HK1192010A - Elisa for vegf - Google Patents

Description

ELISA for VEGF

The present application is a divisional application of an invention application having an application date of 2007, 03/10, and a chinese application No. 200780036803.0, and having an invention name of "ELISA for VEGF".

RELATED APPLICATIONS

This application claims priority and benefit from U.S. provisional application serial No. 60/828,203 filed on 4.10.2006, the specification of which is hereby incorporated herein in its entirety.

Technical Field

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

Background

It is now well established that angiogenesis is involved in the pathogenesis of a variety of conditions. These 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.Chem267:10931-,Annu.Rev.Physiol.53:217-,Vascular diseases in Pathiobiology of annular disease.Garner A, Klintworth GK, edition 2 (Marcel Dekker, NY,1994), page: 1625-. In the case of solid tumors, neovascularization allows tumor cells to acquire a growth advantage and proliferative autonomy compared to normal cells. Thus, a correlation was observed between microvascular density in tumor sections and patient survival for breast cancer and several other tumors (Weidner et al,N Engl J Med324:1-6(1991), Horak et al,Lancet340:1120-,Lancet340:145-146(1992))。

the search for positive regulators of angiogenesis has led to a number of candidates, including, for example, aFGF, bFGF, TGF- α, TGF- β, HGF, TNF- α, angiogenin, IL-8, and the like (Folkman et al, supra, and Klagsbrun et al, supra). Some of the negative regulators identified to date include thrombospondin (Good et al,Proc.Natl.Acad.Sci.USA.87:6624-6628(1990)), a 16kDa N-terminal fragment of prolactin (Clapp et al,Endocrinology133:1292-1299(1993)), angiostatin (O' Reilly et al,Cell79:315-,Cell 88:277-285(1996))。

work done in recent years has established a key role for Vascular Endothelial Growth Factor (VEGF) in the regulation of normal and abnormal angiogenesis (Fer)The rara and the like are adopted,Endocr.Rev.18:4-25(1997)). Even the finding that loss of a single VEGF allele results in embryonic lethality points to the irreplaceable role this factor plays in the development and differentiation of the vascular system (Ferrara et al, supra).

Furthermore, VEGF has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders (Ferrara et al, supra). VEGF mRNA is overexpressed in most human tumors examined (Berkman et al,J Clin Invest91:153-,Human Pathol.26:86-91(1995); Brown et al,Cancer Res.53: 4727. sup. 4735(1993); Mattern et al,Brit.J. Cancer73:931-934(1996), and Dvorak et al,Am J.Pathol.146:1029-1039(1995)). Likewise, the concentration of VEGF in ocular fluids is highly correlated with the presence of active vascular proliferation in patients with diabetic retinopathy and other ischemia-related retinopathies (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 by tissues and does not have to enter the circulation to exert its biological effects, but rather acts locally as a paracrine regulator. The Yang et al,J.Pharm.Exp.Ther.284:103(1998) have found that rhVEGF is cleared from circulation₁₆₅Very rapidly, suggesting that endogenous VEGF in the circulation is most likely the result of constant VEGF synthesis. Furthermore, several studies attempted to correlate circulating VEGF levels with tumor burden and suggested VEGF levels as potential prognostic markers (Ferrari and Scagliotti)Eur.J. Cancer32A, 2368(1996), Gasparini et al,J.Natl.Cancer Inst.89:139(1997);KohnCancer80:2219(1997), Baccala et al,Urology51:327(1998); Fujisaki et al,Am.J. Gastroenterol.93:249(1998)). Clearly, the ability to accurately measure VEGF can be important to understand its potential role in many biological processes, such as maintaining vascular patency, menstrual cycle, ischemia, diabetes, cancer, intraocular disorders, and the like.

There are reports in the literature of very different concentrations of endogenous VEGF in normal and diseased patients, ranging from undetectable levels to high levels. The ability to measure endogenous VEGF levels depends on the availability of sensitive and specific assays. Enzyme-linked immunosorbent assays (ELISA) based on colorimetric, chemiluminescent, and fluorescent assays have been reported for VEGF. Houck et al, supra (1992); Yeo et al,Clin.Chem.38:71(1992), Kondo et al,Biochim.Biophys.Acta1221:211(1994), Baker et al,Obstet.Gynecol.86:815(1995); Hanatani et al,Biosci.Biotechnol.Biochem.59:1958(1995); Leith and MichelsonCell 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,Cancer77:1101(1996), Brekken et al,Cancer Res.58:1952(1998); Obermar et al,Br.J.Cancer77:1870-,Clin.Sci.94:395-404 (1998)。

for example, Houck et al, supra (1992), describe a colorimetric ELISA that exhibits a ng/ml sensitivity that may not be sufficient to detect endogenous VEGF levels. Yeo et al, supra (1992) describe two-site time-resolved immunofluorescence assays, however, VEGF was not detected in normal serum (Yeo et al,Cancer Res.53:2912(1993)). Baker et al, supra (1995), who used a modified version of this immunofluorescence assay, reported detectable levels of VEGF in plasma from pregnant women, with higher levels observed in women with preeclampsia. Anthony et al using radioimmunoassay,Ann.Clin.Biochem.34:276(1997) reported similar data in pregnant women. A chemiluminescent ELISA capable of measuring circulating VEGF was developed by Hanatani et al, supra (1995) and reported VEGF levels in plasma from 30 normal individuals (male and female) at 8-36 pg/ml. Brekken et al, supra (1998) describe ELISA assays using antibodies with binding preferences for VEGF alone or the VEGF: Flk-1 complex.

ELISA kits for VEGF detection are available from R & D Systems (Minneapolis, MN). The R & D VEGF ELISA kit has been used in a sandwich assay where monoclonal antibodies are used to capture the target VEGF antigen and polyclonal antibodies are used to detect VEGF. Webb et al, supra (1998). See also, e.g., Obermair et al, supra (1998).

Keyt et al, J.biol.chem.271:7788-7795(1996); Keyt et al,J.Biol.Chem.271:5638(1996) and Shifren et al, supra (1996) have also developed a dual monoclonal antibody pair-based colorimetric ELISA. While this ELISA is able to detect elevated VEGF levels in cancer patients, it lacks the sensitivity required to measure endogenous levels of VEGF in normal individuals. Rodriguez et al,J. Immunol.Methods219:45(1998) A two-site fluorescence assay VEGF ELISA that describes the sensitivity to produce 10pg/ml VEGF in pure (neat) plasma or serum. However, this fluorometric assay detects the fully intact 165/165 and 165/110 species of VEGF (VEGF 165/165 is reported to be proteolytically cleaved into three other forms: 165/110 heterodimer, 110/110 homodimer, and a 55 amino acid C-terminal fragment (Keyt et al,J.Biol.Chem.271:7788-,Arch.Biochem.Biophys.344:103-113(1997))。

thus, there is a need to develop diagnostic and prognostic assays that detect measurable levels of VEGF in biological samples of animal models or patients that are higher than existing ELISA, and/or that can measure different isoforms of VEGF.

Summary of The Invention

Antibody-sandwich ELISA methods against VEGF as antigen were developed to detect VEGF forms in biological samples. VEGF ELISA are provided herein that are capable of detecting VEGF isoforms and VEGF fragments greater than 110 ("VEGF₁₁₀₊"). Also provides a kit thereof.

For example, for detecting selective Vascular Endothelial Growth Factor (VEGF) forms (VEGF) of greater than 110 amino acids in biological samples₁₁₀₊) The method comprises the following steps: (a) contacting and incubating a biological sample with a capture reagent immobilized to a solid support, wherein theThe capture reagent is an antibody recognizing the same epitope as antibody 5C3 directed against human VEGF, which monoclonal antibody specifically binds to residues greater than 110 in human VEGF; (b) separating the biological sample from the immobilized capture reagent; (c) contacting the immobilized capture agent-target molecule complex with a detectable antibody that binds to the KDR and/or FLT1 receptor binding domains of VEGF; and (d) measuring the VEGF bound by the capture reagent using detection means directed against the detectable antibody₁₁₀₊The level of (c). In certain embodiments, the detectable antibody binds to an epitope in VEGF 1-110. In certain embodiments, a comparative ELISA may be performed to detect different types of VEGF. In certain embodiments, the biological sample (e.g., a tumor sample or tumor lysate, plasma, serum, or urine, etc.) is isolated from a human subject.

In one embodiment, the capture reagent is the 5C3 monoclonal antibody. In one embodiment, the immobilized capture reagent is coated on a microtiter plate. In certain embodiments, the detectable antibody is a monoclonal antibody. In one embodiment, the detectable antibody is a murine monoclonal antibody. In one embodiment, the immobilized monoclonal antibody is mab 5C3 and the detectable antibody is mab a4.6.1. In certain embodiments, the detectable antibody is directly detectable. In one embodiment, the detectable antibody is amplified by a colorimetric reagent. In one embodiment, the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3 ', 5, 5' -tetramethylbenzidine.

In certain embodiments of the invention, the human subject is a vascular, diabetic, or cancer patient, and the measuring step (d) further comprises comparing to a standard curve to determine VEGF levels compared to normal individuals.

Kits are also provided. For example, for detecting Vascular Endothelial Growth Factor (VEGF) forms (VEGF) of greater than 110 amino acids in biological samples₁₁₀₊) The immunoassay kit of (a) may comprise: (a) as capture reagentsThe antibody against human VEGF of (1), wherein the monoclonal antibody specifically binds to residues greater than 110 in human VEGF; and (b) a detectable antibody that binds to KDR and/or FLT1 receptor binding domains of VEGF as a detection reagent. In certain embodiments, the detectable antibody binds to an epitope in VEGF 1-110. In certain embodiments, the kit further comprises a solid support for the capture reagent. For example, the capture reagent may be immobilized on the solid support (e.g., a microtiter plate). In certain embodiments, the kit further comprises a detection means (e.g., colorimetric means, fluorometric means, etc.) that can detect the antibody. In certain embodiments, the kit further comprises purified VEGF as an antigen standard. In certain embodiments of the invention, one or more additional VEGF ELISA may be provided for performing a VEGF-mediated interaction₁₁₀₊Comparative study of ELISA. In one embodiment, the kit comprises a capture reagent monoclonal antibody (which is the murine monoclonal antibody mab 5C 3), and a detectable antibody (which is mab a4.6.1).

In another embodiment, the invention provides anti-VEGF antibody 5C3 (which can be obtained or produced from the hybridoma deposited under ATCC designation PTA-7737). The invention also provides monoclonal antibodies that do not bind VEGF1-110 but bind the same VEGF as the monoclonal antibody produced by hybridoma cell line PTA-7737₁₁₀₊An antibody to the epitope. In certain embodiments, the antibodies of the invention are conjugated to a detectable label. In one embodiment, hybridoma 5c3.1.1, deposited under ATCC accession number PTA-7737, is provided.

Brief Description of Drawings

FIGS. 1A, 1B and 1C show the detection of recombinant VEGF165, VEGF121(1) (truncated, with possible deletion of about 9 amino acids from the carboxy terminus according to the manufacturer R & D Systems), VEGF121(2) (from Pepro Tech), VEGF110 (N-terminal fragment generated by plasmin digestion of VEGF), and VEGF8-109 (artificial VEGF with amino acids 8-109 of VEGF 165) molecules by different VEGF ELISA. (1A) ELISA a, coated with 3.5F8 and detected using biotinylated a4.6.1. (1B) ELISA B, coated with a4.6.1 and detected using biotinylated 2E 3. (1C) ELISA C, coated with 5C3 and detected using biotinylated a4.6.1.

Fig. 2 shows western blots of VEGF produced by a673 cells probed with 3.5F8 (left) or a4.6.1 (right). The samples were VEGF purified from conditioned medium of A673 cells using an A4.6.1 affinity column (lane 1) and recombinant VEGF protein VEGF₁₆₅、VEGF₁₂₁(according to manufacturer R)&D Systems with the possibility of deletion of about 9 amino acids from the carboxy terminus) and VEGF produced by E.coli_8-109(lanes 2,3, 4, respectively).

FIG. 3 shows VEGF₁₆₅、VEGF₁₂₁And VEGF₁₁₀(N-terminal fragment generated by plasmin digestion of VEGF) showing proposed binding sites for the three antibodies of VEGF ELISA.

Detailed Description

Definition of

Before the present invention is described in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

The term "VEGF" as used herein refers to a 165 amino acid VEGF and the related 121, 145, 189 and 206 amino acid VEGF, such as Leung et al,Science246:1306(1989), Houck et al,Mol.Endocrin.5:1806(1991) and Neufeld et al, supra, as well as naturally occurring allelic and processed forms of these growth factors. See also, for example, FIGS. 1A and B of U.S. Pat. No.6,057,428. Active VEGF fragments can be released from ECM-bound VEGF by plasmin cleavage, yielding The first 110 amino acids (see, e.g., KeytBA et al, The carboxyl-terminal domain (111-165) of vascular endothelial growth factor for its clinical susceptibility.J Biol Chem.271:7788-7795(1996))。“VEGF₁₁₀₊"As used herein refers to fragments of VEGF greater than 110 amino acids (from the N-terminus), but not including fragments of the first 110 amino acids or smaller (e.g., VEGF)_8-109）。

The term "detection" is used in the broadest sense to include both qualitative and quantitative measurements of a target molecule. In one aspect, the detection methods described herein are used to identify VEGF only in biological samples₁₁₀₊Or the presence of VEGF. In another aspect, the method is used to test for VEGF in a sample₁₁₀₊Or whether VEGF is at a detectable level. In yet another aspect, the methods can be used to quantify VEGF in a sample₁₁₀₊Or amount of VEGF and further comparing VEGF from different samples₁₁₀₊Or VEGF levels.

The term "biological sample" refers to a body sample from any animal, but preferably from a mammal, more preferably from a human. In certain embodiments, such biological samples are from a vascular, diabetic, or cancer patient. Such samples include biological fluids such as serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, and tissue culture fluid, and tissue extracts (such as homogenized tissue, tumor tissue, and cell extracts). In certain embodiments, the sample is a body sample from any animal, in one embodiment, it is from a mammal, in one embodiment, it is from a human subject. In one embodiment, such biological sample is from a clinical patient.

The term "detectable antibody" refers to an antibody that is capable of being detected directly via a label amplified by a detection means, or is capable of being detected indirectly via, for example, another antibody that is labeled. For direct labeling, the antibody is typically conjugated to a moiety that can be detected by some means. In one embodiment, the detectable antibody is a biotinylated antibody.

The term "detection means" refers to a module or technique used in the ELISA herein to detect the presence of a detectable antibody and includes detection reagents that amplify immobilized labels, such as labels captured on a microtiter plate. In one embodiment, the detection means is a colorimetric detection agent, such as avidin or streptavidin-HRP.

The term "capture reagent" refers to a reagent that is capable of binding to and capturing a target molecule in a sample, such that the capture reagent-target antibody complex can be separated from the remaining sample under suitable conditions. Typically, the capture reagent is immobilized or immobilizable. In a sandwich immunoassay, the capture reagent is preferably an antibody or a mixture of different antibodies directed against the target antigen.

The term "antibody" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

For purposes herein, a "whole antibody" is an antibody comprising a heavy chain variable domain and a light chain variable domain, and an Fc region.

"Natural antibody" generally refers to a polypeptide composed of two identical light chains (L)) And two identical heavy chains (H) of about 150,000 daltons. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (V) at one end_H) Followed by a plurality of constant domains. Each light chain has a variable domain (V) at one end_L) And the other end is a constant domain; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form the interface between the light and heavy chain variable domains.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the invention may be produced by a method initially described by Kohler et al,Nature256:495(1975),

or can be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). "monoclonal antibodies" can also be used such as Clackson et al,Nature352:624-,J.Mol. Biol.222:581-597 (1991).

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins)Leukocyte) (wherein a portion of the heavy and/or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass), and fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al,Proc.Natl.Acad.Sci. USA,81:6851-6855(1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., Old World Monkey (Old World Monkey), such as baboon, rhesus Monkey or cynomolgus Monkey (rhesus or cynomolgus Monkey)) and human constant region sequences (U.S. patent No.5,693,780).

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody comprising minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications were made to further improve antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. Optionally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see Jones et al,Nature321:522-525(1986); Riechmann et al,Nature332:323-329(1988), and PrestaCurr.Op.Struct.Biol.2:593-596(1992). In one embodiment, humanized 5C3 antibodies are provided and usedMethods provided herein.

The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of the antibodies. It is concentrated in three segments called hypervariable regions in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β -sheet conformation, connected by three hypervariable regions that form loops connecting, and in some cases forming part of, the β -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al,Sequences of Proteins of Immunological Interest， release 5, Public Health ServiceNational Institutes of Health, Bethesda, MD, 1991). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

Papain digestion of antibodies produces two identical antigen-binding fragments (called "Fab" fragments, each with a single antigen-binding site), and a remaining "Fc" fragment (the name of which reflects its ability to crystallize readily). Pepsin treatment produced F (ab') with two antigen binding sites and still being able to cross-link the antigen₂And (3) fragment.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to form V_H-V_LAn antigen binding site is defined on the surface of the dimer. The six hypervariable regions together confer antigen-binding specificity to the antibody. However, even a single variable domain (or only three hypervariable domains specific for an antigen)Half of the Fv) also have the ability to recognize and bind antigen, but with a lower affinity than the complete binding site.

The Fab fragment also comprises the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domain carry at least one free thiol group. F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

Antibodies (immunoglobulin) "light chains" from any vertebrate species can be classified into one of two distinct types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences.

Antibodies can be classified into different classes according to their heavy chain constant domain amino acid sequences. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to the different classes of antibodies are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein the domains are present on a single polypeptide chain. Preferably, the Fv polypeptide is at V_HAnd V_LPolypeptide linkers are also included between the domains to enable the scFv to form the desired structure for binding to an antigen. For a review of scFv see Pl ü ckthun, inThe Pharmacology of Monoclonal AntibodiesVol 113, edited by Rosenburg and Moore, Springer-Verlag, New York, p.269-315, 1994.

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding.The hypervariable regions comprise amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34(L1), 50-56(L2) and 89-97(L3) in the light chain variable domain and residues 31-35(H1), 50-65(H2) and 95-102(H3) in the heavy chain variable domain; Kabat et al,Sequences of Proteins of Immunological interest, 5 th edition, Public Health ServiceNational Institutes of Health, Bethesda, MD (1991)) and/or those residues from "hypervariable loops" (e.g., residues 26-32(L1), 50-52(L2) and 91-96(L3) in the light chain variable domain and residues 26-32(H1), 53-55(H2) and 96-101(H3) in the heavy chain variable domain); the genes Chothia and Lesk are,J.Mol.Biol.196:901-917(1987)). "framework region" or "FR" residues refer to those residues in the variable domain other than the hypervariable region residues defined herein.

For the purpose of treatment/management, "mammal" refers to any animal classified as a mammal, including humans, domestic and livestock animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cattle, and the like. Preferably, the mammal refers to a human.

The terms "cancer," "cancerous," or "malignant" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma (carcinoma) (including adenocarcinoma), lymphoma, blastoma, melanoma, sarcoma, and leukemia. More specific 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), peritoneal cancer, hepatocellular cancer, gastric (including gastrointestinal) cancer, gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver (liver cancer) (e.g., hepatoma (hepatoma) and hepatoma (hepatoma)), bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, rectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney (renal) cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, basal cell carcinoma, testicular cancer, esophageal cancer, liver cancer (hepatoma), soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma (carcinoid), mesothelioma, multiple myeloma, And various types of head and neck cancer, and B cell lymphomas including low grade/follicular non-Hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-nucleated NHL, storage disease (bulk disease) NHL, mantle cell lymphoma, AIDS related lymphoma, and Waldenstrom's (Waldenstrom) macroglobulinemia, Hodgkin's lymphoma, Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with scarring nevus (phakomatases), edema (such as that associated with brain tumors), and Meigs' syndrome.

The phrases "vascular" and "cardiovascular" are used interchangeably to describe patients having indications/indications of stimulating angiogenesis and/or cardiovascular formation, and patients having indications/indications of inhibiting angiogenesis and/or cardiovascular formation. Such disorders include, for example, arterial diseases such as atherosclerosis, hypertension, inflammatory vasculitis, raynaud's disease and raynaud's phenomenon, aneurysms, and arterial restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular conditions such as peripheral vascular conditions, AMD, cancers such as vascular tumors, e.g., hemangiomas (capillary and cavernous), glomus, telangiectasia, bacillary angiomatosis, angioendothelioma, angiosarcoma, hemangioepidermoma, Kaposi's sarcoma, lymphangioma, and lymphangiosarcoma, tumor angiogenesis, trauma (trauma) such as wounds (wound), burns, and other injured tissues, implant fixation (implantation), scarring, ischemia reperfusion injury (ischemiperfusion input), rheumatoid arthritis, cerebrovascular diseases, renal diseases such as acute renal failure, and osteoporosis. This may 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 a disease of progressive glucose metabolism involving inappropriate production or utilization of insulin, and is characterized by hyperglycemia and diabetes. The term includes all forms of diabetes, such as type I and type II diabetes and insulin-resistant diabetes (insulin-resistant diabetes), such as Mendenhall's syndrome, warner's syndrome, leprechaunism, lipodystrophy diabetes (lipotrophic diabetes), and other lipoatrophy.

The term "affinity purified" refers to the purification of a substance by eluting it through an affinity chromatography column.

ELISA

Vascular Endothelial Growth Factor (VEGF) is a homodimeric glycoprotein and is a key angiogenic factor in angiogenesis during development and in pathological angiogenesis associated with tumors. VEGF expression is enhanced in response to hypoxia, and potentially other factors such as growth factors, hormones, and oncogenes (see, e.g., Ferrara N: Vascular endothelial growth factor: Basic science and clinical progress).Endocrine Reviews25:581-611(2004)). The human VEGF gene has 8 exons separated by introns. Alternative RNA splicing results in The production of at least 4 major isoforms having 121, 165, 189, and 206 amino acids in monomeric form (see, e.g., Houck KA et al, The molecular growth factor family: identification of a forth molecular species and transformation of alternative splicing of RNA).Mol Endocrinol1806, 1814(1991), and Tischer E et al, The human gene for a vascular endothelial growth factor, multiple protein for an area encoded by a porous alkaline ex-situ hybridization.J Biol Chem266:11947-11954(1991)). Lower frequency isoforms have also been reported, including those with 145 monomeric forms (see, e.g., Poltorak Z et al, VEGF145, a secreted vascular growth factor, which is a host of bits to an extracellular cell)r matrix.J Biol Chem272: 7151-.Invest Ophthalmol Vis Sci40:752-759(1999)) amino acid isoforms. All VEGF isoforms bind to two receptor tyrosine kinases, VEGFR-1 (see, e.g., De Vries C et al, therms-like tyrosine kinase, a receptor for a vascular endothelial growth factor).Science255: 989-.Oncogene6:1677-1683(1991))。VEGF₁₆₅It also interacts with Neuropilin (see, e.g., Soker S et al, Neuropilin-1 is expressed by endothiral and tumor cells as an isoflavone-specific receptor for a vascular endothiral growth factor).Cell 92:735-745(1998))。VEGF₁₈₉And VEGF₂₀₆Binds heparin with high affinity and is largely sequestered in the extracellular matrix (ECM). VEGF₁₆₅Binds heparin with moderate affinity and is partially soluble and partially binds to the cell surface and ECM. VEGF₁₂₁Does not bind heparin and is readily soluble. VEGF was found by reverse transcription-PCR analysis in breast and ovarian cancer tumor specimens and cell lines₁₂₁And VEGF₁₆₅Is a most potently expressed variant, and VEGF₂₀₆Expression was not detected. VEGF found in cell lines₁₈₃And VEG₁₈₉Expression is undetectable or at low levels, and is detected in some tumor specimens (see, e.g., Stimpfl M et al, Vascular Endothelial growth factor plasma variants and the early viral value branch and ocular cancer.Clinical Cancer Research 8:2253-2259(2002))。

Active VEGF fragments can be released from ECM-bound VEGF by plasmin cleavage yielding The first 110 amino acids (see, e.g., Keyt BA et al, The carboxyl-terminal domain (111-165) of vascular endothelial growth factor for its mitogenic potential.J Biol Chem 271:7788-7795(1996)). This may be a mechanism that locally modulates the bioavailability of VEGF during the physiological and pathological processes of angiogenesis. See, for example, Houck KA et al, Dual registration of vascular endothelial growth factor by genetic and proteinaceous mechanisms.J Biol Chem267: 26031-.J Biol Chem271: 7788-.Am Pathology168:670-684(10-12)(2006). However, VEGF in biological samples₁₁₀The concentration has not been reported. Active VEGF fragments can also be released from ECM-bound VEGF by Matrix Metalloproteinase (MMP) cleavage. This is supported by the discovery of degraded VEGF fragments with amino acids other than 1-110 in ascites from ovarian cancer patients. Both plasmin and MMP3 were detected in ascites. See, e.g., Lee S, Shahla MJ et al, processing of VEGF-A by matrix matrices regulation bioloavailability and vaculathatterning in tumors.J Cell Biology 169:681-691(2005)。

Enzyme-linked immunosorbent assays (ELISAs) for various antigens include those based on colorimetric, chemiluminescent, and fluorimetric assays. ELISA has been successfully applied to determine small amounts of drugs and other antigenic components in plasma and urine samples, involves no extraction steps, and is easy to perform. The assays described herein utilize antibodies directed against VEGF and VEGF₁₁₀₊And ELISA for the capture reagent and detectable antibody of (1). In certain embodiments, the ELISA is cell-based. In the first step of the assay, a VEGF-suspected or VEGF-containing sample is assayed₁₁₀₊Is contacted with and incubated with a capture (or coating) antibody such that the capture antibody captures or binds to VEGF or VEGF₁₁₀₊So that it can be detected in the detection step. The detection step involves the use of a detectable antibody that binds to any VEGF or VEGF bound₁₁₀₊Combined with sense of touchThe protein of interest, if present, and detecting the presence of VEGF or VEGF by detecting the marker on the antibody using detection means₁₁₀₊Presence or amount of (a). Such an ELISA can be compared to an ELISA that recognizes total VEGF (e.g., U.S. Pat. No.6,855,508; those ELISAs described herein, and those ELISAs known in the art) or VEGF isoforms to determine the type of VEGF present.

For example, in certain embodiments, the assay utilizes the following steps:

step 1

In step 1 of the assay herein, a biological sample is contacted and incubated with an immobilized capture (or coating) reagent, which is an anti-VEGF monoclonal antibody. The antibody may be from any species, but preferably the monoclonal antibody is a murine or rat or mouse monoclonal antibody, more preferably murine, most preferably selected from the hybridoma-derived monoclonal antibodies 5C3 identified herein. Thus, in a particularly preferred embodiment, the immobilized monoclonal antibody is a murine monoclonal antibody, most preferably mab 5C3. Conventional immobilization is achieved by either insolubilizing the capture reagent prior to the assay protocol, such as by adsorption to a water-insoluble substrate or surface (U.S. Pat. No.3,720,760) or non-covalent or covalent coupling (e.g., using glutaraldehyde or carbodiimide cross-linking, whether with nitric acid for example and U.S. Pat. No.3,645,852 or Rotmans, etc.,J.Immunol.Methods57:87-98(1983) the reducing agent previously activated the support); or insolubilizing the capture reagent after the assay protocol, for example by immunoprecipitation.

The solid phase used for immobilization may be any inert support or carrier that is substantially water insoluble and useful for immunoassays, including supports in the form of, for example, surfaces, particles, porous matrices, and the like. Examples of commonly used supports include small sheets, Sephadex, polyvinyl chloride, plastic beads, and assay plates or tubes (including 96-well microtiter plates) made of polyethylene, polypropylene, polystyrene, and the like, and particulate materials such as filter paper, agarose, cross-linkedDextran, and other polysaccharides. Alternatively, reactive water-insoluble substrates (such as cyanogen bromide activated carbohydrates and reactive substrates described in U.S. Pat. Nos. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4,330,440) are suitable for capture reagent immobilization. In one embodiment, the immobilized capture reagent is coated on a microtiter plate, more specifically, the preferred solid phase used is a multi-well microtiter plate that can be used to analyze several samples at a time, e.g., a micro-test 96-well ELISA plate, such as those sold under Nune Maxisorb or Immulon. In certain embodiments, the plate is MICROTEST^TMOr MAXISORP^TM96-well ELISA plates, such as NUNC MAXISORB^TMOr IMMULON^TMAnd (4) selling.

The solid phase is coated with a capture reagent as defined above, which may be attached by non-covalent or covalent interactions or physical attachment as required. Techniques for attachment include those described in U.S. Pat. No.4,376,110 and the references cited therein. If covalent, the plate or other solid phase is incubated with the crosslinker along with the capture reagent under conditions well known in the art, such as, for example, at room temperature for 1 hour.

Common cross-linking agents for attaching capture reagents to a solid phase substrate include, for example, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxy-succinimide esters (e.g., esters with 4-azido-salicylic acid), homobifunctional imidoesters (including disuccinimidyl esters, such as 3, 3' -dithiobis- (succinimidyl-propionate)), and bifunctional maleimides (such as di-N-maleimide-1, 8-octane). Derivatizing agents such as methyl-3- [ (p-azidophenyl) -dithio ] propioimidate (pro-pioimi-date) produce photoactivatable (photoactivatable) intermediates that are capable of forming crosslinks in the presence of light.

If 96-well plates are utilized, they are typically coated with capture reagents (typically diluted in a buffer such as 0.05M sodium carbonate, by incubation for at least about 10 hours, more preferably at least overnight, at a temperature of about 4-20 ℃, or about 4-8 ℃, and at a pH of about 8-12, or about pH9-10, or about pH 9.6). If a shorter coating time is desired, 96-well plates can be coated, for example, at room temperature for 2 hours. The plates may be stacked and coated well before the assay itself, and then the assay may be performed on several samples simultaneously in a manual, semi-automated, or automated manner (such as by using robotics).

The coated plate is then typically treated with a blocking agent that non-specifically binds to the binding sites and saturates them to prevent unwanted binding of free ligand to too many sites on the wells of the plate. Examples of blocking agents suitable for this purpose include, for example, gelatin, bovine serum albumin, egg albumin, casein, and skim milk. The blocking treatment typically occurs at ambient temperature for about 1-4 hours, preferably about 1-3 hours, or overnight at 0-4 ℃.

After coating and blocking, VEGF standard (purified VEGF) or the biological sample to be analyzed (diluted appropriately) is added to the immobilized phase. The preferred dilution rate is about 1-15%, preferably about 10%, by volume. Buffers that may be used for dilution for this purpose include: (a) contains 0.5% BSA, 0.05% TWEEN 20^TMDetergent (P20), 0.05% PROCLIN^TM300 antibiotic, 5mM EDTA, 0.25% Chaps surfactant, 0.2% beta-gamma globulin, and 0.35M NaCl in PBS, pH 7.4; (b) PBS containing 0.5% bovine serum albumin, 0.05% polysorbate 20, 5mM EDTA, 0.25% CHAPS, 0.2% bovine gamma-globulin, and 0.35M NaCl, ph 7.4; (c) contains 0.5% BSA, 0.05% polysorbate 20(P20), and 0.05% PROCLIN^TM300 PBS, pH 7; (d) contains 0.5% BSA, 0.05% P20, and 0.05% PROCLIN^TM300. 5mM EDTA, and 0.35M NaCl in PBS, pH 6.35; (e) contains 0.5% BSA, 0.05% P20, and 0.05% PROCLIN^TM300. 5mM EDTA, 0.2% beta-gamma globulin, and 0.35M NaCl in PBS, pH 7.4; and (f) contains 0.5% BSA, 0.05% P20, 0.05% PROCLIN^TM300. 5mM EDTA, 0.25% Chaps, and 0.35M NaCl in PBS, pH 7.4. PROCLIN^TM300 acts as a preservative and TWEEN 20^TMActing as a detergent to eliminate non-specific binding.

While the concentration of the capture reagent will generally be determined by the concentration range of interest for VEGF (taking into account any necessary dilution of the biological sample), the final concentration of the capture reagent will normally be determined empirically to maximize the sensitivity of the assay in the range of interest.

The conditions used to incubate the sample and immobilized capture reagent are selected to maximize the sensitivity of the assay and minimize dissociation. Preferably, the incubation is done at a fairly constant temperature, ranging from about 0 ℃ to about 40 ℃, preferably about 20-25 ℃. The incubation time is primarily temperature dependent and is generally no greater than about 10 hours to avoid insensitive assays. Preferably, the incubation time is from about 0.5 to 3 hours, more preferably 1.5-3 hours, at room temperature, to free VEGF₁₁₀₊Or the binding of VEGF to the capture reagent is maximized. If a protease inhibitor is added to prevent the proteases in the biological fluid from degrading VEGF, the duration of the incubation can be longer.

At this stage, the pH of the incubation mixture will generally be in the range of about 4-9.5, preferably in the range of about 6-9, more preferably about 7-8, and the most preferred pH of the assay (ELISA) diluent is pH 7.4. The pH of the incubation buffer is selected to maintain the capture reagent on the VEGF being captured₁₁₀₊Or significant levels of specific binding of VEGF. Various buffers may be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, Tris-HCl or Tris-phosphate, acetate, barbital, and the like. The particular buffer employed is not critical to the present invention, but one buffer may be preferred over another in various assays.

Step 2

In step 2 (which is optional) of the assay methods herein, the biological sample is separated from the immobilized capture reagent (preferably by washing) to remove uncaptured molecules. The solution used for washing is typically a buffer ("wash buffer") whose pH uses the considerations and buffering described above for the incubation stepThe preferred pH range is from about 6 to 9, as determined by the liquid. Three or more washes may be performed. The cleaning temperature is generally from refrigerator temperature to moderate temperature (constant temperature is maintained during the measurement), typically about 0-40 deg.C, more preferably about 4-30 deg.C. For example, wash buffer may be placed in a reservoir at 4 ℃ in ice prior to washing, and a plate washer may be used for this step. A cross-linking agent or other suitable agent may also be added at this stage to allow for newly bound VEGF₁₁₀₊Or the VEGF is covalently attached to a capture reagent if it is bound to the captured VEGF₁₁₀₊Or that VEGF may be dissociated to some extent in subsequent steps.

Step 3

In the next step, the immobilized capture reagent is contacted with a detectable antibody, preferably at a temperature of about 20-40 ℃, more preferably about 20-25 ℃, wherein the exact temperature and time of contact depends primarily on the detection means employed. For example, where streptavidin-peroxidase and 3,3 ', 5, 5' -tetramethylbenzidine are used as the detection means, for example, in one embodiment, the contacting is performed (e.g., for about 1 hour or more) to maximize signal amplification. After washing the plates, it is preferred that the VEGF be compared to free VEGF₁₁₀₊Or the highest expected concentration of VEGF (as described above) molar excess of antibody was added to the plates. The antibody is directly detectable or indirectly detectable. While the detectable antibody may be a polyclonal or monoclonal antibody, for example in certain embodiments, it is a monoclonal antibody, in one embodiment it is murine, and in one embodiment it is mab a4.6.1. Likewise, a detectable antibody may be directly detectable and in one embodiment it has a colorimetric label and in another embodiment it has a fluorometric label. More preferably, the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3 ', 5, 5' -tetramethylbenzidine. The readout of the detection means may be fluorometric or colorimetric. The affinity of the antibody must be high enough that small amounts of free VEGF can be detected₁₁₀₊Or VEGF, but not high enough to cause VEGF₁₁₀₊Or VEGF is dragged out of the capture reagent.

Step 4

In the final step of the assay method, the level of free VEGF that is currently bound to the capture reagent is measured using detection means directed against a detectable antibody. If the biological sample is from a vascular, diabetic, or cancer patient, the measuring step preferably includes comparing the response that occurs as a result of the above three steps to a standard curve to determine VEGF compared to normal individuals₁₁₀₊Or the level of VEGF, or preferably comprises comparing the responses that occur as a result of the above three steps and identifying another VEGF ELISA of a different isoform or total VEGF to determine the level of VEGF type after comparing ELISA and optionally comparing with normal individuals.

Antibody production

Polyclonal antibodies against VEGF are generally generated by multiple subcutaneous (sc) or intraperitoneal (ip) injections of VEGF and an adjuvant in animals. Using bifunctional or derivatizing reagents, e.g. maleimidobenzoyl sulphosuccinimide ester (coupled via cysteine residues), N-hydroxysuccinimide (coupled via lysine residues), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N = C = NR, wherein R and R¹Being different hydrocarbon groups, it may be useful to couple VEGF or a fragment comprising the target amino acid sequence to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

Antibodies used as coating or detectable antibodies may be obtained from any convenient vertebrate source, such as murine, primate, lagomorpha, goat, rabbit, rat, chicken, bovine, ovine, equine, canine, feline, or porcine. Chimeric or humanized antibodies may also be employed, as described, for example, in U.S. Pat. No.4,816,567, Morrison et al,Proc.Natl.Acad.Sci.USA81:6851(1984), Neuberger et al,Nature312:604(1984), Takeda et al,Nature 314:452(1985), and WO 98/45331 published 10, 15, 1998, and other references mentioned above.

Animals were immunized against the immunogenic conjugate or derivative by mixing 1mg or1 μ g of conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals were boosted with an initial amount of 1/5-1/10 of conjugate in Freund's incomplete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, blood was collected from the animals and the serum was assayed for anti-VEGF titer. Animals were boosted until titers reached a plateau (plateaus). Preferably, the animal is fortified with VEGF but conjugates obtained by conjugation to different proteins and/or via different cross-linking agents. Conjugates can also be prepared in recombinant cell culture as protein fusions. Also, coagulants such as alum are used to enhance the immune response. Methods for generating polyclonal antibodies are described in numerous immunological textbooks, such as Davis and the like,microbiology, 3 rd edition(Harper and Row, New York, New York, 1980).

Monoclonal antibodies are prepared by recovering spleen cells from an immunized animal, immortalizing the cells in a conventional manner, for example by fusion with myeloma cells or by Epstein-Barr virus transformation, and screening for clones expressing the desired antibody. See for example Kohler and Milstein,Eur.J. Immunol.6:511(1976). Monoclonal antibodies or antigen binding regions of monoclonal antibodies (such as Fab or (Fab)₂Fragments) may alternatively be generated by recombinant methods.

Examples of suitable antibodies include those antibodies to known RIAs that have been used for the protein in question, for example those directed against VEGF as described in the references given in the introduction herein.

In certain embodiments, anti-VEGF antibody 5C3 (which can be obtained or produced from the hybridoma deposited under ATCC accession number PTA-7737) is used, optionally with another anti-VEGF antibody a4.6.1. The invention also providesThat does not bind VEGF1-110 but binds the same VEGF as the monoclonal antibody produced by hybridoma cell line PTA-7737₁₁₀₊An antibody to the epitope. Hybridoma 5c3.1.1, deposited under ATCC accession number PTA-7737, is provided.

Detection of

The antibody added to the immobilized capture reagent is either directly labeled or indirectly labeled by adding a molar excess of a second labeled antibody to the animal species IgG of the first antibody after washing away the excess of the first antibody. In the latter, indirect assay, labeled antisera to the first antibody is added to the sample such that the labeled antibody is produced in situ.

Whether the label for either the first or second antibody does not interfere with free VEGF₁₁₀₊Or any detectable functionality of VEGF for antibody binding. Examples of suitable labels are those known for use in immunoassays, including moieties that can be detected directly, such as fluorescent dyes, chemiluminescent, and radioactive labels, and moieties that must be reacted or derivatized to be detected, such as enzymes. Examples of such labels include radioisotopes³²P、¹⁴C、¹²⁵I、³H. And¹³¹i, a fluorophore such as a rare earth chelate or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, a luciferase such as firefly luciferase and bacterial luciferase (U.S. Pat. No.4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, a saccharide oxidase such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, a heterocyclic oxidase such as uricase and xanthine oxidase, an enzyme coupled with oxidation of a dye precursor using hydrogen peroxide such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, biotin/streptavidin-beta-galactosidase, and MUG, spin labels, phage labels, stable free radicals, and the like. As has been described above in the context of the present invention,fluorometric detection is one example.

Conventional methods can be used to covalently bind these labels to the protein or polypeptide. For example, coupling agents such as dialdehydes, carbodiimides, dimaleimides, diimides (bis-imidates), diazotized benzidine (bis-diazotized benzidin), and the like may be used to label the antibody with the fluorescent, chemiluminescent, and enzymatic labels described above. See, e.g., U.S. Pat. Nos. 3,940,475 (fluorometric) and 3,645,090 (enzymes); the Hunter et al, the number of the patents,Nature144:945(1962), David et al,Biochemistry13:1014-,J.Immunol.Methods40:219-,J.Histochem.and Cytochem.30:407-412(1982). In certain embodiments, the labels herein are fluorescent to increase amplification and sensitivity to 8pg/ml, more preferably biotin is used with streptavidin- β -galactosidase and MUG to amplify the signal. In certain embodiments, colorimetric labels are used, for example, wherein the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3 ', 5, 5' -tetramethylbenzidine.

Conjugation of such labels (including enzymes) to antibodies is a standard practice in immunoassay technology for the skilled person. See, e.g., O' Sullivan et al, "Methods for the preparation of Enzyme-antibodies Conjugates for Use in Enzyme Immunoassay", inMethods in EnzymologyOne and H.Van Vunakis, volume 73(Academic Press, New York, New York,1981), page 147-.

After addition of the final labeled antibody, the amount of bound antibody is determined by washing to remove excess unbound labeled antibody, measuring the amount of attached label using an appropriate detection method for the label, and comparing the measured amount with the amount of free VEGF in the biological sample₁₁₀₊Or the amount of VEGF. For example, in the case of an enzyme, the amount of color developed and measured would be for VEGF₁₁₀₊Or a direct measurement of the amount of VEGF present. Specifically, if HRP is the label,the color is detected using the substrate 3,3 ', 5, 5' -tetramethylbenzidine at an absorbance of 450 nm.

In one example, after washing the enzyme-labeled second antibody against the first unlabeled antibody from the immobilized phase, color or chemiluminescence is visualized and measured by incubating the immobilized capture reagent with an enzyme substrate. Free VEGF is then calculated by comparison with the color or chemiluminescence produced by a standard VEGF run in parallel₁₁₀₊Or amount of VEGF concentration.

Reagent kit

For convenience, the assay methods of the invention may be provided in kit form. The kit is a kit comprising the following basic elements:

(a) capture reagent consisting of monoclonal antibody against human VEGF molecule, wherein said monoclonal antibody recognizes VEGF₁₁₀₊(ii) a And

(b) a detection reagent consisting of a detectable (labeled or unlabeled) antibody that binds KDR and FLT1 receptor binding domains of VEGF. These basic elements are defined above. In certain embodiments, the detection reagent comprises a detectable antibody that binds to an epitope of VEGF 1-110.

Preferably, the kit further comprises a solid support for the capture reagent, which may be provided as a separate element or on which the capture reagent has been immobilised. Thus, the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such a support included in the kit or provided separately from the kit.

Preferably, the capture reagents are coated on a microtiter plate. The detection reagent may be a labeled antibody for direct detection or an unlabeled antibody detected by a labeled antibody against an unlabeled antibody produced in a different species. If the label is an enzyme, the kit will typically include the cofactors and substrates required for the enzyme, and if the label is a fluorophore, a dye precursor that provides a detectable chromophore. If the detection reagent is unlabeled, the kit may also comprise detection means for a detectable antibody, such as a labeled antibody against the unlabeled antibody, preferably in a fluorometric detection format. If the label is an enzyme, the kit will typically include the cofactors and substrates required for the enzyme, if the label is a fluorophore, a dye precursor that provides a detectable chromophore, and if the label is biotin, an avidin, such as avidin, streptavidin, or streptavidin and MUG coupled to HRP or beta-galactosidase.

In a specific embodiment, the capture reagent is a monoclonal antibody, preferably rodent, more preferably murine or rat or mouse, still more preferably murine, and most preferably mab 5C3. Likewise, in certain embodiments, the detectable antibody is a biotinylated monoclonal antibody, which is rodent, more preferably murine or rat or mouse, still more preferably murine, still more preferably mab a4.6.1. In certain embodiments, the capture reagent is immobilized in such a kit.

In certain embodiments, the kit may comprise a plurality of ELISAs for use in comparative studies described herein for detecting various forms of VEGF and VEGF₁₁₀₊。

Typically, the kit further comprises instructions for performing the assay, and/or VEGF as an antigen standard (e.g., purified VEGF, preferably recombinantly produced VEGF, and VEGF 110), as well as other additives such as stabilizers, wash and incubation buffers, and the like.

An example of a VEGF standard is recombinant human VEGF produced in mammalian cells, which can be obtained from Genentech, Inc. (South San Francisco, California) and those companies and methods described herein.

The components of the kit will be provided in predetermined proportions, with the relative amounts of the reagents being varied appropriately to provide concentrations in the reagent solutions that substantially maximize the sensitivity of the assay. In particular, the reagents may be provided as a dry powder (typically lyophilized, including excipients) that upon dissolution will provide a solution of the reagents in a concentration suitable for combination with the sample to be tested.

Material preservation

The following materials have been deposited at the American Type Culture Collection (ATCC):

5C3.1.1 was deposited at ATCC at 2006, 7/19 and deposited under accession number PTA-7737.

Collection date of classified named collection unit

Splenic cell lines of mice: anti-VEGF 5C3.1.1 ATCC PTA-77372006 year 7 month 19

The following materials have been deposited at the American Type Culture Collection (ATCC):

a4.6.1 was deposited with the ATCC at 1991 at 29.3.month and deposited under accession number HB 10709.

Collection date of classified named collection unit

Hybridoma, A4.6.1 (anti-hVEGF) ATCC HB 107091991 year 3 month 29

The deposit was made according to the provisions of the Budapest Treaty (Budapest treat) and its implementation details, which are internationally recognized for the deposit of microorganisms for the purposes of patent procedure. This ensures that the preserved viable cultures are preserved for 30 years from the date of preservation. The deposit is available through the ATCC under the terms of the Budapest treaty and subject to the agreement between Genentech and ATCC which ensures that the public, upon grant of the pertinent United states patent or publication to the public of any United states or foreign patent application, and whichever is the predecessor, permanently and without limitation, will obtain progeny of the deposited culture, and that progeny of the deposited culture will be available to individuals approved by the United states patent and trademark office under 35 USC 122 and in accordance with its regulatory guidelines (including 37 CFR 1.14, specifically reference 886OG 638).

The assignee of the present application has agreed that if a culture of the deposited material dies, is lost or is damaged when cultured under appropriate conditions, he will be quickly replaced with another portion of the same culture upon notification. The availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The description is deemed to be sufficient to enable one skilled in the art to practice the invention. The scope of the invention is not limited by the deposited constructs, as the deposited embodiments are intended to be a single illustration of certain aspects of the invention, and any functionally equivalent construct is within the scope of the invention. The material deposits herein are not to be construed as an admission that the written description is not sufficient to enable practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific exemplifications presented. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Examples

Example 1:

vascular Endothelial Growth Factor (VEGF), which is expressed in different isoforms due to alternative RNA splicing, is known to be angiogenic in tumorsPlays a key role in the process. We measured VEGF₁₆₅And the concentration of total VEGF, and VEGF was evaluated₁₁₀(which is the active fragment produced by plasmin digestion of VEGF). ELISA A (VEGF165-206 ELISA) detection of VEGF₁₆₅And longer isoforms other than VEGF₁₂₁. ELISA B (VEGF110-206 ELISA) detection of VEGF₁₆₅And isotypic VEGF₁₂₁And VEGF₁₁₀. ELISA C (VEGF121-206ELISA) detection of VEGF₁₆₅And longer isoforms, VEGF₁₂₁And molecular weight greater than VEGF₁₁₀But not VEGF₁₁₀VEGF fragments of (referred to herein as "VEGF₁₁₀₊”）。

Materials and methods

Reagents and cells: recombinant VEGF₁₆₅(Genentech)、VEGF₁₂₁(PeproTech,Rocky Hill,New Jersey)、VEGF_8-109(by VEGF)₁₆₅8-109) and truncated VEGF₁₂₁(R&D Systems, Minneapolis, MN) were generated in E.coli. Truncated VEGF according to Mass Spectrometry₁₂₁Has an intact N-terminus, but has a 26kDa quality, which is consistent with a truncation of approximately 9 amino acids from the carboxy terminus according to the manufacturer. It is in VEGF when analysed by SDS-PAGE under reducing conditions₁₁₀And VEGF₁₂₁And (4) moving between. VEGF₁₁₀Digestion of VEGF by plasmin₁₆₅And (KeytBA et al, The carboxyl-terminal domain (111-165) of The vascular endethical growth for The issues nutritional potential.J Biol Chem271:7788-7795(1996)). The mass spectrometry measurement yielded a molecular weight of 25390, which matched the theoretical mass of 25389. The concentration was determined using bicinchoric acid method (Pierce, Rockford, IL). For VEGF_8-109、VEGF₁₂₁And VEGF₁₆₅The molecular weights calculated for the concentrations were 23.8, 28.9 and 38.2KDa, respectively. Monoclonal anti-VEGF antibodies A4.6.1, 3.5F8, 2E3 and 5C3 were prepared by using VEGF produced in CHO cells₁₆₅Immunization of mice to generate (Kim KJ et al, The molecular expression factor proteins: Identification of biological novel regions by neural stimulating monoclonalnal antibodies.Growth Factors7:53-64(1992)). Mammary cell lines SK-BR-3, BT-474, T-47D and MCF-7 and ovarian cell lines ES-2, OVCAR-3 and SK-OV-3 (American type culture Collection, Rockville, Md.) were cultured in RPMI, 2mM L-glutamine and 10% FBS (except 20% for OVCAR-3) at 37 ℃ in humidified 5% CO₂An incubator.

Purification of VEGF in conditioned medium of a673 cells: a673 cells (American type culture Collection) were cultured in 50: 50F 12/DMEM, 2mM L-glutamine and 5% FBS to 60% confluence, then in serum free medium (Genentech) until confluence. VEGF was purified from the supernatant using an A4.6.1-Sepharose column prepared with CNBr-activated Sepharose (Amersham Biosciences, Piscataway, N.J.). The column eluate and recombinant VEGF controls (0.2. mu.g per lane) were electrophoresed on 18% Tris-glycine gel (Invitrogen, Carlsbad, Calif.) under reducing conditions and blotted onto nitrocellulose. Blots were blocked with 3% bovine serum albumin in 0.5M Tris-HCl pH7.5, 1.5MNaCl, 50mM EDTA, 0.5% Trition100 and probed with 200ng/ml3.5F8 or A4.6.1 followed by 2ng/ml goat anti-mouse Fc- (HRP Jackson ImmunoResearch). The signal was visualized using SuperSignal West Dura (Pierce) and recorded on X-ray film.

VEGF ELISA for measuring VEGF concentration

ELISA A (VEGF165-206 ELISA): unless otherwise indicated, fluorescence assay ELISA a was used to measure VEGF in the samples. Fluorescence assay ELISA A was coated with 3.5F8, detected with biotinylated A4.6.1 followed by streptavidin- β -galactosidase, and using 4-methylumbelliferyl- β -D-galactoside as substrate (Rodriguez CR et al, A sensitive fluoromonomer-linked immunosorbent assay 165in human plasma.J Immunol Methods 219:45-55(1998))。VEGF₁₆₅The standard substance is 1-128pg/mL, or 0.026-3.35 pM. Colorimetric ELISA a was coated with 3.5F8 and detected with biotinylated a4.6.1 following the protocol used for ELISA C described below. VEGF₁₆₅The standard substance is 1.6-200 pg/mL.

ELISA B (VEGF110-206 ELISA) (previously known as VEGF121-206ELISA, Konecny GE et al, Association between HER-2/neu and Vascular endothelial growth Factor Expression preferences Clinical Outcome in primer Breast cancer Patients).Clinical Cancer Research10:1706-1716(2004)): MaxiSorp96 well microplates were coated overnight at 4 ℃ with 100. mu.l/well of 0.5. mu.g/ml antibody A4.6.1 in 50mM carbonate buffer pH 9.6. After this step and between subsequent room temperature incubation steps with PBS containing 0.05% polysorbate 20, ph 7.4. With 0.5% bovine serum albumin, 10ppm Proclin in PBS (150. mu.l/well)^TMPlates were blocked for 1 hour with 300(Supelco, Bellefonte, Pa.). VEGF standard (1.56-200 pg/ml VEGF)₁₆₅Or 0.0409-5.24pM VEGF, in two-fold serial dilutions) and PBS containing 0.5% bovine serum albumin, 0.05% polysorbate 20, 5mM EDTA, 0.25% CHAPS, 0.2% bovine gamma-globulin (Sigma, st. louis, MO), and 0.35M NaCl, ph7.4 (sample buffer) serial diluted samples (minimum 1:10 dilution) in two-fold or three-fold serial dilutions were added to the plates (100 μ l/well) and incubated for 2 hours. Bound VEGF was detected by incubating biotinylated 2E3 (or another antibody capable of binding the receptor binding domain of VEGF) on the plate for 1 hour, followed by streptavidin-HRP (Amersham, Copenhagen, Denmark) for 30 minutes, biotin-tyramide (tyramide) (ELAST ELISA amplification system, Perkin er Life Sciences inc., MA) for 15 minutes, and streptavidin-HRP for 30 minutes. Substrate TMB (3, 3 ', 5, 5' -tetramethylbenzidine) (Kirkegaard) was added&Perry Laboratories) and stopped by adding 1M phosphoric acid. The absorbance was read at 450nm on a Titertek stacked plate reader (ICN, Costa Mesa, Calif.). Titration curves were fitted using a four parameter regression curve fitting program (KaleidaGraph, Synergy software, Reading, PA). The data points falling within the standard curve range are used to calculate the estimated VEGF concentration in the sample. After subtraction of putative 2.1pg/ml endogenous VEGF in 10% plasma used in this study, 1.56-200pg/ml VEGF in 10% human EDTA plasma (Golden West Biologicals Inc., Temecula, Calif.)₁₆₅The recovery of (B) was 92-120%.

ELISA C (VEGF121-206 ELISA): the plates were coated with 1. mu.g/ml anti-VEGF 5C3 antibody and blocked as described above. VEGF standard (4.00-512 pg/ml VEGF)₁₆₅Or 0.105-13.4pM VEGF at 2-fold serial dilutions) and serial diluted samples in sample buffer were added to the plates. The plates were incubated for 2 hours. Bound VEGF was detected by addition of biotinylated a4.6.1 followed by streptavidin-HRP and TMB as substrate. The plate was read and the data analyzed as described above. 4.00-512pg/ml VEGF in 10% plasma after subtraction of 1.6pg/ml putative endogenous VEGF in 10% plasma used in this study₁₆₅The recovery of (B) was 77-101%.

Results and discussion

VEGF ELISA the aforementioned ELISA A was coated with 3.5F8 and detected with biotinylated A4.6.1 (Rodriguez CR et al, A sensitive fluorometric enzyme-linked immunological assay and substrates were used with human plasma 165 in.J Immunol Methods219:45-55,1998). It detects VEGF165 (VEGF)₁₆₅) But not VEGF121(1) (VEGF)₁₂₁(1) From R&D Systems with deletion of about 9 amino acids from the carboxy terminus), and VEGF121(2) (VEGF₁₂₁(2) Commercially available from PeproTech) (fig. 1A). Binding of VEGF according to BIAcore, 3.5F8₁₆₅But not VEGF₁₂₁. A4.6.1 binding to all isoforms and VEGF₁₁₀The receptor binding domain (Kim KJ et al, The vascular endothelial growth factor: Identification of biological legacy regions by neutral monoclonal antibodies).Growth Factors7:53-64,1992). 3.5F8 probably at amino acids 116 and 118 (which are not present in VEGF)₁₂₁Medium) near binding. 5C3 may be at amino acids 111-113 (which are not present in VEGF)₁₁₀Medium) near (fig. 3). ELISA A may detect VEGF-containing samples₁₆₅VEGF isoforms of sequence, including VEGF₁₈₃、VEGF₁₈₉And VEGF₂₀₆(see, e.g., Stimpfl M et al, Vascular Endothelial growth factor splice variants and their prognostic value inbreast and ovarian cancer.Clinical Cancer Research8:2253-2259,2002). ELISA B (previously known as VEGF121-206ELISA, Konecny GE et al, Association beta amino HER-2/neu and Vascular Endothelial Growth Factor prediction scientific Outcome in primer Breast Cancer Patents).Clinical Cancer Research10:1706-1716,2004) was coated with A4.6.1 and detected using biotinylated 2E 3. A4.6.1 and 2E3 bind to the receptor binding domain present in all three molecules. See, for example, Kim KJ et al, the vascular intrinsic growth factor proteins, Identification of biological viral regions by neutral monoclonal antibodies.Growth Factors7:53-64(1992), and Muller YA et al, molecular end growth factor, crystalline structure and functional mapping of the kinase domain receiver binding site.Proc Natl Acad Sci USA94:7192-7197(1997). Other antibodies that bind in these regions may also be used. This ELISA detects VEGF equally₁₆₅、VEGF₁₂₁Truncated VEGF₁₂₁(deletion of about 9 amino acids from the carboxy terminus), VEGF₁₁₀And VEGF_8-109(FIG. 1B). This ELISA can detect total VEGF, including greater than VEGF produced by matrix metalloproteinase digestion₁₁₀A fragment of (a). ELISA C described herein (which was coated with 5C3 and detected using biotinylated A4.6.1) detects VEGF equally₁₆₅、VEGF₁₂₁And truncated VEGF₁₂₁But not detecting VEGF₁₁₀Or VEGF_8-109(FIG. 1C). Binding of VEGF according to BIAcore, 5C3₁₂₁But not VEGF_8-109. This ELISA allows the detection of VEGF_110-206All VEGF molecules detected, except VEGF₁₁₀And smaller fragments.

For VEGF in samples using a minimum of 1:10 dilution, the sensitivity of ELISA A, ELISA B and ELISA C were 10, 16 and 40pg/ml VEGF, respectively₁₆₅(or 0.26, 0.41 and 1.05p for different VEGF isoforms and fragments, respectivelyM). ELISA B and ELISA C were reproducible (tables 1 and 2). ELISA B and ELISA C are specific for VEGF (VEGF-A). VEGF-B, VEGF-C and VEGF-D at concentrations as high as 50ng/ml gave only background signals. Insulin-like growth factor1, growth hormone, recombinant nerve growth factor, tumor necrosis factor (Genentech), platelet-derived growth factor AB, placental growth factor, transforming growth factor beta 1 (R)&D Systems) (up to 200ng/ml) gave only background signal. Heparin (Leo Laboratories, Bucks, UK and Dublin, Ireland) (up to 100U/ml) had no significant effect on the assay.

Table 1: ELISA B (VEGF)_110-206ELISA). The standard range is 1.56-200pg/ml VEGF in buffer₁₆₅(0.0409-5.24pM VEGF). The OD ratio of 1.56pg/ml standard to blank was 1.37. + -. 0.11. CV is the coefficient of variation.

^aBy combining recombinant VEGF₁₆₅Intermediate and high controls were prepared by incorporation into human EDTA plasma. By introducing VEGF₁₆₅Low controls were prepared by spiking into 70% plasma, as the plasma contains endogenous VEGF. Control 1:10 was diluted and assayed in duplicate in 34 independent assays.

Table 2: ELISA C (VEGF)_121-206ELISA). The standard range is 4.00-512pg/ml VEGF₁₆₅(0.105-13.4pM VEGF). The OD ratio of 4pg/ml standard to blank was 2.72. + -. 0.37. CV is the coefficient of variation.

^aBy combining recombinant VEGF₁₆₅Controls were prepared by incorporation into human EDTA plasma. They were diluted 1:10 and assayed in duplicate in 15 independent assays。

VEGF in conditioned medium of cell lines: measurement of VEGF from the used VEGF by three ELISAs using non-glycosylated VEGF produced in E.coli as standard₁₆₅conditioned media of six stable CHO clones transfected with cDNA (Meng et al, 2000). Glycosylated recombinant VEGF in conditioned Medium from six Stable CHO clones₁₆₅Very similar concentrations are given in the three ELISAs. The concentrations measured by ELISA B were 28, 63, 64, 43, 3.8 and 3.2nM, respectively. The ratio of the VEGF concentration measured by ELISAA and ELISA C compared to that measured by ELISA B was 0.90. + -. 0.08 and 1.08. + -. 0.10, respectively. Thus, the three ELISAs quantitated equally the glycosylated VEGF, and rarely the VEGF under culture conditions₁₆₅The protein of (3) is hydrolyzed.

The VEGF concentrations in a673 cell conditioned medium measured by ELISA a, ELISA B and ELISA C were 0.15, 0.29 and 0.24nM VEGF, respectively. The concentrations measured by ELISA A were lower, indicating VEGF₁₂₁Is present. When VEGF was purified from conditioned media using an A4.6.1 affinity column and analyzed by Western blotting, two bands, possibly glycosylated and non-glycosylated VEGF, were detected by 3.5F8₁₆₅. The lower band had a similar profile to the purified VEGF produced in E.coli₁₆₅Same mobility (fig. 2, left). N-glycanase treatment shifted the higher band to the lower band. A4.6.1 detected two additional lower molecular weight bands, possibly glycosylated (which are identical to the putative non-glycosylated VEGF)₁₆₅Bands partially overlapping) and non-glycosylated VEGF₁₂₁(FIG. 2, right). The lower band had a similar profile to the purified VEGF produced in E.coli₁₂₁The same mobility, and the N-glycanase treatment shifted the higher band to the lower band.

VEGF concentrations in conditioned media from mammary cell lines SK-BR-3, BT-474, T-47D and MCF-7 as measured by ELISA B were 3.6, 16, 13 and 13pM, respectively. The ratios of VEGF concentration measured by ELISAA to VEGF concentration measured by ELISA B were 0.49, 0.42, 0.43 and 0.38 (or 49%, 42%, 43% or 38%), respectively,this correlates with 43, 35, 40 and 41% VEGF in these corresponding cell lines₁₆₅Expression was consistent (Stimpfl M et al, Vascular Endothelial growth factor plasma variants and the viral value in break and ocular cancer.Clinical Cancer Research8:2253-2259,2002). For these cell lines, the ratio of VEGF concentration measured by ELISA C to VEGF concentration measured by ELISA B was 1.1-1.2, indicating that little VEGF was present₁₁₀. The VEGF concentrations measured by ELISA B in conditioned media from ovarian cell lines ES-2, OVCAR-3 and SK-OV-3 were 32, 11 and 20pM, respectively. The ratios of the VEGF concentrations measured by ELISA A to those measured by ELISA B were 0.24, 0.20, and 0.32 (or 24%, 20%, and 32%), respectively, which compared to 38, 42, and 24% of VEGF in these corresponding cell lines₁₆₅Expression was compared (Stimpfl et al, supra). For these cell lines, the ratio of VEGF concentration measured by ELISA C to VEGF concentration measured by ELISA B was 0.64-0.79, indicating VEGF₁₁₀(or smaller fragments) may be present.

Claims (25)

1. A method for detecting selective Vascular Endothelial Growth Factor (VEGF) forms in biological samples₁₁₀₊) The method comprises the following steps:

(a) contacting and incubating a biological sample with a capture reagent immobilized to a solid support, wherein the capture reagent is an antibody that recognizes the same epitope as antibody 5C3 directed against human VEGF, and the monoclonal antibody specifically binds to residues greater than 110 of human VEGF;

(b) separating the biological sample from the immobilized capture reagent;

(c) contacting the immobilized capture agent-target molecule complex with a detectable antibody that binds to a KDR and/or FLT1 receptor binding domain of VEGF or binds to an epitope in VEGF 1-110; and are

(d) Measuring VEGF bound by the capture reagent using a detection means directed against the detectable antibody₁₁₀₊The level of (c).

2. The method of claim 1, wherein the biological sample is isolated from a human subject.

3. The method of claim 2, wherein the human subject is a vascular, diabetic, or cancer patient and the measuring step (d) further comprises comparing to a standard curve to determine the level of VEGF compared to normal individuals.

4. The method of claim 1, wherein said biological sample is a tumor lysate, plasma, serum, or urine.

5. The method of claim 1, wherein the capture reagent is the 5C3 monoclonal antibody.

6. The method of claim 1, wherein the immobilized capture reagent is coated on a microtiter plate.

7. The method of claim 1, wherein the detectable antibody is directly detectable.

8. The method of claim 7, wherein the detectable antibody is amplified by a fluorometric reagent.

9. The method of claim 8 wherein said detectable antibody is biotinylated and said detection means is avidin or streptavidin-peroxidase and 3,3 ', 5, 5' -tetramethylbenzidine.

10. The method of claim 1, wherein the detectable antibody is a monoclonal antibody.

11. The method of claim 10, wherein the detectable antibody is a murine monoclonal antibody.

12. The method of claim 11, wherein the immobilized monoclonal antibody is mab 5C3 and the detectable antibody is mab a4.6.1.

13. Method for detecting VEGF in biological sample₁₁₀₊The immunoassay kit of (1), the kit comprising:

(a) an antibody directed against human VEGF as a capture reagent, wherein the monoclonal antibody is capable of specifically binding to residues greater than 110 in human VEGF; and

(b) a detectable antibody that is a detection reagent that binds to KDR and/or FLT1 receptor binding domains of VEGF or binds to an epitope in VEGF 1-110.

14. The kit of claim 13, further comprising a solid support for the capture reagent.

15. The kit of claim 14, wherein the capture reagent is immobilized on the solid support.

16. The kit of claim 15, wherein the capture reagent is coated on a microtiter plate.

17. The kit of claim 16, further comprising a means for detecting said detectable antibody.

18. The kit of claim 17, wherein said detection means is colorimetric.

19. The kit of claim 13, further comprising purified VEGF as an antigen standard.

20. The kit of claim 13, wherein the capture reagent antibody is the murine monoclonal antibody mab 5C3 and the detectable antibody is mab a4.6.1.

21. An antibody 5C3 obtainable from (or produced by) hybridoma 5C3.1.1 deposited as PTA-7737.

22. The antibody of claim 21 conjugated to a detectable label.

23. A hybridoma 5c3.1.1 deposited with the ATCC and having accession number PTA-7737.

24. An antibody that does not bind VEGF110 but binds to the same epitope as the monoclonal antibody produced by hybridoma cell line PTA-7737.

25. The monoclonal antibody of claim 24 conjugated to a detectable label.