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 method and kits for VEGF as an antigen were developed to
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 method and kits for VEGF as an antigen were developed to detect VEGF levels in biological samples from animal models and human patients and are used as a diagnostic/prognostic index.
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1. A method for detecting multiple isoforms of vascular endothelial growth factor (VEGF) in a biological sample, comprising:(a) incubating a biological sample with a capture reagent immobilized on a solid support to bind multiple isoforms of VEGF to the capture reagent, wherein the capture reagent c
1. A method for detecting multiple isoforms of vascular endothelial growth factor (VEGF) in a biological sample, comprising:(a) incubating a biological sample with a capture reagent immobilized on a solid support to bind multiple isoforms of VEGF to the capture reagent, wherein the capture reagent comprises a mixture comprising a polyclonal antibody that binds VEGF and a monoclonal antibody that specifically binds to amino acid residues 111-165 of human VEGF; and (b) detecting VEGF bound to the immobilized capture reagent by contacting the bound VEGF with a detectable antibody that binds to N-terminal amino acid residues 1-110 of VEGF. 2. The method of claim 1, wherein the biological sample is isolated from a human.3. The method of claim 2, wherein the human is a vascular, diabetic, or cancer patient.4. The method of claim 3, further comprising:(c) measuring an amount of VEGF detected in (b), wherein the amount is quantitated using a standard curve. 5. The method of claim 1, wherein the biological sample is plasma, serum, or urine.6. The method of claim 1, wherein the capture reagent is immobilized in a weight ratio of about 0.8:1 to about 1.2:1 of monoclonal to polyclonal antibody.7. The method of claim 6, wherein the weight ratio is about 1:1 of monoclonal to polyclonal antibody.8. The method of claim 7, wherein the amount of monoclonal antibody immobilized is about 0.4 μg/ml and the amount of polyclonal antibody immobilized is about 0.4 μg/ml.9. The method of claim 1, wherein the solid support is a microtiter plate.10. The method of claim 1, wherein the detectable antibody is fluorescently labeled.11. The method of claim 1, wherein the capture reagent monoclonal antibody is a murine monoclonal antibody.12. The method of claim 11, wherein the capture reagent monoclonal antibody is MAb 3.5F8 produced by a hybridoma having ATCC Accession No. HB PTA-3499, or progeny thereof.13. The method of claim 1, wherein the capture reagent polyclonal antibody is a rabbit or goat polyclonal antibody.14. The method of claim 1, wherein the capture reagent polyclonal antibody is affinity purified.15. The method of claim 1, wherein the detectable antibody is a monoclonal antibody.16. The method of claim 15, wherein the detectable monoclonal antibody is a murine monoclonal antibody.17. The method of claim 16, wherein the detectable monoclonal antibody is MAb A4.6.1 produced by a hybridoma having ATCC Accession No. HB 10709, or progeny thereof.18. An immunoassay kit for detecting multiple isoforms of vascular endothelial growth factor (VEGF) in a biological sample, the kit comprising:(a) as a capture reagent, a mixture comprising a polyclonal antibody that binds VEGF and a monoclonal antibody that specifically binds to amino acid residues 111-165 of human VEGF; and (b) as a detection reagent, a detectable antibody that binds to N-terminal amino acid residues 1-110 of VEGF. 19. The kit of claim 18, further comprising purified VEGF as an antigen standard.20. The kit of claim 18, wherein the capture reagent is immobilized on a solid support in a weight ratio of about 0.8:1 to about 1.2:1 of monoclonal to polyclonal antibody.21. The kit of claim 20, wherein the weight ratio is about 1:1 of monoclonal to polyclonal antibody.22. The kit of claim 21, wherein the amount of monoclonal antibody immobilized is about 0.4 μg/ml and the amount of polyclonal antibody immobilized is about 0.4 μg/ml.23. The kit of claim 20, wherein the solid support is a microtiter plate.24. The kit of claim 18, wherein the detectable antibody is fluorescently labeled.25. The kit of claim 18, wherein the capture reagent monoclonal antibody is a murine monoclonal antibody.26. The kit of claim 25, wherein the capture reagent monoclonal antibody is MAb 3.5F8, produced by a hybridoma having ATCC Accession No. HB PTA-3499, or progeny thereof.27. The kit of claim 18, wherein the capture reagent polyclonal antibody is a rabbit or goat polyclonal antibody.28. The kit of claim 18, wherein the capture reagent polyclonal antibody is affinity purified.29. The kit of claim 18, wherein the detectable antibody is a monoclonal antibody.30. The kit of claim 29, wherein the detectable monoclonal antibody is a murine monoclonal antibody.31. The kit of claim 30, wherein the detectable monoclonal antibody is MAb A4.6.1, produced by a hybridoma having ATCC Accession No. HB 10709, or progeny thereof.
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