Detection and quantification of complexed and free soluble human vascular endothelial growth factor receptor-1 (sVEGFR-1) by ELISA

Vascular endothelial growth factor (VEGF) is an important factor for endothelial cell proliferation and a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Its biological activity is mediated by two receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (KDR). In contrast to VEGFR-2, a naturally occurring soluble form of the VEGFR-1 (sVEGFR-1) is produced by endothelial cells by differential splicing of the flt-1 gene, and it is a secreted gene product. In order to develop a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of sVEGFR-1, we established five anti-human receptor antibodies and characterized them in detail. These antibodies recognize different epitopes located within the seven Ig-like domains of the extracellular receptor protein but have no neutralizing activity in ligand binding assays. Together with a polyclonal antiserum, a specific human sVEGFR-1 ELISA was developed using the mAb #190.11. The ELISA can detect human sVEGFR-1 with a minimum detection limit of 1 ng/ml. The ELISA does not show any cross-reactivity with other related soluble receptors. Using this assay, human sVEGFR-1 was measured in the supernatant of different VEGFR-1 expressing cell types. No sVEGFR-1 protein was detectable after heparin Sepharose treatment or size-exclusion filtration (