106D)that plays an essential role in the processes stop bleeding (hemostasis), namely in the reactions of vascular-platelet and plasma coagulation hemostasis components. [Barkagan SS Hemorrhagic diseases and syndromes., M., 1988. s-234; Papayan, L.P. Modern problems of clinical coagulatory., L., 1985., p.21-27; T.S. Zimmerman, Z.M. Ruggeri Hum. Path., 1987, 18:140-152]. This protein is synthesized in the vascular endothelium and megakaryocytes in the bone marrow. The synthesized protein is found in the intracellular granules of endothelial cells and platelets (formed from megakaryocytes) and is secreted to the blood plasma.

One of the main function PV is providing adhesion (attachment) of platelets to the damaged area of the vascular wall. PV plays the role of a molecular glue, interacting simultaneously with subendothelial structures of the vessel and its main receptor on the surface of the platelet GP Ib. On the surface of activated platelets PV can also connect Atisa with another receptor GP IIb-IIIa. Through this interaction, along with fibrinogen, participates in the formation of molecular bonds between activated platelets, i.e. in their aggregation.

Participation in PE in the coagulation hemostasis is ensured by its ability to form plasma complex with coagulation factor VIII, in which the PV plays the role of carrier protein and provides a smaller size factor VIII stability during circulation in the bloodstream, increasing the time of his life in the bloodstream and facilitating its transportation to the place of damaged blood vessels.

Hereditary disorders by reducing the number or change the functional properties of PE are the names of Willebrand's disease. This disease represents one of the most common abnormalities of the hemostatic system. [Cm. reviews: Barkagan SS Hemorrhagic diseases and syndromes., M., 1988, s-234; shiffman PD Pathophysiology of blood, "Publishing Binom" - "Nevsky dialect)-SPb., 2000, s-8; Sandler J.E., Davie E.W. Von Willebrand's factor and von Willebrand's disease. In "The Molecular basis of Blood Disease", eds. Stamatoyannopoulos, S., P.W. Majerus, Perlmutter R., Varmus H. W.B. Saunders Company., Philadelphia, London, New York, St. Louis, Sydney, Toronto., 2001, p.697-718; Z.M. Ruggeri Thromb. Haemost, 1999, 82:576-84]. The main manifestations of reducing the level and/or functional activity of PE in patients with von Willebrand's disease are expressed human performance, ha is acarisuga viability of platelet-vascular hemostasis (increase bleeding time, the reduction in the adhesion of platelets and their aggregation in the presence of antibiotic ristocetin), as well as profuse bleeding from mucous membranes due to defective formation of the primary platelet plug. Observation of patients with von Willebrand's disease have also shown that they can have reduced activity of factor VIII in connection with quantitative and/or qualitative defects of carrier protein - PV. Clinically it is manifested by bleeding, which resemble those observed in patients with hemophilia A, with the true defect of synthesis of factor VIII.

It is also known that elevated levels of PV may be a risk factor for thrombotic acute coronary heart disease such as myocardial infarction and unstable angina [Thompson S.G., et al. N. Engl. J. Med., 1995, 332:635-41; Voskoboi IV and other Cardiology, 2002, 42:4-11]. The increase in the concentration of PE in the blood plasma is one of the markers of activation and/or damage to endothelial cells and platelets, which is regarded as one of the significant factors causing the development of thrombotic complications in cardiovascular diseases.

Based on the foregoing, the determination of the plasma level PV is important for the diagnosis of von Willebrand disease and differentiation of some forms of this disease with hemophilia a, as well As for prediction of development of thrombosis. In connection with this representation is aetsa actual creation of systems for the quantitative determination of PE in the blood plasma.

At the present time to determine the PV uses a wide range of methods. So, the content of PE in plasma can be determined by measuring its so-called ristocetin-cofactor activity, i.e. the ability of this protein to induce platelet aggregation by interaction with GP Ib in the presence of antibiotic ristocetin. In various embodiments, this method uses native or fixed platelets and various ways to measure aggregation. In addition, to determine the PV using various immunochemical techniques such as rocket electrophoresis by Laurell and options radioimmunoassay and enzyme immunoassay using polyclonal and monoclonal antibodies (monot) [shiffman PD Pathophysiology of blood, "Publishing Binom" - "Nevsky dialect)-SPb., 2000, s-8: Sandier J.E., Davie E.W. Von Willebrand's factor and von Willebrand's disease in "The Molecular basis of Blood Disease", Eds. Stamatoyannopoulos, S., P.W. Majerus, Perimutter R., Varmus H. W.B. Saunders Company., Philadelphia, London, New York, St. Louis, Sydney, Toronto., 2001, p.697-718].

Definition ristocetin-cofactors activity is a time-consuming method, requiring receipt of donor platelets, the use of special equipment to measure their aggregation and do not allow for simultaneous testing of a large number of samples. In addition, this method is significantly below the sensitivity of most immunochemical IU the W, and get with it, the results can be judged more about activity PE (ability to communicate with GP Ib), but not its concentration. Therefore, when variants of von Willebrand disease with impaired function of this protein, this method can only be used in combination with immunochemical methods.

In recent years, several methods have been developed to study the PV, based on the interaction of soluble antigen-specific antibodies and precipitation of the formed complex. In particular, we have proposed immunoelectrophoresis and immunodiffusion methods, the disadvantages of which are the duration of the study (up to several days), a complex evaluation of the results and most importantly low sensitivity (limit of detection of PV 5-10% of normal values in blood plasma). Immunoradiometric and immunoassay methods are deprived of these defects and allow you to catch a tiny protein PV is less than 1% of the amount present in plasma of healthy donors [Casonato, A., Girolami A. Folia haemat (Lps.), 1986, 113:670-684; Sandier J.E., Davie E.W. Von Willebrand's factor and von Willebrand's disease in "The Molecular basis of Blood Disease", Eds. Stamatoyannopoulos, S., P.W. Majerus, Perimutter R., Varmus H. W.B. Saunders Company., Philadelphia, London, New York, St. Louis, Sydney, Toronto., 2001, p.697-718]. However, the most effective and convenient is the enzyme-linked immunosorbent assay (ELISA), as it is less time consuming and unlike radioimmunoassay methods is e requires the use of radioactively labelled reagents.

Closest to the claimed method according to technical essence and the achieved effect is the method of determining the PV ELISA based on the use of monot from hybridoma strain 380 F2. [Toropova V.G. and other lab. case, 1990, 12:52-55]. The proposed method ELISA includes fixation holes in plastic tablet polyclonal rabbit antibodies to PV production Dakopetts" (Denmark), laundering of phosphate-saline buffer solution, sequential entry in the wells studied plasma at various dilutions, mouse monot to PE (in the form of cultural liquid strain 380 F2) and peroxidase conjugate (antibody to mouse IgG, horseradish peroxidase), incubation tablet and determining the activity of peroxidase, which is part of the conjugate, using a chromogenic substrate at a wavelength of 490 nm. Control of nonspecific binding is performed with normal mouse immunoglobulins. The disadvantages of this method is the use of polyclonal antibodies, because the properties of polyclonal antibodies from batch to batch can vary. In addition, when using Monat possible not only to improve the reproducibility of the method, but also to avoid time-consuming and costly procedure affinity purification of specific polyclonal antibodies from serum.

Along with those mentioned in the previous work is known and the other is the development of manaat to PV. In particular, the well-known Monat MA-82D1E1 and MA-82D6A3, capable of selectively reacting with PV [V I. et al. Haemostasis, 1991, 21:125-134]. Monot were obtained by conventional methods and purified from ascitic fluid of mice BALB/c mice, after growing in the peritoneal cavity cells-producers, using affinity chromatography on protein a-Sepharose. It is shown that during the IFA they appeared to be more promising than polyclonal antibodies, but their characteristics in detail is not described. Also close antibodies and strains to declare are Monat MAb 53 and MAb D7 [Bradley L. et al., Clin. Chem., 1984, 30: R-92), which were obtained by a method that includes immunization FVIIIR:Ag (factor VIII related antigen - previously used the name of the antigen PV) mice of BALB/c, hybridization of spleen cells of the immune mice, selection and cloning hybridoma cells producing antibodies MAb D7 and MAb 53. Detailed description of strains producing and cleaned monot to PV in this work is missing.

Problem to be solved in the framework of the present patent, was the creation of a more effective way of determining PV by ELISA. It has been suggested that the sensitivity of the method can be substantially increased when it is used to determine the PV at the same time three monot to it - one for immobilization and two labeled Biotin for detection of immobilized antigen. For e the CSOs all three antibodies used should not compete with each other for binding to the designated antigen The PV.

The offered method is that as immovable PV antibodies used Monat SS, and as the detecting reagent is a mixture of labeled Biotin Monat N and 7D12.

The use of a mixture of these Monat (N and 7D12) for detection of immobilized PV allows to increase the sensitivity of the method. However, fundamentally it is possible in some cases that do not require high detection sensitivity, be used as the detecting reagent each of these antibodies separately.

Used with monot C, N and 7D12 were obtained from a murine hybridoma cell strains S (NO. RCCC (P) D), 2H2 NO. RCCC (P) 683 D) 7D12 (NO. RCCC (P) 685 D), respectively.

The basic properties of monot ST are:

- belongs to the class of IgGI;

- associated with PV;

- does not compete for binding with PE with monot N and 7D12.

The basic properties of monot N are:

- belongs to the class of IgG2b;

- associated with PV,

- does not compete for binding with PE with monot S and 7D12.

The basic properties of monot 7D12 are:

- belongs to the class of IgGI:

- associated with PV:

- does not compete for binding with PE with monot N and Z.

The ability of the above monot to bind with the PV and the lack of competition between them for binding to this antigen (see example 1) call the lilo to use all three antibodies to create a system ELISA to determine the PV. Antibody IS used as immovable, and labeled with Biotin antibodies N and 7D12 - as detector. The use of a mixture of two antibodies (N and 7D12) for detection of immobilized PV has improved the sensitivity of the ELISA (see example 2). The proposed method allows to determine the PV in the plasma of patients with von Willebrand's disease to diagnose this pathology (see example 3).

To obtain the above Monat used hybrid strains of cultured animal cells Mus. Musculus 5C3, N and 7D12, respectively.

All strains are products merge cells, mouse myeloma X-63.Ag8.653 (subclan P3About1and spleen cells of a mouse immunized with purified PV. Immunization of mice was performed as follows: 25 μg PV intraperitoneally in complete Freund's adjuvant: 2 weeks - 25 µg intraperitoneally in PE in Freund's adjuvant, after 2 weeks intraperitoneally 25 mcg PV, 2 weeks later (3 days before hybridization) - intravenous 5 µg PV. Merging and cloning were carried out according to the method of Keller and Milstein [Kohler g, Milstein C. Nature, 1975, 256:495-7]. Fusion was performed using poly (ethylene glycol) PEG-1500, and selection of hybrid cells by selective environment GAT (gipoksantin - aminopterin - thymidine).

Strains are characterized by the following properties.

The hybrid strain of cultured animal cells Mus. Musculus S.

Line 5 is 3 - the product of the fusion of myeloma cells X.63.Ag8.653 (subclan P3O1) and spleen cells mouse BALB/c mice immunized with PV. The merger carried out using polyethylene glycol PEG-1500. Selection of hybrid cells is carried out using the selective environment GAT (gipoksantin - aminopterin - thymidine). The strain was 3 cloning, positive clones not less than 100%. Now after the last cloning strain was 3 passage.

STANDARD CULTURING CONDITIONS: Medium RPMI-1640 with 20% fetal bovine serum, 4 mm L-glutamine, 1 mm sodium pyruvate, 100 u/ml penicillin and 100 μg/ml streptomycin.

CULTURAL PROPERTIES: For cultivation of strain, you can use the culture flasks. In a bottle size of 25 cm2in 5 ml of medium seeded with 1×106cells. The passage is at a culture density - 1×106cells in 1 ml of medium. For growing cells in the abdominal cavity of mice suitable mouse BALB/c mice. 10 days before the injection of strain mice intraperitoneally injected with 0.5 ml of the Wharf. Strain injected intraperitoneally on 1-5×106cells in the mouse. Ascitic fluid is taken in 10-12 days with the maximum volume of ascites.

KARYOLOGICAL CHARACTERISTICS of STRAIN: a Modal chromosome number 66. Marker chromosome was not detected.

CONTAMINATION: Bacteria and fungi in culture are not found in long-term care the Institute. Test for Mycoplasma negative.

The BIOSYNTHESIS of the USEFUL PRODUCT: the Secretion of monot S in vitro is 5-10 µg/ml, in the ascitic fluid of 2-5 mg/ml when determining the binding to immobilized on plastic PE (see below for selection monot).

CRYOPRESERVATION: the Cells of strain resuspended in fetal bovine serum containing 10% dimethyl sulfoxide at a concentration of 3×106cells in 1 ml, poured into plastic vials at 4°C, is placed in a low temperature refrigerator at -70°C for 24 hours and then transferred into liquid nitrogen. Cells are rapidly thawed at 37°C, diluted in 10 ml of medium without serum precipitated by centrifugation, resuspended in 5 ml of the same medium containing 20% serum, and transferred into a culture flask. Cell viability, determined by the inclusion Trypanosoma blue, is more than 80%.

The strain is deposited in the collection RCCC NO. RCCC (P) D.

The hybrid strain of cultured animal cells Mus. Musculus N.

Line N - product merge cells, P X.63.Ag8.653 (subclan P3About1and spleen cells mouse BALB/c mice immunized with PV. The merger carried out using polyethylene glycol PEG-1500. Selection of hybrid cells is carried out using the selective environment GAT (gipoksantin - aminopterin - thymidine). The strain was 3 cloning, positive clones not less than 100%. At the present time the I after the last cloning strain was 3 passage.

STANDARD CULTURING CONDITIONS: Medium RPMI-1640 with 20% fetal bovine serum, 4 mm L-glutamine, 1 mm sodium pyruvate, 100 u/ml penicillin and 100 μg/ml streptomycin.

CULTURAL PROPERTIES: For cultivation of strain, you can use the culture flasks. In a bottle size of 25 cm2in 5 ml of medium seeded with 1×106cells. The passage is at a culture density - 1×106cells in 1 ml of medium. For growing cells in the abdominal cavity of mice suitable mouse BALB/c mice. 10 days before the injection of strain mice intraperitoneally injected with 0.5 ml of the Wharf. Strain injected intraperitoneally on 1-5×106cells in the mouse. Ascitic fluid is taken in 10-12 days with the maximum volume of ascites.

KARYOLOGICAL CHARACTERISTICS of STRAIN: a Modal chromosome number 66. Marker chromosome was not detected.

CONTAMINATION: Bacteria and fungi in culture were not detected during long-term observation. Test for Mycoplasma negative.

The BIOSYNTHESIS of the USEFUL PRODUCT: the Secretion of monot N in vitro is 5-10 µg/ml, in the ascitic fluid of 2-5 mg/ml when determining the binding to immobilized on plastic PE (see below for selection monot).

CRYOPRESERVATION: the Cells of strain resuspended in fetal bovine serum containing 10% dimethyl sulfoxide at a concentration of 3×106cells in 1 ml, once is more in plastic vials at 4° C, is placed in a low temperature refrigerator at -70°C for 24 hours and then transferred into liquid nitrogen. Cells are rapidly thawed at 37°C, diluted in 10 ml of medium without serum precipitated by centrifugation, resuspended in 5 ml of the same medium containing 20% serum, and transferred into a culture flask. Cell viability, determined by the inclusion Trypanosoma blue, is more than 80%.

The strain is deposited in the collection RCCC NO. RCCC (P) 683 D.

The hybrid strain of cultured animal cells Mus. Musculus 7D12.

Line 7D12 - product of the fusion of myeloma cells X-63.Ag8.653 (subclan P3About1and spleen cells mouse BALB/c mice immunized with PV. The merger carried out using polyethylene glycol PEG-1500. Selection of hybrid cells is carried out using the selective environment GAT (gipoksantin - aminopterin - thymidine). The strain was 3 cloning, positive clones not less than 100%. Now after the last cloning strain was 3 passage.

STANDARD CULTURING CONDITIONS: Medium RPMI-1640 with 20% fetal bovine serum, 4 mm L-glutamine, 1 mm sodium pyruvate, 100 u/ml penicillin and 100 μg/ml streptomycin.

CULTURAL PROPERTIES: For cultivation of strain, you can use the culture flasks. In a bottle size of 25 cm2in 5 ml of medium seeded with 1×106cells. The passage is made upon the achievements the Institute of culture density - 1×106cells in 1 ml of medium. For growing cells in the abdominal cavity of mice suitable mouse BALB/c mice. 10 days before the injection of strain mice intraperitoneally injected with 0.5 ml of the Wharf. Strain injected intraperitoneally on 1-5×106cells in the mouse. Ascitic fluid is taken in 10-12 days with the maximum volume of ascites.

KARYOLOGICAL CHARACTERISTICS of STRAIN: a Modal chromosome number 66. Marker chromosome was not detected.

CONTAMINATION: Bacteria and fungi in culture were not detected during long-term observation. Test for Mycoplasma negative.

The BIOSYNTHESIS of the USEFUL PRODUCT: the Secretion of monot 7D12 in vitro is 5-10 µg/ml, in ascitic fluid 3-6 mg/ml when determining the binding to immobilized on plastic PE (see below for selection monot).

CRYOPRESERVATION: the Cells of strain resuspended in fetal bovine serum containing 10% dimethyl sulfoxide at a concentration of 3×106cells in 1 ml, poured into plastic vials at 4°C, is placed in a low temperature refrigerator at -70°C for 24 hours and then transferred into liquid nitrogen. Cells are rapidly thawed at 37°C, diluted in 10 ml of medium without serum precipitated by centrifugation, resuspended in 5 ml of the same medium containing 20% serum, and transferred into a culture flask. Cell viability, determined by the inclusion of Trifanov the th blue, is more than 80%.

The strain is deposited in the collection RCCC NO. RCCC (P) D.

The selection of specific monot, communicating with PV was performed using ELISA. For this bottom polystyrene 96-well plates were coated with purified PV - 5 µg/ml in PE in phosphate-buffered saline (FSB), pH 7.4, 100 μl per well, 1 hour at 37°C. Not contacting plastic protein was washed FSB containing 0.05% tween-20 (FSB/twin), and blocked designated non-specific binding with 1% bovine serum albumin (BSA) in FSB/Twin 150 μl per well, 1 hour at 37°C. Then the wells were made on 50-100 μl of culture medium containing products secreting hybrid cells (Monat), incubated for 30 min at room temperature, washed wells FSB/Twin, making them peroxidase labeled antibodies goat against mouse IgG (BioRad, USA) (100 μl per well in 1% BSA/FSB/Twin, breeding, proposed by the manufacturer) and incubated for 30 min at room temperature. The wells were washed FSB/Twin and registered the binding of the antibody using a chromogenic substrate (100 µg/ml ortho-phenylenediamine, 0.006% hydrogen peroxide in citrate buffer, pH 4.5, 100 μl per well). The reaction was stopped by adding 50 μl of 50% sulfuric acid and record the optical density in the wells at a wavelength of 492 nm (A).

To obtain preparative amounts of antibodies S, N and 7D12 cell line is adequate producer strains are cultivated in the abdominal cavity of mice BALB/c mice. 10 days prior to injection of cells of mice injected intraperitoneally 0.5 ml of the Wharf. Cells injected intraperitoneally on 1-5×106on the mouse. Ascitic fluid is taken in 10-12 days with the maximum volume of ascites. The selection manout of ascitic fluid is carried out by precipitation with ammonium sulfate and subsequent purification using ion-exchange chromatography on ToyoPearl DEAE 650-M

These strains and produced their Monat are part of the inventive method, combined with it to form a single inventive concept and is aimed at solving common tasks - creating ELISA to determine the PV. This allows us to consider these inventions as part of a group of inventions.

The essence of this group of inventions is illustrated by the following examples.

Example 1. Getting treated Monat C, N and 7D12, linking them with the PV and the lack of competition between them for binding to the antigen.

Obtain preparative amounts of antibodies. Cells of strains S, N and 7D12 was administered intraperitoneally to mice of BALB/c 5×1061 mouse. 10 days prior to the introduction of cells, mice were injected intraperitoneally 0.5 ml of the Wharf. Ascitic fluid was collected after 10-12 days after injection of cells with the maximum volume of ascites. Purification of antibodies from ascitic fluid was performed using salt sulfate precipitation, shumilkin) and subsequent chromatography on ToyoPearl-DEAE 650 M (Tosoh Biosep, USA). To one volume of ascitic fluid was added drip 1 volume of saturated solution of ammonium sulfate and stirred for 1 hour at room temperature. The precipitate was besieged at 10,000 g for 20 min, dissolved in 10 mm phosphate buffer, pH 8.1, and were dialyzed against 10-fold volume of the same buffer (3 shifts for 5 h at 4°). After dialysis and precipitation of insoluble material (10000 g, 15 min, at 4° (C) the antibody solution was applied onto a column of DEAE-Toyo Soda-650 M (not more than 10 mg per 1 ml sorbent), washed with column buffer coating (2 volume of the column) and suirable antibodies linear gradient of phosphate buffer 10-150 mm, pH 8.1 (total volume of eluent 10 column volumes). Protein in the fractions was determined by absorption at a wavelength of 280 nm. Fractions containing protein were tested for antibodies using ELISA using immobilized on plastic PE (see above, selection of monot). Fractions containing antibody were pooled, were dialyzed against 10-fold volume of phosphate-saline buffer (FSB), containing 0.15 M NaCI, 10 mm sodium phosphate, pH 7.4 (3 shifts for 5 h at 4°). After dialysis besieged insoluble material (10000 g, 15 min at 4° (C) and protein content was determined in the solution of antibodies. Antibodies in the aliquot of 1-2 ml were stored at -70°C.

Labelling of antibodies with Biotin. Peeled Monat N and 7D12 was labelled with Biotin using N-Succinimidyl the (Sigma, USA). Antibodies (2-3 mg in 1-2 ml FSB) were dialyzed against 100-fold volume of 100 mm carbonate buffer, pH 8.0 (2 shifts of 5 hours at 4°). After dialysis the solution of antibody was added N-Succinimidyl (freshly prepared solution in DMSO) in a molar ratio of antibody:N-Succinimidyl - 1:40 (volume of added solution of N-Succinimidyl not more than 1/20 of the volume of the solution of antibodies) and incubated for 40 min at 4°and With constant stirring). After incubation, the antibodies were dialyzed against 100-fold volume of the FSB, pH 7.4 (2 shifts of 5 hours at 4°). To labeled Biotin antibody was added sodium azide (final concentration of 0.05%) and kept at 4°With in 2-3 months.

Study the competition between antibodies S, N and 7D12 for binding to PV. Unlabeled antibodies C and N at a concentration of 5 µg/ml in the FSB, pH 7.4 was made in the wells of 96-well polystyrene plates (100 μl well) and incubated 1 hour at 37°C. Rezorbirovanny antibodies were washed FSB/Tween and blocked for nonspecific binding sites with 1% BSA/FSB/Twin room (150 μl per well, 1 hour at 37°). After this was made in the wells pooled plasma of healthy donors (receive pool - see below, example 3) in different dilutions (figa) or purified PE in various concentrations (figb) in 1% BSA/ FSB/Twin (100 μl per well), incubated for 30 min at room temperature and washed nesv is sasisa with antibodies PV FSB/Twin room. Then labeled with Biotin antibody N was made in the wells adsorbed antibody C, and labeled with Biotin antibody 7D12 - in wells with adsorbed antibodies C and 2H2 labeled with Biotin antibody was added at a concentration of 10 μg/ml in 1% BSA in FSB/Twin 100 μl per well), incubated for 30 min at room temperature and washed unbound labeled antibodies FSB/Twin room. After this well was added 100 μl of streptavidin-peroxidase (IMTEK, Moscow), in 1% BSA/FSB/Twin in breeding, suggested by the manufacturer (100 μl per well) incubated for 30 min at room temperature and washed wells FSB-Twin. Antibody binding was detected using a chromogenic substrate as described above (see selecting monot). The results are shown in figure 1, where figa presents the results obtained during the titration of plasma, and PIGB - during the titration of purified PE (both Fig. curves 1-S sorbed + N-Biotin, curves 2 - S sorbed + 7D12-Biotin, curves 3 - N sorbed + 7D12-Biotin). As can be seen from the graphs labeled with Biotin antibody 7D12 effectively communicates with PV, immobilized as using antibodies C and N, and labeled with Biotin antibody N with PV, immobilized with antibodies 7D12. Similar results were obtained using plasma as the source PE and cleansing the aqueous PV. These results indicate that all three antibodies do not compete with each other for binding to PV, i.e. directed against different epitopes in the molecule of the antigen.

Example 2. Enzyme-linked immunosorbent assay was performed according to the method of example 1, using immobilization PV antibody S, and for detection: (1) a mixture of two labeled Biotin antibodies, 2H2 5 μg/ml) and 7D12 (5 μg/ml), (2) labeled with Biotin Monat 2H2 10 μg/ml), (3) labeled with Biotin Monat 7D12 (10 µg/ml). As PV source used pooled plasma of healthy donors (receive - see below, example 3). Figure 2 presents the results of determining the PV used for the detection of a mixture of two Monat (curve 1-N-Biotin (5 mg/ml) + 7D12-Biotin (5 mg/ml)) or each antibody separately (curve 2 - N-Biotin (10 mg/ml) and curve 3 - 7D12-Biotin (10 mg/ml)). As can be seen from this drawing, the definition of PV using two labeled antibodies increases the sensitivity of the method is approximately in 2 times in comparison with the definition that was conducted using only one labeled antibodies, despite the fact that the total antibody concentration in all cases was the same.

Thus, from these data suggest the possibility of using to determine the PV as a mixture of labeled Biotin two monot, N and 7D12, and each of these antibodies separately (which in some cases is up to the adequate and at the same time, it is more profitable).

Example 3. The definition of PE in the plasma of healthy donors and patients with von Willebrand's disease.

Obtaining plasma of healthy donors and patients with von Willebrand's disease. The blood of healthy donors and patients with von Willebrand's disease (all patients were monitored in Hematological scientific center of the Russian Academy of medical Sciences) were collected, using as an anticoagulant in 5% EDTA (pH 7.4) in the ratio of blood:anticoagulant to 9:1. To obtain plasma, the blood was centrifuged at 1500 g for 20 min at room temperature. Selected plasma, for which a complete removal of platelets again centrifuged in microcentrifuge at 10,000 g, 5 min at 4°C. the Plasma was divided into aliquots of 0.1-0.2 ml and frozen at -70°C. Immediately before the definition of PV plasma was rapidly thawed at 37°in the water thermostat. Plasma obtained from 12 healthy donors, were United in a common pool and then divided into aliquots and frozen.

The definition of PV using ELISA. ELISA was performed according to the method of example 1, using for immobilization of antigen Monat C, and for detecting a mixture of two labeled Biotin antibody - N and 7D12. To construct the calibration curve used purified PV. Determined the content of PE in a pool of plasmas of healthy donors, which was taken as 100%, and plasmas 4 patients with von Willebrand's disease.

Measurement ristocetin-cofactor the activity of the studied plasmas. Washed from the plasma of a healthy donor platelets were obtained by the method ([A.I. Vinogradov et al. Biochemistry, 1991, 56, s-97) and suspended at a concentration of 5×108ml In the cuvette of aggregometry (Biola, Moscow) was introduced into 150 μl of washed platelets and 150 µl of plasma in various breeding and then 1.5 mg/ml (final concentration) of ristocetin (Rena, Moscow). Aggregation was detected by the change of transmittance of the suspension of platelets for 3 min 37°and under stirring with a speed of 800 Rev/min Ristocetin-cofactor activity in each sample was estimated by the maximum level of light transmission in the registration process of aggregation. To construct the calibration curve used pooled plasma of healthy donors in various dilutions, ristocetin-cofactor activity which was taken as 100%. Thus, carried out a determination of ristocetin-cofactors activity in plasmas 4 patients with von Willebrand's disease, which also determined the content of the PV using ELISA. Comparison of measurement results PV using ELISA and ristocetin-cofactors activity in patients with von Willebrand's disease is given in the table.

The results of the PV measurement using ELISA and measurement ristocetin-cofactors activity in patients with disease Villebrun the A.
Plasma samplePV (ELISA)Ristocetin-tofactory activity, %
mg/ml%
A pool of plasma donors14,0100100
Patient M (BV* 1 type)7,75550
Patient F. (BV 3 types)1,0718
Patient L. (BV 3 types)2,11512
Patient O. (BV 3 types)0,14113
* VWD - von Willebrand's disease.

From the above data it is seen that as the content of the PV, measured by ELISA, and ristocetin-tofactory activity were reduced in all 4 patients with von Willebrand's disease. 2 patients with von Willebrand's disease type 1 contents PV and ristocetin-tofactory activity were reduced by approximately 50% compared with the pooled plasma of healthy donors, and in 3 patients with von Willebrand's disease type 3, these values were below 20%. It should be noted that two patients (F. and O.) concentration PV was reduced to a greater extent than ristocetin-kovacina activity, possibly due to the appearance in the blood of large multimers of this protein, the result is what these patients, the reduction in the number of PV can be combined with the increase in its functional activity [shiffman FD Pathophysiology of blood, "Publishing Binom" - "Nevsky dialect)-SPb., 2000, s-238].

The obtained results indicate the possibility of application of the developed ELISA for the diagnosis of von Willebrand disease and the feasibility of combining this method with the definition of ristocetin-cofactors activity.

1. Immunoassay method for the determination of von Willebrand factor, which includes the sorption of antibodies to von Willebrand factor in the wells, washing nesorbiruyushchegosya antibody buffer solution, introduction into the wells of the analyzed sample containing von Willebrand factor, immobilization of von Willebrand factor, making the sample detection reagent is an antibody-based, processing samples of a chromogenic substrate and measuring results using spectroscopy, characterized in that as immovable antibodies using monoclonal antibody S, and as the detecting reagent mixture labeled with Biotin monoclonal antibodies N and 7D12.

2. The method according to claim 1, characterized in that as the detecting reagent is used labeled with Biotin antibody N.

3. The method according to claim 1, characterized in that as the detecting reagent is used labeled with Biotin antibody 7D12.

4. Monoclonal antibody S, characterized by the fact that is produced by a strain gibertoni cultured cells Mus musculs L. 5C3, belongs to the class IgG1 directed against von Willebrand factor and does not compete for the binding of von Willebrand factor with the antibody N and 7D12.

5. Monoclonal antibody N, characterized by the fact that is produced by a strain gibertoni cultivated cells of Mus musculus L. 2H2, belongs to the class of IgG2b directed against von Willebrand factor and does not compete for the binding of von Willebrand factor with the antibody S and 7D12.

6. Monoclonal antibody 7D12, characterized by the fact that is produced by a strain gibertoni cultivated cells of Mus musculus L. 7D12 belongs to the class IgG1 directed against von Willebrand factor and does not compete for the binding of von Willebrand factor with the antibody S and N.

7. The hybrid strain of cultured cells of Mus musculus L. 5C3 deposited in RCCC (NO. RCCC (P) D), used as a producer of monoclonal antibody 5C3 against von Willebrand factor.

8. The hybrid strain of cultured cells of Mus musculus L. 2H2 deposited in RCCC (NO. RCCC (P) D), used as a producer of monoclonal antibodies 2H2 against von Willebrand factor.

9. The hybrid strain of cultured cells of Mus musculus L. 7D12 deposited in RCCC (NO. RCCC (P) D), used as a producer of monoclonal antibody 5C3 against von Willebrand factor.



 

Same patents:

FIELD: medicine, mammology.

SUBSTANCE: additionally to cytological research one should study the content of mammary gland due to a solid-phase immunoenzymatic assay for the presence of endometrial protein alpha-2-microglobulin of fertility (AMGF). Detection of AMGF in cystic content enables to prove cytological diagnosis (at 80% potential) of intracystic mammary cancer. Absence of AMFF in the content of mammary cysts enables to prove cytological diagnosis of fibrous-cystic disease at 99% potential.

EFFECT: higher efficiency verification.

2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: method involves bringing antitoxin monoclonal antibody in interaction with a toxic protein, building clusters and analyzing their volume. Toxic proteins are detected when finding clusters exceeding toxic protein molecules in size.

EFFECT: high sensitivity of method.

11 cl

FIELD: medicine, clinical laboratory diagnostics.

SUBSTANCE: at terms of 6-12 wk of gestation one should detect relative content of HLA-DR+ monocytes in peripheral venous blood in pregnant women and at its value being either equal or below 57.9% it is possible to predict the onset of the syndrome of delayed fetal development. The method suggested is sensitive and specific and enables to conduct the complex of curative-prophylactic means simultaneously with predicting techniques.

EFFECT: higher accuracy of prediction.

3 ex, 1 tbl

FIELD: medicine, clinical laboratory diagnostics.

SUBSTANCE: in the sample of peripheral venous blood one should determine relative content of CD45RO+ lymphocytes and at its value being equal to 31% or lower it is possible to diagnose external genital endometriosis. The method is atraumatic and enables to diagnose external genital endometriosis at high accuracy.

EFFECT: higher efficiency of diagnostics.

3 ex, 1 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: during the 1st trimester of pregnancy (6-13 wk) in peripheral venous blood in women at risk of failed pregnancy one should detect relative content of CD16+CD56- lymphocytes and at its value being either equal or below 11% it is possible to predict the development of infectious diseases in full-term neonatals during the first 7-10 d of their lives. The innovation enables to predict the development of local form of infectious-inflammatory diseases in full-term neonatals.

EFFECT: higher accuracy of prediction.

3 ex, 1 tbl

FIELD: medicine, immunological laboratory diagnostics.

SUBSTANCE: at terms from 6 to 12 wk of gestation one should study relative content of CD3+CD16+ lymphocytes in peripheral venous blood and at its values being either equal or above 5.4% one should predict the development of light-degree gestosis to carry out the complex of curative-prophylactic means.

EFFECT: higher efficiency of prediction.

3 ex, 1 tbl

FIELD: medicine, gynecology.

SUBSTANCE: invention relates to a method for diagnosis of internal endometriosis in peripheral venous blood of women wherein the relative content of lymphocytes CD25+ is determined. Internal endometriosis is diagnosed at values of this index 6% or above. Proposed method provides carrying out diagnosis of internal endometriosis in women with high precision, sensitivity and specificity that allows carrying out the correct and well-timed necessary complex of curative-prophylactic treatment.

EFFECT: improved method for diagnosis.

1 tbl, 3 ex

FIELD: medicine, pediatrics.

SUBSTANCE: in 5-10-d-aged neonatals one should detect relative content of CD8+HLA-DR+ lymphocytes in peripheral blood and at its value being below 2.4% it is possible to predict the healing at different types of neonatal infections.

EFFECT: higher accuracy of prediction.

3 ex, 1 tbl

FIELD: medicine, obstetrics.

SUBSTANCE: one should detect during pregnancy-free period relative content of CD38+ lymphocytes in peripheral blood, at its value being either equal to or above 12% one should predict efficient restoration of reproductive function. The method is very simple in application.

EFFECT: higher accuracy and sensitivity of detection.

3 ex, 1 tbl

FIELD: medicine, therapy, obstetrics.

SUBSTANCE: at gestation terms of 20-28 wk in peripheral blood one should detect the index for the ratio of relative content of CD4+ to CD8+ lymphocytes. At values of CD4+/CD8+ being equal to or below 2.4 it is possible to predict positive effect of common therapy, and at values being above 2.4 one should predict thorough and prolonged therapy. The method enables to match another therapeutic tactics in due time.

EFFECT: higher accuracy of prediction.

3 ex, 1 tbl

FIELD: biotechnology, medicinal virology, medicine.

SUBSTANCE: invention describes isolated strain of human hepatitis B virus "S"-133 Ooh comprising genome of subtype adr that is deposited in the European collection of cellular cultures at № P 97121504 or P 97121505 or P 97121506 and encoding polypeptide of basic surface antigen of human hepatitis B virus. Abovementioned antigen comprises mutation in amino acid sequence (polypeptide), namely, methionine at 133 position is replaced with threonine. Invention discloses variants of isolated nucleic acids encoding the mutated polypeptide and variants of isolated nucleic acids encoding peptides showing properties of the indicated polypeptide. The description text gives nucleotide sequences of nucleic acid variants encoding abovementioned polypeptide and peptides. Also, invention describes variants of vectors used in cloning DNA fragments of mutant human hepatitis B virus comprising isolated nucleic acid and isolated nucleic acid bound functionally with RNA transcription promoter respectively and wherein each isolated nucleic acid encodes indicated polypeptide. Invention discloses methods for producing polypeptide and peptide and methods for preparing the purified polypeptide and peptide by using indicated vectors. Also, invention describes purified polypeptides and peptides of mutated antigen retaining antigenic properties of polypeptides of the natural human hepatitis B virus, and methods for preparing antibodies raised to them and anti-antibodies, among them, monoclonal antibodies. Also, invention discloses different methods for using abovementioned isolated nucleic acids, polypeptides and peptides for aim for diagnosis and treatment of diseases associated with human hepatitis B virus. Using the proposed invention provides the development of diagnosis schedules and treatment of diseases associated with the mutated human hepatitis B virus.

EFFECT: valuable medicinal properties of mutant virus.

66 cl, 2 dwg, 4 ex

FIELD: biotechnology, medicine, in particular viral disease treatment.

SUBSTANCE: invention relates to recessive dividing retroviral vector useful in inhibition of wild-type retrovirus replication. Vector contains retroviral long terminal repeat sequences; retroviral packing signal; nucleotide sequence encoding (expressing) genetic antiviral agent; and optionally the second nucleotide sequence. Disclosed are method for production of said vector and reproduction thereof. Further isolated and purified nucleic acid (NA) molecule providing of selective advantage in regard to viral generation packing into virions is disclosed. Uses of retroviral vector in particular for specific antibody production are described.

EFFECT: new genetic antiviral agents generating prolonged and stable immunological responses in regard, for example, to AIDS and cancer viruses.

97 cl, 11 ex

FIELD: biotechnology, virology.

SUBSTANCE: invention relates to preparing a new strain of hybrid cells of Mus musculus L., NIIMB-280 (9E2), as a producer of monoclonal antibodies to the West Nile virus (WNV) protein E. West Nile virus (strain WNV/LEIV-VIg99-27889) is isolated in Volgograd district in 1999 year from a patient. Producing monoclonal antibodies can be used effectively for detection of the strain WNV/LEIV-VIg99-27880 of WNV that causes human diseases in Russia territory. New hybrid strain of cells is obtained by fusion of murine myeloma cells p3-X63/Ag8.653 (NS0/1) with murine splenocytes BALB/c immunized with the purified and inactivated preparation WNV (strain WNV/LEIV-VIg99-27889). The strain of hybrid cells Mus musculus L., NIIMB-280 (9E2), is deposited in Collection of cellular cultures of NII cellular cultures GNTS VB "VEKTOR" at number № NIIMB-280. Author's name of hybridoma cellular strain is 9E2. Using hybridoma allows preparing specific monoclonal antibodies raised to the West Nile virus protein E that, in turn, gives a possibility for identification of WNV and to standardize the content of protein E in immunodiagnostics.

EFFECT: valuable properties of strain.

1 dwg, 3 ex

FIELD: biotechnology, hybridoma technology.

SUBSTANCE: hybridoma strain is prepared by fusion of murine plasmocytoma Sp2/0-Ag.8 and B-lymphocytes of murine spleen of the inbred strain BALB/c immunized with protein-polysaccharide complex from Y. enterocolitica. Hybridoma produces monoclonal antibodies of isotype IgG to Y. enterocolitica O3 and O9 serovars used as components of IFA-test-system for identification of indicated serovars that are isolated most often in European areas from sick humans, agricultural animals and from objects of environment. The usage of monoclonal antibodies producing by hybridoma allows carrying out the identification of Y. enterocolitica strains of indicated serovars representing the most epidemic danger among other intestine-persistent microorganisms. Invention can be used in the development of diagnostic test-systems for identification of Y. enterocolitica strains O3 and O9 serovars for aims laboratory diagnosis in the public health, veterinary science and in carrying out scientific investigations.

EFFECT: valuable properties of strain.

1 tbl, 2 ex

FIELD: immunology; treatment of mediated diseases IL-1 and failures.

SUBSTANCE: bonding molecule IL-1β which is antibody to human IL-1β and especially human antibody to human IL-1β where hypervariable sections CDRs of heavy and light chains have definite amino acid sequences. Antibody may be used for treatment of mediated disease IL-1, for example osteoarthritis, osteoporosis and other inflammatory processes of bones of rheumatism or podagra nature. Constructions of deoxyribonucleic acid are described which code heavy and light chains or their fragments and expressive vectors which may be replicated in cells including deoxyribonucleic acid constructions. Method of obtaining bonding molecule IL-1β by means of cell transformed by vector is described. Proposed antibody may be used both in prophylactic and treatment of diseases.

EFFECT: enhanced efficiency.

15 cl, 3 dwg, 5 ex

FIELD: medicine, immunobiology, pharmacy.

SUBSTANCE: humanized monoclonal antibody (monAb) or its fragments comprises heavy and/or light chain with the binding rate constant with AILIM 1.0 x 103 (1/M x s) and above, and the dissociation rate constant between monAb and AILIM 1.0 x 10-3 (1/s) or less. MonAb shows also a nucleotide sequence encoding variable region of light and/or heavy chain and corresponding amino acid sequences. Invention relates to DNA and it part encoding monAb or its fragments, and vectors comprising nucleotide sequences encoding antibody or its fragments. The humanized monAb can be prepared by using a genetically recombinant host. MonAb is comprised as a component of pharmaceutical compositions used for inhibition or induction of AILIM-mediated transfer of signal into cell for induction of antibody-dependent cytotoxicity against AILIM-expressing cell and others. Invention can be effective in treatment of different autoimmune diseases associated with AILIM-mediated transfer of co-stimulating signal. Invention can be used in medicine for treatment of diseases associated with AILIM-mediated transfer of co-stimulating signal.

EFFECT: valuable medicinal properties of antibody.

75 cl, 78 dwg, 14 ex

FIELD: biology, hybridoma technology.

SUBSTANCE: invention represents a new strain of mammalian hybrid cells C3/S-3E5 of Mus musculus L. producing monoclonal antibodies (MCAb) to Bernet's coxiellas (strain "Grita") in cell cultures and abdominal cavity of syngenic animals. Hybridoma C3/S-3E5 producing MCAb to this pathogen is obtained by fusion of murine myeloma of strain Sp-2/0 and murine splenocytes of strain BALB/c immunized with the concentrated and purified Bernet's coxiella preparation (strain "Grita) inactivated with formalin using polyethylene glycol of molecular mass 1000 Da as a fusing agent and the following cloning by method of maximal dilutions. Specificity of prepared MCAb: absence of cross-reactions in IFA with Provacheck's rickettsia antigen and with the non-infected accumulation substrate. Using prepared MCAb it is possible to carry out specific detection of Bernet's coxiellas by method IFA (direct and indirect variants). IFA sensitivity based on these MCAb is 2.0 x 103 ID50 x cm-3 for white rats. Applying the present invention allows detecting and identifying pathogens of rickettsial etiology.

EFFECT: improved method preparing, valuable properties of strain.

3 tbl, 1 dwg, 1 ex

FIELD: biology, biotechnology, medicine.

SUBSTANCE: strain of murine hybridoma cells is prepared by fusion of murine splenocytes immunized with cell lysates of lymphoblastoid line RAMOS with cells of murine myeloma. Hybridoma secrets monoclonal antibodies directed to antigen of molecular mass 110 kDa located on microtubules in cell cytoplasm and detected in nuclear cells of different species. Using the invention allows studying pathogenesis of cell dividing and state of mitotic activity of health and malignant cells, and evaluation of anti-mitotic (anti-tumor) effect of different chemopreparations and substances. Invention can be used for identifying phase of mitotic activity of cells.

EFFECT: valuable properties of strain.

2 dwg

Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

Up!