ELISA for VEGF

Abstract

The vascular endothelial growth factor (VEGF) activity in a patient's bloodstream or other biological sample can serve as a diagnostic and prognostic index for cancer, diabetes, heart conditions, and other pathologies. Antibody-sandwich ELISA methods and kits for VEGF as an antigen are provided to detect types of VEGF levels in biological samples from animal models and human patients and can be used as a diagnostic/prognostic index.

1 . A method for detecting selective vascular endothelial growth factor (VEGF) forms (VEGF 110+ ) in a biological sample comprising the steps of:
 (a) contacting and incubating the biological sample with a capture reagent immobilized to a solid support, wherein the capture reagent is a antibody that recognizes same epitope as antibody 5C3 against human VEGF, said monoclonal antibody binding specifically to residues greater than 110 of human VEGF;   (b) separating the biological sample from the immobilized capture reagents;   (c) contacting the immobilized capture reagent-target molecule complex with a detectable antibody that binds to the KDR and/or FLT1 receptor binding domains of VEGF or that binds to an epitope in VEGF1-110; and   (d) measuring the level of VEGF 110+  bound to the capture reagents using a detection means for the detectable antibody.   
     
     
         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 a comparison with a standard curve to determine the level of VEGF compared to a normal individual. 
     
     
         4 . The method of  claim 1 , wherein the biological sample is tumor lysates, plasma, serum or urine. 
     
     
         5 . The method of  claim 1 , wherein the capture reagent is 5C3 monoclonal antibody. 
     
     
         6 . The method of  claim 1 , wherein the immobilized capture reagents are 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 fluorimetric reagent. 
     
     
         9 . The method of  claim 8 , wherein the detectable antibody is biotinylated and the detection means is avidin or streptavidin-peroxidase and 3,3′,5,5′-tetramethyl benzidine. 
     
     
         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 . An immunoassay kit for detecting VEGF 110+  in a biological sample, the kit comprising:
 (a) as capture reagent, an antibody against human VEGF, wherein the monoclonal antibody binds specifically to the residues greater than 110 of human VEGF; and   (b) as detection reagent, a detectable antibody that binds to the KDR and/or FLT1 receptor binding domains of VEGF or that binds to an epitope in VEGF1-110.   
     
     
         14 . The kit of  claim 13 , further comprising a solid support for the capture reagents. 
     
     
         15 . The kit of  claim 14 , wherein the capture reagents are immobilized on the solid support. 
     
     
         16 . The kit of  claim 15 , wherein the capture reagents are coated on a microtiter plate. 
     
     
         17 . The kit of  claim 16 , further comprising a detection means for the detectable antibodies. 
     
     
         18 . The kit of  claim 17 , wherein the 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 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 deposit number PTA-7737. 
     
     
         22 . The antibody of  claim 21  conjugated to a detectable label. 
     
     
         23 . A hybridoma 5C3.1.1 deposited under ATCC deposit number PTA-7737. 
     
     
         24 . An antibody that does not bind VEGF 110 and which binds to the same epitope as the monoclonal antibody produced by the hybridoma cell line PTA-7737. 
     
     
         25 . The monoclonal antibody of  claim 24  which is conjugated to a detectable label.