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 (30 kDa). The ELISA assay for sVEGFR-1 was also used to measure the amount of the soluble receptor in amniotic fluid samples of patients undergoing amniocentesis during the course of normal pregnancies. The concentration of the samples was in the range of 5-35 ng/ml. This ELISA could be useful powerful tool for investigations concerning the physiological function of the soluble receptor under normal and pathophysiological conditions.Furthermore, it may facilitate studies of the mechanisms of receptor production.