Detection and Quantification of VEGF Isoforms by ELISA.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays an important role in physiological and tumor angiogenesis. The human VEGF gene has eight exons. Different VEGF isoforms are expressed via alternative RNA splicing and VEGF121 and VEGF165 are the major isoforms present in human tissues. The exact roles of these different VEGF isoforms are not totally clear. Assays to detect specific VEGF isoforms in biological samples are needed to understand the biological functions of these different VEGF isoforms and to better assess their potential use as predicative biomarkers for anti-angiogenic therapy. Because monoclonal antibodies specific to different VEGF isoforms are lacking, we used antibodies directed to different epitopes on VEGF165 in a set of three enzyme-linked immunosorbent assays (ELISAs) to assess the amount of VEGF121 and VEGF165 as well as VEGF110, which can be generated by plasmin cleavage in vivo. The first ELISA detects VEGF165. The second ELISA detects both VEGF121 and VEGF165. The third ELISA detects VEGF165, VEGF121, and VEGF110. The concentrations of VEGF121 can be assessed from the difference in VEGF concentrations measured by the second and the first ELISAs; the concentrations of VEGF110 can be assessed from the difference in VEGF concentrations measured by the third and the second ELISAs. The same assay strategy may be used to assess the amount of other VEGF isoforms if antibodies directed against the desired amino acids in those isoforms can be obtained.