Solid phase enzyme-immunoassay (eliza) for vascular endothelial growth factor (vegf)

FIELD: medicine.

SUBSTANCE: invention relates to the field of immunology, namely to enzyme-immunoassay, in particular to a method of detecting forms of vascular endothelial growth factor (VEGF) with a size more than 110 amino acids in a biological sample. The method includes the following stages: contact and incubation of the biological sample with an uptake reagent, immobilised on a solid substrate, where the uptake reagent contains a monoclonal antibody, which recognises and specifically binds with residues, in quantity more than 110, from human VEGF; separation of the biological sample from the immobilised uptake reagents; contact of the immobilised molecular complex of the reagent of the uptake-target with detected antibody, which binds with VEGF domains, responsible for binding with KDR and/or FLT1 receptor, or which binds with an epitope in VEGF_1-110; measurement of the level of VEGF₁₁₀₊, bound with reagents of the uptake, with application of means of detection for the detected antibody. Set of immune assay reagents for detection of VEGF₁₁₀₊ forms in the biological sample. An antibody 5C3, obtained from hybridoma 5C3.1.1 with a depositary number PTA-7737, with the said antibody 5C3 binding VEGF₁₁₀₊ forms, including VEGF₁₂₁₊. Hybridoma 5C3.1.1, deposited in ATCC with the depositary number PTA-7737, to obtain the monoclonal antibody 5C3.

EFFECT: application of the claimed invention makes it possible to increase accuracy of detecting VEGF isoforms, which must not include isoform VEGF₁₁₀ and must obligatory include isoform VEGF_121.

25 cl, 3 dwg, 2 tbl, 1 ex

A RELATED APPLICATION

This application claims the priority and benefit of Provisional Application U.S. No. 60/828203, filed October 4, 2006, the description of which is included in this description completely.

The technical FIELD

The present invention relates to immunoassays for the detection of certain populations of VEGF, which can be used as diagnostic and prognostic methods for patients with cancer, cardiovascular or other diseases.

The LEVEL of TECHNOLOGY

By now well established that angiogenesis is involved in the pathogenesis of various disorders. These violations include solid tumors, intraocular neovascular syndromes, such as proliferative retinopathy or age-related macular degeneration (AMD), rheumatoid arthritis and psoriasis (Folkman et al. J. Biol. Chem. 267:10931-10934 (1992); Klagsbrun et al. Annu. Rev. Physiol. 53:217-239 (1991); and Garner A, Vascular diseases. In: Pathobiology of ocular disease. A dynamic approach. Garner A, Klintworth GK, Eds. 2^ndEdition (Marcel Dekker, NY, 1994), pp. 1625-1710). In the case of solid tumor neovascularization provides the ability of tumor cells to acquire advantage of the growth and proliferative autonomy in comparison with normal cells. Consequently, there was a correlation between the density of microvessels in the tumor sites and survival of patients suffering from breast cancer, and also elshimi some other types of tumors (Weidner et al. N Engl J Med 324: 1-6 (1991); Horak et al. Lancet 340: 1120-1124 (1992); and Macchiarini et al. Lancet 340: 145-146 (1992)).

Search positive regulators of angiogenesis has led to many candidates, including, for example, aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-α, angiogenin, IL-8, etc. (Folkman et al., above, and Klagsbrun et al., above). Some negative regulators identified to date include thrombospondin (Good et al. Proc. Natl. Acad. Sci. USA. 87: 6624-6628 (1990)), the N-terminal fragment of prolactin with a molecular mass of 16 kilodaltons (Clapp et al. Endocrinology, 133: 1292-1299 (1993)), angiostatin (O'reilly et al. Cell 79: 315-328 (1994)), and endostatin (O'reilly et al. Cell 88: 277-285 (1996)).

Using the work done in the last few years, have established the key role of growth factor vascular endothelial (VEGF) in the regulation of normal and abnormal angiogenesis (Ferrara et al. Endocr. Rev. 18: 4-25 (1997)). The discovery that the loss of even a single VEGF allele results in fatal cases, embryos, points to the essential role played by this factor in the development and differentiation of the vascular system (Ferrara et al., above).

In addition, it was shown that VEGF is a key mediator of neovascularization associated with tumors and intraocular disorders (Ferrara et al., above). mRNA VEGF sverkhekspressiya most studied human cancers (Berkman et al. J Clin Invest 91: 153 to 159 (1993); Brown et al. Human Pathol. 26: 86-91 (1995); Brown et al. Cancer Res. 53: 4727-4735 1993); Mattern et al. Brit. J. Cancer. 73: 931-934 (1996); and Dvorak et al. Am J. Pathol. 146: 1029-1039 (1995)). The concentration of VEGF in ocular fluids significantly correlated with the presence of active proliferation of blood vessels in patients with diabetic and other related ischemia with retinopathy (Aiello et al. N. Engl. J. Med. 331: 1480-1487 (1994)). In addition, studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients with acute macular degeneration (AMD) (Lopez et al. Invest. Ophtalmo. Vis. Sci. 37: 855-868 (1996)).

VEGF is produced in the tissues, and it is not necessary to enter the bloodstream for the manifestation of its biological effect, but rather it acts locally as a paracrine regulator. In a recent study, described in the publication Yang et al. J. Pharm. Exp. Ther. 284: 103 (1998), it was found that the removal of rhVEGF₁₆₅from the bloodstream very rapidly, suggesting that the presence of endogenous VEGF in the blood stream, most likely, is the result of continuous synthesis of VEGF. In addition, in some studies, attempts were made to correlate the levels of circulating VEGF with the severity of the tumor, and it was proposed to use the levels of VEGF as a potential prognostic marker (Ferrari and Scagliotti Eur. J. Cancer 32A: 2368 (1996); Gasparini et al. J. Natl. Cancer Inst. 89: 139 (1997); Kohn Cancer 80: 2219 (1997); Baccala et al. Urology 51: 327 (1998); Fujisaki et al. Am. J. Gastroenterol. 93: 49 (1998)). Obviously, the ability to accurately measure VEGF is important for understanding its potential role(s) in many biological processes, such as maintaining the patency of blood vessels, menstrual cycle, ischemia, diabetes, cancer, intraocular disorders, etc.

In publications concentrations of endogenous VEGF is widely varied in healthy and ill patients, being in the range from netdetective to a high level. The ability to measure the level of endogenous VEGF depends on the availability of sensitive and specific assays. Published options enzyme-linked immunosorbent assays (ELISA) for VEGF-based colorimetric, chemiluminescent and fluorescent methods. Houck et al., above, (1992); Yeo et al. Clin. Chem. 38: 71 (1992); Kondo et al. Biochim. Biophys. Acta 1221: 211 (1994); Baker et al. Obstet. Gynecol. 86: 815 (1995); Hanatani et al. Biosci. Biotechnol. Biochem. 59: 1958 (1995); Leith and Michelson Cell Prolif. 28:415 (1995); Shifren et al. J. Clin. Endocrinol. Metab. 81: 3112 (1996); Takano et al. Cancer Res. 56: 2185 (1996); Toi et al. Cancer 77: 1101 (1996); Brekken et al. Cancer Res. 58: 1952 (1998); Obermair et al. Br. J. Cancer 77: 1870-1874 (1998); Webb et al. Clin. Sci. 94: 395-404 (1998).

For example, in the publication Houck et al., higher (1992) described a colorimetric ELISA method, which, obviously, has a sensitivity of the order of ng/ml, which is not sufficiently sensitive for detection of endogenous levels of VEGF. In the publication Yeo et al., higher (1992) described dvuhsvetny immunofluorescence analysis with a resolution on the straps, however, in normal serum VEGF is not detected (Yeo et al. Cancer Res. 53: 2912(1993)). In the publication Baker et al., above (1995)described that using a modified version of this immunofluorescent analysis was watching detected levels of VEGF in the plasma of pregnant women, with higher levels in women with preeclampsia. Similar data for pregnant women were described in the publication of Anthony et al. Ann. Clin. Biochem. 34: 276 (1997) using radioimmunoassay. Hanatani et al., above (1995) developed a chemiluminescent ELISA method capable of measuring the level of circulating VEGF, and in the publication described that the level of VEGF in the serum of 30 healthy individuals (men and women) is from 8-36 PG/ml In the publication Brekken et al above (1998) described the ELISA assays using antibodies preferably bind or separate VEGF, or VEGF complex:F1k-1.

The ELISA kit for the detection of VEGF is commercially available from the firm R&D Systems (Minneapolis, MN). Set R&D VEGF ELISA was also used in sandwich assays, where a monoclonal antibody is used to capture the target in the form of VEGF antigen and polyclonal antibody is used for detection of VEGF. Webb et al. above (1998). Cm. also, for example, Obermair et al. above (1998).

Keyt et al. J. Biol. Chem. 271: 7788-7795 (1996); Keyt et al. J. Biol. Chem. 271: 5638 (1996); Shifren et al., above (1996) also developed a colorimetric ELISA method based on the dual is ary monoclonal antibodies. Although this method ELISA was capable of detecting elevated levels of VEGF in patients with malignancy, it lacks the sensitivity required for the measurement of endogenous VEGF levels in healthy individuals. Rodriguez et al. J. Immunol. Methods 219:45 (1998) described dvuhsvetny fluorescent method VEGF-ELISA, which gives the sensitivity, component 10 PG/ml VEGF in the initial plasma or serum. However, this fluorescence analysis detects fully intact samples 165/165 and 165/110 VEGF (it has Been described that VEGF 165/165 can be proteoliticeski split into three other forms: heterodimer 165/110, glycosilated 110/110 and C-terminal fragment of 55 amino acids (Keyt et al. J. Biol. Chem. 271: 7788-7795 (1996); Keck et al. Arch. Biochem. Biophys. 344: 103-113 (1997))).

Thus, there is a need to develop diagnostic and prognostic analysis, which would have detected higher measured levels of VEGF in a biological sample experimental animal models or patients than current ELISA methods, and/or could measure the different VEGF isoforms.

The INVENTION

Methods ELISA antibody-sandwich-type VEGF as antigen have been developed for detection of forms of VEGF in a biological sample. The way VEGF ELISA offered by the present invention, capable of detecting the VEGF isoforms and fragments of VEGF with a size of more than 110 amino acids ("VEGF 110+"). Also available sets.

For example, methods of detection of selective forms of growth factor vascular endothelial (VEGF) with a size of more than 110 amino acids (VEGF₁₁₀₊) in a biological sample involves the following stages: (a) contacting and incubating a biological sample with a capture reagent immobilized on a solid substrate, where the capture reagent is an antibody that recognizes the same epitope as the antibody 5C3 against human VEGF, and the specified monoclonal antibody specific contacts with residues in more than 110 of human VEGF; (b) separating a biological sample from the immobilized capture reagents; (c) contacting the immobilized molecular complex of the capture reagent-target detektivami antibody which binds to the domains of VEGF, which is responsible for binding to receptor KDR and/or FLT1; and (d) measuring the level of VEGF₁₁₀₊associated with the capture reagents, using a detection means for the detected antibodies. In certain embodiments detective antibody binds to the epitope in VEGF 1-110. In certain embodiments of the comparative analysis of the ELISA can be performed for detection of different types of VEGF. In certain embodiments the biological sample (for example, samples of tumor or tumor lysates, plasma, serum or urine, etc.) select the person.

In one embodiment, the capture reagent is a monoclonal antibody 5C3. In one embodiment, the immobilized capture reagent is applied as a coating on microtiter plate. In certain embodiments detective antibody is a monoclonal antibody. In one embodiment detective antibody is a mouse monoclonal antibody. In one embodiment, the immobilized monoclonal antibody is an antibody MAb 5C3 and detective antibody is an antibody MAb A4.6.1. In certain embodiments detective antibody is directly detektivami. In one embodiment detective antibody amplificates using the colorimetric reagent. In one embodiment detective antibody biotinylated and means of detection is avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethylbenzidine.

In certain embodiments of the invention the person is a patient with vascular, diabetes or cancer, and stage of measurement (d) further includes comparison with a standard curve to determine the level of VEGF in comparison with the level in a healthy individual.

Also available sets. For example, the kit immunoassay for detection form factor vascular endothelial growth (VEGF), containing b is more than 110 amino acids (VEGF ₁₁₀₊), in a biological sample comprises: (a) the anti-VEGF antibody of the person, as the capture reagent, where a monoclonal antibody specific contacts with residues in more than 110 of VEGF person; and (b) detective antibody as a detection reagent that binds to domains of VEGF, which is responsible for binding to the receptor, KDR and/or FLT1. In certain embodiments detective antibody binds to the epitope in VEGF 1-110. In certain embodiments the kit further includes a solid substrate reagent capture. For example, the capture reagents can be immobilized on a solid substrate (for example, the microtiter plate). In certain embodiments the kit further includes a detection means (for example, a colorimetric agent, fluorescent agent, etc.) for detecting antibodies. In certain embodiments the kit further includes a purified VEGF as a standard antigen. In certain embodiments of the invention additional analysis of VEGF-ELISA or more may be offered for comparative studies using VEGF₁₁₀₊-ELISA. In one embodiment the kit includes a capture reagent in the form of monoclonal antibodies, which is a mouse monoclonal antibody MAb 5C3 and detective antibody, which is a MAb A4.6.1.

In another embodiment of the invention features an antibody 5C3 against VEGF (derived from hybridoma deposited in the ATCC under number PTA-7737 or produced by the specified hybridomas). The invention also features an antibody that does not bind to VEGF 1-110, and binds to the same epitope of VEGF₁₁₀₊that and a monoclonal antibody produced by hybridoma cell line PTA-7737. In certain embodiments, the antibody of the invention conjugated with a detectable label. In one embodiment of the proposed hybridoma 5C3.1.1 deposited in the ATCC under number PTA-7737.

BRIEF DESCRIPTION of DRAWINGS

Fig. 1. Panels A, B and C illustrate the detection of molecules of recombinant VEGF 165, VEGF 121 (1) (a shortened, with the likely absence of about 9 amino acids at the C-end, according to the manufacturer R&D systems), VEGF 121 (2) (Pepro Tech), VEGF 110 (N-terminal fragment produced by cleavage by plasmin VEGF) and VEGF8-109 (synthetic VEGF with amino acids 8-109 VEGF 165) using different analyses of VEGF-ELISA. (A). ELISA using a 3.5F8 for coating and biotinylated A4.6.1 for detection. (B). ELISA B A4.6.1 for coating and biotinylated 2E3 for detection. (C). ELISA C using 5C3 for coating and biotinylated A4.6.1 for detection.

Fig. 2 illustrates a protein blot of VEGF produced by A673 cells, used in the tion as probes 3.5F8 (left) or A4.6.1 (right). The samples represent VEGF, purified from the conditioned medium of A673 cells using A4.6.1-affinity column (lane 1)and recombinant VEGF proteins: VEGF₁₆₅, VEGF₁₂₁(with the possible lack of about 9 amino acids at the C-end, according to the manufacturer R&D systems), and VEGF_8-109produced using E. coli (lane 2, 3 and 4, respectively).

Fig. 3 illustrates a diagram of VEGF₁₆₅, VEGF₁₂₁and VEGF₁₁₀(N-terminal fragment produced by cleavage by plasmin VEGF), showing putative binding sites of the antibodies used in the three analyses of VEGF-ELISA.

DETAILED DESCRIPTION

Definitions

Before a detailed description of the present invention should be understood that the present invention is not limited to particular compositions or biological systems, which, without doubt, can vary. You should also understand that it uses the terminology only for the purpose of describing particular embodiments, and it is implied that she is not a limitation. When used in this description and appended claims, the singular number include the plural, unless the content clearly indicates otherwise. Thus, for example, the designation of a molecule optionally includes a combination of two or more molecules, and so forth.

the ri using here the term "VEGF" refers to a growth factor vascular endothelium of 165 amino acids and related factors vascular endothelial growth from 121, 145, 189 and 206 amino acids, as described in publications Leung et al. Science 246: 1306 (1989), Houck et al. Mol. Endocrin. 5: 1806 (1991), and Neufeld et al., above, together with natural allelic and progressirovanii forms of these growth factors. Cm. also, for example, Fig. 1 A and B of U.S. Patent No. 6057428. Active fragment of VEGF may be released from the ECM-bound VEGF with cleavage by plasmin, obtaining the first 110 amino acids (see, for example, Keyt BA, et al.: The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem. 271: 7788-7795 (1996)). When used in the present description, the term "VEGF₁₁₀₊" means fragments of VEGF, which contain more than 110 amino acids (from N-end), but does not include the first 110 amino acids or smaller fragments (e.g., VEGF_8-109).

The term "detection" is used in a broad sense, including both measurements of a target molecule - qualitative and quantitative. In one aspect of the described method of detection is used to identify only the presence of VEGF₁₁₀₊or VEGF in a biological sample. In another aspect, the method is used to test whether VEGF₁₁₀₊or VEGF in the sample on detektiruya level. In another aspect, the method can be used to quantify the amount of VEGF₁₁₀₊or VEGF in the sample, and, in addition, to compare the levels of VEGF₁₁₀₊or VEGF from different samples.

The term "biologist is ical sample" means a sample from an organism, obtained from any animal, preferably a mammal, more preferably human. In certain such embodiments the biological sample receive from a patient with vascular, diabetes or cancer. Such samples include biological fluids such as serum, plasma, vitreous fluid of the body, lymphatic fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebrospinal fluid, saliva, sputum, lacrimal fluid, the fluid in sweat, mucus, tumor lysates, and the environment of tissue cell cultures, and tissue extracts, such as gomogenizirovannogo tissue, tumor tissue and cell extracts. In certain embodiments the sample is a sample from an organism obtained from any animal, in one embodiment, from a mammal, in another embodiment from the man. In one embodiment of such a biological sample receive from clinical patients.

The term "detective antibody" means an antibody that can be detected either directly by using labels, amplified by means of detection, or indirectly using, for example, another labeled antibody. For direct labeling of the antibody is usually conjugated with compo is entom, which is detected by some means. In one embodiment detective antibody is a biotinylated antibody.

The term "detection means" refers to a component or method used for detection of the presence of detected antibodies in the present analysis, ELISA, and includes agents detection, which immobilized amplified a label such as label, captured on titration the microplate. In one embodiment the detection means is an agent for the colorimetric detection, such as avidin or streptavidin-HRP.

The term "capture reagent" means a reagent capable of binding and capture of target molecules in the sample, so that under suitable conditions, the complex in the form of reagent capture and target molecules could be separated from the rest of the sample. Typically, the capture reagent is immobilized or can be immobilized. In the immunoassay of the sandwich-type capture reagent preferably is an antibody or mixture of antibodies against the target antigen.

The term "antibody" is used in the present description in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies)formed by at least two intact antibodies, and fragments the coefficients of antibodies, provided that they exhibit the desired biological activity.

The term "antibody fragments" includes part of an intact antibody, preferably comprising its antigen-binding or variable plot. Examples of fragments of antibodies include Fab fragments, Fab', F(ab')₂and Fv; dimeric antibodies; linear antibodies; single-chain molecule antibodies; and multispecific antibodies formed from fragments of antibodies.

For the purposes of this invention "intact antibody" is an antibody comprising the variable domains of the heavy and light chains, as well as the plot of Fc.

"Native antibodies" are usually heterotetrameric glycoproteins with a molecular mass of approximately 150000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of immunoglobulins of various isotypes. Each heavy and light chain also contains disulfide bridges within the chain, spaced at regular intervals. Each heavy chain contains at one end a variable domain (V_H), following a number of constant domains. Each light chain contains at one end variabeln the th domain (V _L) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain variable domain and the light chain is aligned with the variable domain of the heavy chain. It is assumed that the specific amino acid residues forming the interface between the variable domains of the light chain and heavy chain.

When used here, the term "monoclonal antibody" refers to an antibody obtained from a population essentially homogeneous antibodies, i.e the individual antibodies included in the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to conventional drugs (polyclonal) antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinants. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized using hybridoma cultures containing no other immunoglobulins. The designation of "monoclonal" indicates the character of the anti-Christ. ate, which is derived from essentially homogeneous population of antibodies, and this designation should not be understood as the necessity of obtaining the antibody by any particular method. For example, a monoclonal antibody used according to the present invention can be obtained by using the hybridoma method first described in the publication Kohler et al., Nature, 256: 495 (1975), or may be obtained using methods of recombinant DNA (see, for example, U.S. Patent No. 4816567). "Monoclonal antibodies" may also be isolated from phage libraries of antibodies using the methods described, for example, in the publications of Clackson et al. Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991).

In the present description monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins)in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies isolated from a particular species or belonging to a specific class or subclass of antibody, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies isolated from another species or belonging to another class or subclass antibodies as well as fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. Patent No. 486567; Morrison et al., Proc. Natl. Acad. Sci. USA. 81: 6851-6855 (1984)). Here, the chimeric antibodies of interest include "primaryservername" antibodies, including antigen-binding sequence of the variable domain selected from the Primate, non-human (e.g., from the monkeys of the old world, such as the baboon, macaque-rhesus or macaque-having), and including the sequence of the constant part of a human (U.S. Patent No. 5693780).

"Humanized" forms are not human (e.g. murine) antibodies are chimeric antibodies that contain minimal sequence selected from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody)in which residues from a hypervariable area of the recipient are replaced by residues from a hypervariable area of a species other than human (donor antibody)such as mouse, rat, rabbit, or Primate, non-human, and these antibodies have the desired specificity, affinity and capacity. In some cases, the remains of the skeleton of the plot (FR) of a human immunoglobulin replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues that are not found in the recipient antibody or in the donor an the. These modifications produce additional for improving functions of antibodies. As a rule, humanitariannet antibody includes, essentially, all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to such sequences from non-human immunoglobulin and all or substantially all of the FR plots represent such sequences from a human immunoglobulin. Humanitariannet antibody also optionally includes at least part of the constant part of the immunoglobulin (Fc), typically of a human immunoglobulin. For additional details, see the publication of Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992). In one embodiment of the proposed humanitariannet antibody 5C3, and apply this proposed methods.

The term "variable" refers to the fact that certain parts of the variable domains differ in sequence among antibodies and are used for binding and specificity of each particular antibody for its particular antigen. However, the variability is represented unevenly throughout the sequence of the variable domains of antibodies. It is concentrated in three segments called hypervariable sites is variable in both domains - light chain and heavy chain. The most highly conserved part of the variable domains are called the frame sections (FR). The variable domains of native heavy and light chains each comprise four FR plot, mostly taking a configuration of a β-sheet connected by three hypervariable sites, which form loops connecting, and in some cases forming part of the structure of β-sheet. Hypervariable sites in each chain are supported together in close proximity by using FR, and together with hypervariable sites from another chain that contributes to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5^thEd. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding the antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

By papain cleavage of antibodies have two identical antigen-binding fragment, called " fragments "Fab", each of which contains a single antigen-binding site, and a residual fragment "Fc", whose name reflects its ability to easily crystallize. Using processing pepsin get the fragment F(ab')₂, catalysterror two antigen-binding site, and is still capable of cross-linking antigen.

"Fv" is an antibody fragment that contains a complete antigen-recognition and antigen-binding site. This site consists of a dimer of one variable domain of the heavy chain and one variable domain of the light chain, which are dense non-covalent Association. The fragment is in this configuration that the three hypervariable section of each variable domain interact to define an antigen-binding site on the surface of the dimer V_H-V_L. Together the six hypervariable sites give the antibody antigen-binding specificity. However, even a single variable domain (or half of an Fv fragment comprising only three hypervariable site specific for an antigen) has the ability to bind antigen, although with lower affinity than the whole binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fragments, Fab' differ from Fab fragments by inserting a few residues at the carboxy-end of the CH1 domain of the heavy chain comprising one or more cysteines from the hinge area antibodies. Fab'-SH in the present description represents the designation for Fab'in which the residue(s) of cysteine in the constant domains bear, m is Nisha least one free Tilney group. Fragments F(ab')₂antibodies of the source were obtained in the form of pairs of fragments, Fab', which contained between cysteine hinge area. Also known other chemical condensation of fragments of antibodies.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two completely different types, called Kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domains of the heavy chain, which correspond to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

Fragments of antibodies, "single-chain Fv" or "scFv" include domains V_Hand V_Lantibodies, where these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further includes a polypeptide linker between domains V_Hand V_Lthat PR is delivers the ability of scFv to form the desired structure for antigen binding. As an overview on the topic of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

When used here, the term "hypervariable area" refers to amino acid residues of an antibody which are responsible for binding to the antigen. Hypervariable site comprises amino acid residues from a "plot that defines complementarity" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain; Kabat et al., Sequences of Proteins of Immunological Interest, 5^thEd. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The remains of the "framework" or "FR" are those variable domain residues other than residues of the hypervariable region as defined here.

"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo animals, sports animals, or domestic animals, such as dogs, horses, cats, sheep, pigs, cows, etc. Preferred mammal is a human is.

The terms "cancer", "cancer" and "cancerous" refer to or describe the physiological disease in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung), cancer of the peritoneum, liver cell cancer, gastric or stomach cancer (including gastrointestinal cancer), gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (e.g., hepatic carcinoma and hepatoma), bladder cancer, hepatoma, breast cancer, colorectal cancer, colorectal cancer, rectal cancer, endometrial or uterine carcinoma, carcinoma of the salivary gland, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, basal cell carcinoma, testicular cancer, esophageal cancer, hepatic carcinoma, sarcoma of soft tissue Kaposi's sarcoma, carcinoid carcinoma, mesothelioma, multiple myeloma, and various types of head and neck cancer, as well as B-cell lymphoma (including nscoder linzirovannye/follicular nahodkinskuju lymphoma (NHL); NHL small lymphocytes (SL); sredneperesechennoy/follicular NHL; sredneperesechennoy diffuse NHL; high differentiation immunoblastic NHL; well-differentiated lymphoblastoid NHL; well-differentiated small cell lymphoma NHL with unsplit core; the massive defeat of the NHL; the lymphoma cells of the mantle tissue; AIDS-associated lymphoma; and Macroglobulinemia waldenstrom); Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL)leukemia hair cells; chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as edema associated with brain tumors), and Meigs syndrome.

The expression "vascular" and "cardiovascular" are used interchangeably and describe patients with signs that stimulate angiogenesis and/or cardiovasculardisease, and with characteristics that inhibit angiogenesis and/or cardiovasculardisease. Such violations include, for example, arterial disease, such as atherosclerosis, hypertension, inflammatory vasculitis, Raynaud's disease and phenomenon, Raynaud's disease, aneurysms and arterial restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitic and impenema; and other vascular disorders such as peripheral vascular disease, AMD, cancer, such as tumor vessels, e.g., hemangioma (capillary and cavernous), glamurnye tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma and lymphangiosarcoma, tumor angiogenesis, trauma such as wounds, burns and other tissue damage, the fixation of the implant, scar, ischemic reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. These violations also include angina, myocardial infarction such as acute myocardial infarction, cardiac hypertrophy, and heart failure such as congestive heart failure (CHF).

The term "diabetes" refers to progressive disease of carbohydrate metabolism, including inadequate production or utilization of insulin and is characterized by hyperglycemia and glycosuria. This term includes all forms of diabetes, such as diabetes type I and type II, and insulinotherapy diabetes, such as the Mendenhall syndrome (Mendenhall), Werner syndrome, leprechaunism, lipotropics diabetes and other lipoatrophy.

The term "affinity purified" refers to the purification of substances by using it in the elution through a column for affinity chromatography.

ELISA

Growth factor vascular endothelial (VEGF) is homodimeric glycoprotein and is a key angiogenic factor in the formation of blood vessels during development and in pathological angiogenesis associated with tumors. The expression of VEGF increases in response to hypoxia, and potentially in response to other factors, such as growth factors, hormones and oncogenes (see, for example, Ferrara N: Vascular endothelial growth factor: Basic science and clinical progress. Endocrine Reviews 25: 581-611 (2004)). Gene VEGF person contains eight exons, separated by introns. Alternative RNA splicing results in obtaining at least four major isoforms containing monomer 121, 165, 189 and 206 amino acids (see, for example, publish, Houck KA, et al.: The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 5: 1806-1814 (1991); and Tischer E, et al.: The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 266: 11947-11954 (1991)). Also described are less represented isoforms include isoforms containing monomer 145 amino acids (see, for example, Poltorak Z., et al.: VEGF145, a secreted vascular endothelial growth factor isoform that binds to increasing interest among matrix. J Biol Chem 272: 7151-7158 (1997)) and 183 amino acids (see, for example, the publication Jingjing L, et al.: Human Muller cells express VEGF 183, a novel spliced variant of vascular endothelial growth factor. Invest Ophthalmol Vis Sci 40: 752-759 (1999)). All isoforms VEG associated with two receptor tyrosine kinases, VEGFR-1 (see, for example, De Vries C, et al.: The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 255:989-991 (1992)) and VEGFR-2 (see, for example, Terman BI, et al.: Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene 6: 1677-1683 (1991)). VEGF₁₆₅it also interacts with neuropilins (see, for example, Soker S. et al.: Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92: 735-745 (1998)). VEGF₁₈₉and VEGF₂₀₆bind heparin with high affinity, and are the most connected in the extracellular matrix (ECM). VEGF₁₆₅binds to heparin with an average degree of affinity, and is partly associated with the cell surface and ECM. VEGF₁₂₁not associated with heparin and easily soluble. It was found that VEGF₁₂₁and VEGF₁₆₅are the most dominant variants, expressed in samples and cell lines of breast cancer and ovarian cancer, obtained through analysis of reverse transcription-PCR, while the expression of VEGF₂₀₆not detected. It was found that the expression of VEGF₁₈₃and VEG₁₈₉not detected or were at a low level in cell lines and detected in some samples from tumors (see, for example, Stimpfl M, et al.: Vascular Endothelial growth factor splice variants and their prognostic value in breast and ovarian cancer. Clinical Cancer Research 8: 2253-2259 (2002)).

Active fragment of VEGF m the may release of ECM-bound form of VEGF through cleavage by plasmin with getting the first 110 amino acids (see, for example, Keyt BA, et al., The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem. 271: 7788-7795 (1996)). This may be a mechanism for local regulation of the bioavailability of VEGF during physiological and pathological processes of angiogenesis. See, for example, the publication Houck KA, et al. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem 1992; 267:26031-26037 (1992); Keyt BA, et al. The carboxy-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem. 271:7788-7795 (1996); and Roth D, et al. Plasmin modulates vascular endothelial growth factor-A-mediated angiogenesis during wound repair. Am Pathology 168: 670-684. (10-12) (2006). However, the concentration of VEGF₁₁₀biological samples were not described. Active fragments of VEGF can also be released from the ECM-bound form of VEGF by splitting the matrix metalloproteinases (MMP). This assertion is supported by the discovery of degraded fragments of VEGF containing amino acids in addition to 1-110 present in the ascites of patients with ovarian cancer. Both protein - plasmin and MMP3 were detected in the ascites. See, for example, Lee S, Shahla MJ, et al. Processing of VEGF-A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors. J Cell Biology 169:681-691 (2005).

Enzyme-linked immunosorbent assays (ELISA) for different antigens include assays based on colorimetry, chemiluminescence and fluorescence. ELISA has been successfully applied in the determination of low amounts of the drugs and other antigenic components in samples of plasma and urine not including the extraction step and being easy to use. The analysis described here is an ELISA that uses antibodies as capture reagents and detected antibodies to VEGF and VEGF₁₁₀₊. In certain embodiments, the ELISA is performed on a cell basis. In the first stage of analysis of the biological sample potentially containing VEGF or VEGF₁₁₀₊were contacted and were incubated with antibody capture (or printed in the form of a coating antibodies), antibodies to capture grasped or contacted with VEGF or VEGF₁₁₀₊so that they are detected at the stage of detection. Detection stage includes using the detected antibodies, which is in contact with any of the related VEGF or VEGF₁₁₀₊associated with the protein of interest, if present, and uses the detection means for detecting the label on the antibody, and therefore, detection of the presence or quantity is present VEGF or VEGF₁₁₀₊. This ELISA can be compared with ELISA, which detects total VEGF (e.g., U.S. Patent No. 6855508; publications described herein, and publishing, known from the prior art) or VEGF isoforms to determine the presence of type VEGF.

For example, in certain embodiments in the analysis are the following stage.

The first stage

On what erway stage of this analysis of the biological sample in contact or incubated with immobilized (or applied in the form of a coating) a capture reagent, which is a monoclonal antibody against VEGF. This antibody can be isolated from any species, but preferably, the monoclonal antibody is a murine or mouse monoclonal antibody, more preferably a mouse, and most preferably, the monoclonal antibody is an antibody MAb 5C3, selected from hybridoma identified in the present invention. Therefore, in a particular preferred embodiment, the immobilized monoclonal antibody is a murine monoclonal antibody, most preferably, the antibody is MAb 5C3. Immobilization is performed in an appropriate manner or by transferring the capture reagent in an insoluble form before the assay procedure, or through adsorption of water-insoluble matrix or surface (U.S. Patent No. 3720760), or are not covalently or through covalent condensation (for example, using cross-linking glutaraldehyde or carbodiimide, together, or in the absence, prior to activation of the substrate by using, for example, nitric acid and a reducing agent as described in U.S. Patent No. 3645852 or in the publication Rotmans et al. J. Immunol. Methods 57:87-98 (1983)), or after the procedure of analysis, for example, by using thus.

The solid phase used for immobilization, can be any inert under the oku or media which, essentially, is insoluble in water and applied in immunodeficiency analyses, including the substrate, for example, in the form of surfaces, particles, porous matrices, etc. are Examples of commonly used substrates include small plates, Sephadex, polyvinyl chloride, plastic granules and tablets for analysis or test tubes made from polyethylene, polypropylene, polystyrene and the like, including 96-well microtiter plates, as well as zernistie materials such as filter paper, agarose, crosslinked dextran, and other polysaccharides. Alternatively, for immobilization of the capture reagent appropriately applied reactive insoluble in water matrices, such as carbohydrates, activated CYANOGEN-bromide, and the reactive substrates described in U.S. Patent№ 3969287; 3691016; 4195128; 4247642; 4229537 and 4330440. In one embodiment, the immobilized capture reagent is applied as a coating on microtiter tablet, and used particularly preferred solid phase is advance microtiter tablet that can be used to analyze multiple samples simultaneously, for example, 96-well ELISA tablet for microanalysis, such as provided by the company Nune Maxisorb or Immulon. In certain embodiments, the tablet is the way the th 96-well ELISA tablet from MICROTEST ^TMor MAXISORP^TMsuch as provided by the company NUNC MAXISORB^TMor IMMULON^TM.

For solid phase coating is applied to the capture reagent as defined above, which may be associated with non-covalent or covalent interactions, or by physical connection, as described in the literature. Methods of joining include the methods described in U.S. Patent No. 4376110 and in the references cited here. If the covalent joining, tablet or other solid phase is incubated with a cross-linking agent, together with the capture reagent under conditions well known in the prior art, such as within 1 hour at room temperature.

Forming cross-linking agents commonly used to attach the capture reagent to the substrate of the solid phase include, for example, 1,1-bis(diazoacetate)-2-Penilaian, glutaraldehyde, N-hydroxysuccinimide esters, for example esters with 4-azidoaniline acid, homobifunctional complex imidiately, including disuccinimidyl, such as 3,3'-dithio-bis-(succinimidylester), and bifunctional maleimides, such as bis-N-maleimido-1,8-octane. Using modified agents such as methyl-3-[(p-azidophenyl)dithio]propionamide get photoactivated intermediate compounds are capable of forming cross is Sivok in the presence of light.

If you are using 96-well plates, it is usually as a coating applied to the capture reagent (typically diluted in a buffer, such as 0.05 M sodium carbonate, by incubation for at least 10 hours, more preferably, during the night when temperatures are in the approximate range 4-20 º C, or in the approximate range of 4-8°C and at pH values that are in the approximate range of 8-12, or in the approximate range of pH 9-10, or about pH 9,6). If it is expedient to carry out the coating in a short time, the coating on 96-well tablets may be conducted by a specialist, for example, for two hours at room temperature. The tablets can be stacked, and can be conducted by coating before the analysis can then be conducted analysis on several samples manually, semi-automatic or automatic means, such as with the use of robotics.

Tablets coated then usually treated with a blocking agent, which is not specific binds and fills the binding sites to prevent unwanted binding of the free ligand with an excess of sites on the wells. Examples of suitable blocking agents for these purposes include, for example, gelatin, bovine serum and albumin, egg albumin, casein and nonfat milk. Block processing is usually conducted under conditions of ambient temperature for about 1-4 hours, preferably about 1-3 hours, or overnight at 0-4°C.

After coating and blocking the VEGF standard (purified VEGF) or biological sample that you want to analyze, appropriately diluted, added to the immobilized phase. The preferred level of dilution by volume is about 1-15%, preferably about 10%. Buffers for breeding, which can be used for this purpose include (a) PBS containing 0.5% BSA, 0.05% detergent TWEEN 20^TM(P20), 0.05% antibiotic PROCLIN^TM300, 5 mm EDTA, 0.25% of a surfactant Chaps, 0.2% of the beta-gamma globulin, and 0,35M NaCl, pH 7,4; (b) PBS containing 0.5% bovine serum albumin, 0.05% Polysorbate 20, 5 mm EDTA, and 0.25% CHAPS, and 0.2% bovine γ-globulins, and 0.35 M NaCl; pH of 7.4 (c) PBS containing 0.5% BSA, 0.05% Polysorbate 20 (P20), and 0.05% PROCLIN^TM300, pH 7; (d) PBS containing 0.5% BSA, 0.05% of P20, of 0.05% PROCLIN^TM300, 5 mm EDTA, and 0.35 M NaCl, pH 6.35mm; (e) PBS containing 0.5% BSA, 0.05% of P20, of 0.05% PROCLIN^TM300, 5 mm EDTA, and 0.2% beta-gamma globulin, and 0.35 M NaCl, pH 7,4; and (f) PBS containing 0.5% BSA, 0.05% of P20, of 0.05% PROCLIN^TM300, 5 mm EDTA, and 0.25% Chaps, and 0.35 M NaCl, pH 7,4. PROCLIN^TM300 acts as a preservative, and TWEEN 20^TMacts as a detergent to eliminate non-specific tie is found.

While the concentration of capture, as a rule, is determined according to the interval of interest concentrations of VEGF, taking into account any necessary dilution of the biological sample, the final concentration of the capture reagent is usually determined empirically to obtain maximum sensitivity analysis in the interval of interest concentrations.

Conditions of incubation of the sample and the immobilized capture reagent is chosen to maximize the sensitivity analysis and the minimum dissociation. Preferably, the incubation is carried out by a fairly constant temperatures are in the range of from about 0°to about 40°C., preferably from about 20 to 25°C. the incubation Time depends mainly on temperature, which usually does not exceed more than about 10 hours, to avoid insensitive analysis. Preferably, the incubation time is from about 0.5 to 3 hours, and more preferably, 1.5 to 3 hours at room temperature to obtain maximum binding of free VEGF₁₁₀₊or VEGF with capture reagents. The incubation period may be greater if you add proteiny inhibitor to prevent by degradation of VEGF in a biological fluid.

At this stage the pH of the incubation sm is si, as a rule, is in the approximate range 4-9,5, preferably in the approximate range 6-9, more preferably in the approximate range of 7-8, and most preferably, the pH of the diluent for the assay (ELISA) is 7.4. the pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagent with captured VEGF₁₁₀₊or VEGF. You can apply a variety of buffers to achieve and maintain during this stage the desired pH values, including borate, phosphate, carbonate, Tris-HCl or Tris-phosphate, acetate, barbitala and the like. Specific buffer used is not critical to the invention, but in the individual analyses, one buffer may be preferable to another.

The second stage

In the second stage, represented here by way of analysis, which is optional, the biological sample is separated (preferably by washing) from the immobilized capture reagent for removal / her non-captured molecules. The solution used for washing, as a rule, represents a buffer ("washing buffer")having a pH value determined using analyses and buffers described above for the stage of incubation, with a preferred pH in the approximate range 6-9. Washing may be performed three or more times. Temperature premiun what I as a rule, is in the range from low temperature to medium temperature with a constant temperature to be maintained during the analysis, which is typically in the approximate range 0-40°C, more preferably in the approximate range 4-30°C. for Example, the washing buffer may be before washing placed in a container on ice at 4°C, and for this stage can be used the device for washing tablets. A crosslinking agent or other suitable agent may also be added at this stage to give an opportunity currently associated VEGF₁₁₀₊or VEGF covalently join the capture reagent, if there is any doubt that captured VEGF₁₁₀₊or VEGF may to some extent be dissociativity in subsequent stages.

The third stage

At the next stage, the immobilized capture reagent is in contact with detectivesyme antibodies, preferably, at a temperature which is in the approximate range of 20-40°C, more preferably in the approximate range of 20-25°C, with the exact value of temperature and time of contact, and these two parameters depend mainly on the means of detection. For example, when used as a tool for detection by streptavidin-peroxidase and 3,3',5,5'-tetramethylbenzidine, for example, in one embodiment the contact which the testing is carried out (for example, about 1 hour or more) for the amplification of the signal to the maximum. Preferably, a molar excess of the antibody relative to the maximum expected concentration of free VEGF₁₁₀₊or VEGF (as described above) is added to the plate after washing. This antibody is directly or indirectly detected. Along with the fact that detective antibody can be a polyclonal or monoclonal antibody, for example, in certain embodiments it is a monoclonal antibody, in one embodiment is a mouse, and in one embodiment the antibody MAb A4.6.1. Also detective antibody can be directly detektivami and, in one embodiment contains a colorimetric label, and in another embodiment includes a fluorescent label. More preferably, detective antibody is biotinylated, and the detection means is an avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethylbenzidine. The reading means of detection may be fluorescent or colorimetric. The affinity of antibodies should be high enough could be detected small amounts of free VEGF₁₁₀₊or VEGF, but not so high that VEGF₁₁₀₊or VEGF was away from the capture reagents.

The fourth stage

At the last stage of the method of analysis the level of free VEGF, in which the point p is associated with a capture reagent, is measured using a detection means for the detected antibodies. If the biological sample obtained from a patient with vascular, diabetes or cancer, the stage of measurement, preferably, includes a comparison of the reaction, which occurs as a result of the above three stages, with a standard curve to determine the level of VEGF₁₁₀₊or VEGF compared with their level in healthy individuals, or, preferably, includes a comparison of the reaction, which occurs as a result of the above three stages, with the results of another analysis of VEGF-ELISA detection of different isoforms of VEGF or total VEGF, to determine the level of types VEGF when compared to the ELISA results, and, optionally, compared with levels in healthy individuals.

The production of antibodies

Polyclonal antibodies to VEGF, as a rule, receive from animals with multiple subcutaneous (sc) or intraperitoneal (ip) injections of VEGF and adjuvant. It may be convenient to konjugierte VEGF or a fragment containing the amino acid sequence of the target protein that is immunogenic in the species that you want to immunize, for example, hemocyanin lymph snails, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or modifying agent, n is the sample, maleimidophenylmethacrylates ether (conjugated through cysteine residues), N-hydroxysuccinimide (conjugated through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR, where R and R1 are different alkyl groups.

The antibody used for coating or as detectable antibodies can be obtained from any suitable source in the form of a spine, such as a mouse, Primate, lagomorphs, goat, rabbit, rat, chicken, bull, sheep, horse, dog, pet the cat family, or pig. Can be used chimeric or humanized antibodies, as described, for example, in U.S. Patent No. 4816567; Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851 (1984); Neuberger et al. Nature 312: 604 (1984); Takeda et al. Nature 314:452 (1985); and WO 98/45331 from October 15, 1998, and additional links provided above.

Animals can be immunized against the immunogenic conjugates or derivatives by combining 1 mg or 1 μg of conjugate (for rabbits or mice, respectively) with 3 volumes complete adjuvant's adjuvant, and injected the solution intradermally at multiple points. One month animals stimulated by the introduction of 1/5 - 1/10 of the original amount of conjugate in incomplete Freund's adjuvant using subcutaneous injection at multiple points. After 7-14 days the animals on which Bireli blood, and analyzed on serum titer of antibodies to VEGF. Animals stimulated as long as the value of title is not stabilized. Preferably, the animal is stimulated with VEGF conjugate, but conjugated to a different protein and/or through a different cross-linking agent. The conjugates can also be obtained in the culture of recombinant cells in the form of a fused protein. The aggregation agents such as alum are used to enhance the immune response. Methods of producing polyclonal antibodies are described in numerous textbooks on immunology, such as Davis et al. Microbiology, 3^rdEdition, (Harper & Row, New York, New York, 1980).

Monoclonal antibody is produced by extraction of spleen cells from immunized animals and the immortalization of the cells in a suitable manner, for example, by fusion with myeloma cells or by using the transformation by Epstein-Barr,and screening for clones expressing the desired antibody. See, for example, Kohler and Milstein, Eur. J. Immunol. 6:511 (1976). Alternatively, monoclonal antibodies or antigennegative plot monoclonal antibodies, such as Fab fragments or (Fab)₂can be obtained using recombinant methods.

Examples of suitable antibodies include antibodies that are already known in the RIA analyses for proteins, referred to, for example, antibodies etc is against VEGF, as described in the links provided here in the introduction.

In certain embodiments of the applied antibody 5C3 against VEGF, which is produced, or which is produced by using hybridoma deposited in the ATCC under number PTA-7737, not necessarily with another antibody A4.6.1 against VEGF. The invention also features an antibody that does not bind to VEGF 1-110 and binds to the same epitope of VEGF₁₁₀₊that and a monoclonal antibody produced by hybridoma cell line PTA-7737. Offered hybridoma 5C3.1.1 deposited in the ATCC under Deposit number PTA-7737.

Detection

The antibody is added to the immobilized capture reagents, is directly labeled or detected indirectly by adding, after washing the excess of the first antibody molar excess of the second labeled antibody directed against the IgG of the animal species to which belonged the first antibody. In recent indirect analysis of tagged anticavity against the first antibody is added to the sample so as to obtain the labeled antibody in situ.

Used label for the first or second antibody is any detektiruya functional group which does not interfere with the binding of free VEGF₁₁₀₊or with VEGF antibody. Examples of suitable labels are numerous labels are known for use the application in immunoassay, includes components that can be detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as components, such as enzymes, for which detection should react or modified. Examples of such labels include the radioisotopes³²P,¹⁴C,¹²⁵I³H and¹³¹I, fluorophores such as rare earth chelates metals or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, such as Firefly luciferase and bacterial luciferase (U.S. Patent No. 4737456), luciferin, 2,3-dihydropteridine, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saridakis, for example, glucose oxidase, galactosidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, condensed with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microbiocides, Biotin/avidin, Biotin/streptavidin, Biotin/streptavidin-β-galactosidase with MUG, spin-labels, bacteriophobia labels, stable free radicals, and the like. As mentioned above, fluorescence detection is only one example.

For covalent binding of these labels to proteins or polypeptid the AMI available suitable methods. For example, to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels can be used binding agents, such as dialdehyde, carbodiimide, timelimit, bis-imidate, bis-diazotized benzidine, and the like. See, for example, published U.S. Patent No. 3940475 (fluorescence) and 3645090 (enzymes); Hunter et al. Nature 144: 945 (1962); David et al. Biochemistry 13: 1014-1021 (1974); Pain et al. J. Immunol. Methods 40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem. 30: 407-412 (1982). In certain embodiments of the labels used here are fluorescent labels to increase amplification and sensitivity to 8 PG/ml, more preferably, are Biotin with streptavidin-β-galactosidase and MUG for the amplification of the signal. In certain embodiments uses a colorimetric label, for example in the case where detective antibody is biotinylated, and the detection means is an avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethylbenzidine.

For professionals in the field of immunoassay methods conjugation of such labels include enzymes, with the antibody is a standard ongoing procedure. See, for example, the publication O 'Sullivan et al. "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay", Methods in Enzymology, ed. J.J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166.

After adding on the last labeled antibody, the amount of bound antibody is determined by removing excess unbound labeled antibody by washing and then measuring the amount of label attached using detection method appropriate to the label, and correlating the measured amount with the amount of free forms of VEGF₁₁₀₊or VEGF in a biological sample. For example, in the case of enzymes, manifested and measured the amount of staining is a direct measurement of the number of present forms of VEGF₁₁₀₊or VEGF. Specifically, if the label is HRP staining detected using the substrate 3,3',5,5'-tetramethylbenzidine with absorption at 450 nm.

In one example, after washing from the immobilized phase, enzyme-labeled second antibody directed against the first unlabeled antibody staining or chemiluminescence were measured using incubation of immobilized capture reagent with the enzyme's substrate. Then the number of free forms of VEGF₁₁₀₊or VEGF in the form of concentration was calculated by comparison with staining or chemiluminescence obtained by parallel analysis using standard forms of VEGF.

Sets

For convenience, the method of analysis according to the present invention may be offered in the form of a set. This kit is a packaged combination of basic elements:

(a) a capture reagent comprising a monoclonal antibody against the molecule VEGF human monoclonal antibody from the onset of VEGF ₁₁₀₊;

(b) detection reagents, consisting of a detected (labeled or unlabeled) antibodies that are associated with domains of VEGF, which is responsible for binding to the receptor KDR and/or FLT1. These basic elements are defined hereafter. In a particular embodiment, the detection reagents include detective antibody(a), which binds to the epitope VEGF1-110.

Preferably, the kit further includes a solid substrate reagent capture, which may be offered as a separate element, or which already immobilized capture reagents. Therefore, antibody capture kit can be immobilized on a solid substrate, or they can be immobilized on a substrate, which is included in the kit or available separately from the set.

Preferably, the capture reagents applied in the form of a coating on microtiter plate. The detection reagent may be a labeled antibody, detected directly, or unlabeled antibodies that are detected using labeled antibodies directed against unlabeled antibodies originating from other species. In the case where the label is an enzyme, the kit generally include substrates and cofactors required for the enzyme, and in the case where the label is a fluorophore, a set usually includes a precursor of the dye, with which the CSOs receive the detected chromophore. In the case where the detection reagent is unlabeled, the kit may further include detection means for detecting antibodies such as a labeled antibody directed against the unlabeled antibody, preferably, in the format of fluorescent detection. In the case where the label is an enzyme, the kit generally include substrates and cofactors required for the enzyme, in the case where the label is a fluorophore, a set usually includes a dye precursor, which receives the detected chromophore and, in the case where the label is a Biotin, a set usually includes avidin, such as avidin, streptavidin or streptavidin conjugated with HRP, or β-galactosidase with a MUG.

In one particular embodiment, the capture reagent is a monoclonal antibody, preferably a rodent antibody, more preferably a mouse or rat, more preferably a mouse, and most preferably the antibody MAb 5C3. In specific embodiments detective antibody is a biotinylated monoclonal antibody which is an antibody rodent, more preferably a mouse or mouse, even more preferably a mouse, and most preferably, the antibody is MAb A4.6.1. In certain embodiments, the capture reagent in the kit is immobilizovana is.

In certain embodiments, the kit can contain reagents for multiple ELISA analysis for comparative studies, as described herein, for the detection of various forms of VEGF and VEGF₁₁₀₊.

The set also typically includes instructions for analysis and/or VEGF as a standard antigen (for example, a purified form of VEGF, preferably recombinante received VEGF and VEGF110), as well as other additives, such as stabilizers, buffers for washing and incubation, and the like.

Examples of standards for VEGF are recombinant human VEGF obtained in mammalian cells, the standards are available from the company Genentech, Inc., South San Francisco, California, and from those companies and with the help of such methods, which are described here.

The components of the kit are available in a certain ratio, the relative amounts of the various reagents that are appropriate vary with getting in solution concentrations of reagents, which essentially maximizes the sensitivity analysis. Specifically, reagents may be offered in the form of dry powders, usually liofilizovannyh, including excipients, which when dissolved receive a reagent solution having the appropriate concentration for Association with the tested sample.

Deposition of materials

Following the e materials deposited in the American Collection Model Cell Cultures, 10801 University Boulevard, Manassas, VA. 20110-2209, USA (ATCC): 5C3.1.1 deposited in the ATCC under the number PTA-7737 from 19.07.2006.

The deposition was carried out on the basis of the provisions of the Budapest Agreement on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure and Regulations in accordance with this (the Budapest Agreement). This assures maintenance of a viable culture of cells on Deposit for 30 years from the date of Deposit. Deposits are available through ATCC based on the definitions of the Budapest Agreement, and subject to agreement between Genentech, Inc. and ATCC, which guarantee a constant and uninterrupted availability of the progeny of the culture to the scientific community with the publication of the pertinent U.S. patent or upon laying out to the scientific community of any patent of the United States or foreign patent, whichever was first, and assures availability of the progeny of the culture professionals, op is edeleny with the Commissioner of patents and trademarks U.S. rights to the offspring of culture according to 35 USC § 122 and in accordance with the rules of the Commission (including 37 CFR § 1.14 with special reference to 886 OG 638).

The assignee of this application agrees that if the culture of the materials on Deposit will perish or be lost or destroyed when cultivated under suitable conditions, when the notification materials will be immediately replaced by others the same. Availability of the deposited material should not be construed as a license for the practical application of the invention in contravention of the rights granted by the authority of any government in accordance with its patent laws.

It is believed that the description is sufficient to enable a person skilled in the field of practical application of the invention. The invention is not limited to the deposited structure, since the deposited embodiment of the invention is intended to illustrate certain aspects of the invention, and any functionally equivalent designs are within the invention. Deposition of this material does not permit the recognition that the written description is insufficient to enable the practical application of any aspect of the invention, including even more of it, and it should not be understood as a limitation of the framework of the claims defined illustrations, which she presents. Indeed, various modifications in addition the s to the what is shown and described herein will be obvious to experts in this field from the preceding description, and will fall into the scope of the attached claims.

It is clear that the examples and embodiments described herein are presented only for the purpose of illustration and that, in this light, specialists in this area will be treated with various modifications or changes, and the examples and embodiments described herein are included in the essence and scope of this application and the scope of the attached claims. All publications, patents and patent applications, cited here, is actually fully introduced here by reference.

EXAMPLES

Example 1:

It is known that the growth factor vascular endothelial (VEGF), which is expressed in different isoforms due to alternative RNA splicing plays a key role in tumor angiogenesis. We have measured the concentration of VEGF₁₆₅and total VEGF and evaluated the relative amount of VEGF₁₁₀who is an active fragment obtained by cleavage by plasmin VEGF. ELISA A (VEGF165-206 ELISA) detects VEGF₁₆₅and the longer isoform, but does not detect VEGF₁₂₁. ELISA B (VEGF110-206 ELISA) detects VEGF₁₆₅and isoforms of VEGF₁₂₁and VEGF₁₁₀. ELISA C (VEGF121-206 ELISA) detects VEGF₁₆₅and the longer isoforms, VEGF₁₂₁and fragments of VEGF with mole is Blarney weighing more than VEGF ₁₁₀but not VEGF₁₁₀(designated here as "VEGF₁₁₀₊").

Materials and methods

Reagents and cells: Recombinant VEGF₁₆₅(Genentech), VEGF₁₂₁(PeproTech, Rocky Hill, New Jersey), VEGF_8-109(consisting of amino acids 8-109 of VEGF₁₆₅and shortened VEGF₁₂₁(R&D Systems, Minneapolis, MN) was produced in E. coli. Shortened VEGF₁₂₁contains an intact N-end defined using mass spectrometry, but has a mass of 26 kDa, in accordance with the truncation of about nine amino acids from the carboxy-end as described by the manufacturer. He migrates between VEGF₁₁₀and VEGF₁₂₁in the analysis using SDS-PAGE under reducing conditions. VEGF₁₁₀received via cleavage by plasmin VEGF₁₆₅(Keyt BA, et al.: The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem. 271: 7788-7795 (1996)). The molecular mass measured by mass spectrometry, was 25390, consistent with theoretical mass, component 25389. The concentration was determined using the method of bicinchoninic acid (Pierce, Rockford, IL). Molecular weight, used to calculate the concentrations of VEGF_8-109, VEGF₁₂₁and VEGF₁₆₅made up 23.8, 28,9 and 38.2 kDa, respectively. Monoclonal antibodies against VEGF, A4.6.1, 3.5F8, 2E3 and 5C3 was obtained by immunization of mice with VEGF₁₆₅produced in CHO cells (Kim KJ, et al.: The vascular endothelil growth factor proteins: Identification of biologically relevant regions by neutralizing monoclonal antibodies. Growth Factors 7: 53-64 (1992)). Cell lines of breast cells SK-BR-3, BT-474, T-47D and MCF-7 as well as cell lines of ovarian ES-2, OVCAR-3 and SK-OV-3 (American Collection of Typical Cell culture, Rockville, MD) were grown in RPMI medium in the presence of 2 mm L-glutamine and 10% FBS (except 20% for cells OVCAR-3) in a humidified incubator in the presence of 5% CO₂at 37°C.

Purification of VEGF in the conditioned medium of cells A673: A673 cells (American Collection Model of Cell Cultures) were grown in an environment of 50:50 F12/DMEM in the presence of 2 mm L-glutamine and 5% FBS up to 60% of confluences, and then in an environment free from serum, (Genentech) to complete confluently. VEGF was purified from supernatant using A4.6.1-Separatey speakers I got from Sepharose activated with CNBr (Amersham Biosciences, Piscataway, NJ). The eluate of the column and controls recombinant VEGF (0.2 ug per lane) were driven on the gels with a content of 18% Tris-Glycine (Invitrogen, Carlsbad, CA) under reducing conditions and transferred to nitrocellulose blot. The blot was blocked with a solution of 0,5M Tris-HCl, pH 7.5, 1,5M NaCl, 50 mm EDTA, 0,5% Triton containing 3% bovine serum albumin, and hybridized with 200 ng/ml 3.5F8 or A4.6.1 followed by hybridization with 2 ng/ml of goat antibodies to mouse Fc-HRP (Jackson ImmunoResearch). Signals were displayed using SuperSignal West Dura (Pierce) and recorded on x-ray film.

VEGF ELISA to measure the concentrations of VEGF/u>

ELISA A(VEGF165-206 ELISA). If not specified differently, to measure VEGF in the samples used fluorescent ELISA A. In fluorescent ELISA A antibody 3.5F8 used for coating and biotinylated antibody A4.6.1 used after the streptavidin-β-galactosidase for detecting and used 4-methylumbelliferyl-β-D-galactoside as substrate (Rodriguez CR, et al.: A sensitive fluorometric enzyme-linked immunosorbent assay that measures vascular endothelial growth factor 165 in human plasma. J Immunol Methods 219: 45-55 (1998)). Standards VEGF₁₆₅was a concentration 1-128 PG/ml, or 0,026-at 3.35 PM. In the colorimetric ELISA A antibody 3.5F8 used for coating and biotinylated antibody A4.6.1 used for detection according to the Protocol used for ELISA C, described below. Standards VEGF₁₆₅represented a concentration of 1.6-200 PG/ml.

ELISA B(VEGF110-206 ELISA) analysis, previously known as VEGF121-206 ELISA, Konecny GE, et al.: Association between HER-2/neu and Vascular Endothelial Growth Factor Expression Predicts Clinical Outcome in Primary Breast Cancer Patients. Clinical Cancer Research, 10: 1706-1716 (2004)): 96-microwell tablets MaxiSorp was coated with 0.5 μg/ml antibody A4.6.1 in 50 mm carbonate buffer, pH 9.6 volume of 100 μl/well at 4°C over night. The tablets were washed after this stage and between subsequent stages of incubation at room temperature with buffer PBS, pH 7,4, steriade is about 0.05% Polysorbate 20. The plates were blocked with 0.5% of bovine serum albumin, 10 ppm Proclin^TM300 (Supelco, Bellefonte, PA) in PBS (150 μl/well) for 1 h Standards VEGF (1.56 to 200 PG/ml VEGF₁₆₅or 0,0409-5,24 PM VEGF two-fold serial dilution) and serially diluted samples (with a minimum dilution of 1:10) in two-or three-fold serial dilution in PBS buffer, pH of 7.4, containing 0.5% bovine serum albumin, 0.05% Polysorbate 20, 5 mm EDTA, and 0.25% CHAPS, and 0.2% bovine γ-globulin (Sigma, St. Louis, MO) and 0.35 M NaCl (buffer for sample) was added to the plates (100 μl/well) and incubated for 2 hours Associated VEGF were detected using incubation of the biotinylated antibodies 2E3 (or other antibody that binds to domain VEGF responsible for binding to the receptor) on the tablets for 1 h followed by incubation with streptavidin-HRP (Amersham, Copenhagen, Denmark) for 30 min, with biotinyl-tramadom (ELAST ELISA amplification System, Perkin Elmer Life Sciences Inc., MA) for 15 min, and with streptavidin-HRP for 30 minutes was Added to the substrate TMB (3,3',5,5'-tetramethylbenzidine) (Kirkegaard &Perry Laboratories) and the reaction was stopped by adding 1 M phosphoric acid. The absorption was read at 450 nm on secerna the reader Titertek (ICN, Costa Mesa, CA). Titration curves were selected using the selection curve regression using even the PEX parameters (KaleidaGraph, Synergy software, Reading, PA). Measuring point, which were in the range of the standard curve used for the calculation of the estimated concentrations of VEGF in the samples. Getting 1.56 to 200 PG/ml VEGF₁₆₅in 10% EDTA-plasma (Golden West Biologicals, Inc., Temecula, CA) was 92-120% after deducting the estimated concentration of endogenous VEGF, part of 2.1 PG/ml in 10% plasma used for this study.

ELISA C(VEGF121-206 ELISA): on the microwell plates were coated with 1 μg/ml antibody 5C3 against VEGF and were blocked as described above. Standards VEGF (4-512 PG/ml VEGF₁₆₅or 0,105-13,4 PM VEGF in 2-fold serial dilution) and serially diluted samples in dilution buffer was added to the tablets. The plates were incubated for 2 hours Associated VEGF were detected by adding biotinylated antibody A4.6.1 with the subsequent addition of streptavidin-HRP and TMB as substrate. Data from tablet to read and analyzed as described above. Getting 4-512 PG/ml VEGF₁₆₅in 10% plasma was 77-101% after subtraction of 1.6 PG/ml expected concentrations of endogenous VEGF in 10% plasma used for this study.

Results and discussion

VEGF ELISAIn the previously described ELISA A used antibody 3.5F8 for coating and biotinylated antibody A4.6.1 for de is ecchi (Rodriguez CR, et al.: A sensitive fluorometric enzyme-linked immunosorbent assay that measures vascular endothelial growth factor 165 in human plasma. J Immunol Methods 219: 45-55, 1998). This analysis detects VEGF165 (VEGF₁₆₅), but not VEGF121(1) (VEGF₁₂₁(1)), which is obtained from R&D systems, and it is missing about 9 amino acids from the carboxy-end, and VEGF121(2) (VEGF₁₂₁(2)), which is obtained from PeproTech (Fig. 1A). Antibody 3.5F8 associated with VEGF₁₆₅but is not associated with VEGF₁₂₁according to the data obtained using BIAcore. The antibody A4.6.1 associated with the domain responsible for binding to the receptor (Kim KJ, et al.: The vascular endothelial growth factor proteins: Identification of biologically relevant regions by neutralizing monoclonal antibodies. Growth Factors 7: 53-64, 1992), which is present in all isoforms and VEGF₁₁₀. Antibody 3.5F8, probably associated with the neighboring amino acids 116 and 118, which are not present in VEGF₁₂₁. Antibody 5C3, probably associated with the neighboring amino acids 111 and 113, which are not present in VEGF₁₁₀(Fig. 3). The ELISA may be used to detect VEGF isoforms, which contain sequences of VEGF₁₆₅including VEGF₁₈₃, VEGF₁₈₉and VEGF₂₀₆(see, for example, Stimpfl M, et al.: Vascular Endothelial growth factor splice variants and their prognostic value in breast and ovarian cancer. Clinical Cancer Research 8: 2253-2259, 2002). ELISA B (analysis, previously known as VEGF121-206 ELISA, Konecny GE, et al., Association between HER-2/neu and Vascular Endothelial Growth Factor Expression Predicts Clinical Outcome in Primary Breast Cancer Patients. Clinical Cancer Research, 10: 1706-1716, 2004) uses the antibody A4.6.1 for NAS is the basis of coating and biotinylated antibody 2E3 for detection. Antibody A4.6.1 and 2E3 contact the domain responsible for binding to the receptor, which is present in all three molecules. See, for example, the publication of Kim KJ, et al. The vascular endothelial growth factor proteins: Identification of biologically relevant regions by neutralizing monoclonal antibodies. Growth Factors 7: 53-64 (1992); and, Muller YA, et al. Vascular endothelial growth factor: Crystal structure and functional mapping of the kinase domain receptor binding site. Proc Natl Acad Sci USA 94: 7192-7197 (1997). They may also use other antibodies that bind to these sites. This ELISA detects VEGF₁₆₅, VEGF₁₂₁, shortened VEGF₁₂₁(absence of about 9 amino acids from the carboxy-end), and with the same success VEGF₁₁₀and VEGF_8-109(Fig. 1B). This ELISA can detect total VEGF, including fragments of more than VEGF₁₁₀obtained by hydrolysis of matrix metalloproteinases. The ELISA C, described here, which uses the antibody 5C3 for coating and biotinylated antibody A4.6.1 for detection, detects VEGF₁₆₅, VEGF₁₂₁and with the same success truncated VEGF₁₂₁but will not detect VEGF₁₁₀or VEGF_8-109(Fig. 1, C). Antibody 5C3 associated with VEGF₁₂₁but is not associated with VEGF_8-109according to the data obtained using BIAcore. This ELISA can detect all molecules VEGF, detected using VEGF_110-206except VEGF₁₁₀and shorter fragments.

Chu the reality of ELISA assays A, ELISA B and ELISA C was 10, 16, and 40 PG/ml VEGF₁₆₅(or of 0.26, 0.41 and 1,05 PM for different isoforms and fragments of the VEGF) VEGF in the sample using a minimum dilution of 1:10, respectively. Tests ELISA ELISA B & C are reproducible (Table 1 and 2). Analyses and ELISA ELISA B C specific for VEGF (VEGF-A). VEGF-B, VEGF-C and VEGF-D at concentrations up to 50 ng/ml gave only background signals. Insulin-like growth factor 1, growth hormone, recombinant nerve growth factor, tumor necrosis factor (Genentech), platelet growth factor AB, placental growth factor, transforming growth factor-β1 (R&D Systems) (up to 200 ng/ml) gave only background signals. Heparin (Leo Laboratories, Bucks, UK and Dublin, Ireland) (100 U/ml) had no significant effect on the analysis.

VEGF in the conditioned medium of cell lines: Air-conditioned environment of the six stable CHO clones, transfected with cDNA of VEGF₁₆₅(Meng et al., 2000), was measured using three ELISA assays, which used non-VEGF produced in E. coli as a standard. Glycosylated recombinant VEGF₁₆₅the air-conditioned environment of the six stable clones gave similar concentrations in three ELISA assays. Concentrations measured by ELISA B, 28, 63, 64, 43, of 3.8 and 3.2 nm, respectively. The ratio of the concentrations of VEGF measured by ELISA A and ELISA C, compared with concentrations measured by ELISA B, was 0.90±0.08 and 1,08±0,10, respectively. Thus, three ELISA, with the same success quantitatively evaluated glycosylated VEGF, and there was a small proteolysis VEGF₁₆₅under the conditions of cultivation.

Concentric and VEGF in the conditioned medium of A673 cells, measured using ELISA A, ELISA ELISA B & C, amounted to 0.15, 0.29 to 0.24 nm VEGF, respectively. Concentration measured by ELISA A, was below that indicates the presence of VEGF₁₂₁. When VEGF was purified from conditioned medium using A4.6.1-affinity column and analyzed using protein blotting, using antibodies 3.5F8 were detected two strips, these likely correspond to glycosylated and is not glycosylated form of VEGF₁₆₅. Lower strip had the same mobility, and purified VEGF₁₆₅produced in E. coli (Fig. 2, left). Treatment with N-glycanase turned higher strip in the lower. Using antibody A4.6.1 were detected two additional strips with a lower molecular weight, probably corresponding glycosylated (partially overlapping with the proposed line does not glycosylated forms of VEGF₁₆₅and not glycosylated form of VEGF₁₂₁(Fig. 2, right). Lower strip had the same mobility, and purified VEGF₁₂₁produced in E. coli, and treatment with N-glycanase turned higher strip in the lower.

The concentration of VEGF in the conditioned media from cell lines of breast cells SK-BR-3, BT-474, T-47D and MCF-7, as measured by ELISA B, was 3.6, 16, 13 and 13 PM, respectively. The ratio of conc is traci VEGF, measured using ELISA A, and the concentrations measured by ELISA B, was 0.49, 0.42 and of 0.43 and 0.38 (or 49%, 42%, 43% and 38%), respectively, which is consistent with the percentage of expression of VEGF₁₆₅in these respective cell lines constituting 43, 35, 40, and 41% (Stimpfl M, et al.: Vascular Endothelial growth factor splice variants and their prognostic value in breast and ovarian cancer. Clinical Cancer Research 8: 2253-2259, 2002). The ratio of the concentrations of VEGF measured using ELISA C, the concentrations measured by ELISA B, amounted to 1.1-1.2 for these cell lines, indicating a small presence of VEGF₁₁₀. The concentration of VEGF in the conditioned media from cell lines of ovarian ES-2, OVCAR-3 and SK-OV-3, as measured by ELISA B was 32, 11, and 20 PM, respectively. The ratio of the concentrations of VEGF measured by ELISA A, and the concentrations measured by ELISA B, amounted to 0.24, 0.20 0,32 (or 24%, 20% and 32%, respectively, compared with the percentage of expression of VEGF₁₆₅in these respective cell lines, components38, 42 and 24% (Stimpfl et al., above). The ratio of the concentrations of VEGF measured using ELISA C, the concentrations measured by ELISA B, made 0.64-0.79, which for these cell lines, indicating the possible presence of VEGF₁₁₀(or shorter fragments).

1. Method detection forms with size more than 110 amino acids (VEGF₁₁₀₊) factor vascular endothelial growth (VEGF) in the biological the immediate vicinity of the sample, where forms include VEGF₁₂₁that includes stage:
(a) contacting and incubating a biological sample with a capture reagent immobilized on a solid substrate, where the capture reagent contains a monoclonal antibody that recognizes and associated with specific residues in more than 110 of human VEGF;
(b) separating a biological sample from the immobilized capture reagents;
(c) contacting the immobilized molecular complex of the capture reagent-target detektivami antibody which binds to the domains of VEGF, which is responsible for binding to the receptor KDR and/or FLT1, or which binds to the epitope in the VEGF1-110; and
(d) measuring the level of VEGF₁₁₀₊associated with the capture reagents, using a detection means for the detected antibodies.

2. The method according to claim 1, where the biological sample is selected in humans.

3. The method according to claim 2, where the person is a patient with vascular, diabetes or cancer, and stage of measurement (d) further includes comparison with a standard curve to determine the level of VEGF in comparison with the level in a healthy individual.

4. The method according to claim 1, where the biological sample is a tumor lysates, plasma, serum or urine.

5. The method according to claim 1, where the capture reagent recognizes the same epitope as the antibody 5 is 3, and preferably is a monoclonal antibody S.

6. The method according to claim 1, where the immobilized capture reagents is applied as a coating on microtiter plate.

7. The method according to claim 1, where detective antibody is directly detektivami.

8. The method according to claim 7, where detective antibody amplificates using fluorescent reagent.

9. The method of claim 8, where detective antibody is biotinylated, and the detection means is an avidin or streptavidin-peroxidase and 3,3',5,5'-tetramethylbenzidine.

10. The method according to claim 1, where detective antibody is a monoclonal antibody.

11. The method according to claim 10, where detective antibody is a mouse monoclonal antibody.

12. The method according to claim 11, where the immobilized monoclonal antibody is an antibody MAb 5C3 and detective antibody is an antibody MAb A4.6.1.

13. The method according to claim 1, where the specified method further selectively detects the shape of VEGF₁₆₅.

14. The reagent kit immunoassay for the detection of forms of VEGF₁₁₀₊in the biological sample, where the forms include VEGF₁₂₁including:
(a) a monoclonal antibody against human VEGF, as capture reagent, where a monoclonal antibody specific contacts with residues in more than che is 110, of VEGF person; and
(b) detective antibody as a detection reagent that binds to domains of VEGF, which is responsible for binding to the receptor KDR and/or FLT1, or which binds to the epitope in the VEGF1-110.

15. Set on 14, further comprising a solid substrate reagent capture.

16. Set on 15, where the capture reagents immobilized on a solid substrate.

17. Set in clause 16, where the capture reagents is applied as a coating on microtiter plate.

18. Set on 17, further comprising detection means for detecting antibodies.

19. Set p, where the tool is a colorimetric detection.

20. Set on 14, additionally comprising purified VEGF as a standard antigen.

21. Set in 14, where the capture reagent to form antibody is a mouse monoclonal antibody MAb 5C3 and detective antibody is an antibody MAb A4.6.1.

22. The reagent kit immunoassay for 14, where a specified set of reagents for immunoassay additionally selectively detects the shape of VEGF₁₆₅.

23. Antibody 5C3 derived from hybridoma 5C3.1.1 with a Deposit number of MOUTH-7737 (or produced specified hybridomas), with the indicated antibody 5C3 connects forms of VEGF₁₁₀₊including VEGF₁₂₁.

24. The antibody according to item 23, conjugated with a detectable label.

25. G is britama S, deposited in ATSS with a Deposit number of MOUTH-7737, to obtain monoclonal antibodies S.