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 endoth elial cell proliferation and a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Its biological activity is medi ated by two receptor tyrosine kinases, VEGFR-1 (Flt-l) and VEGFR-2 (KDR). I n contrast to VEGFR-2, a naturally occurring soluble form of the VEGFR-1 (s VEGFR-1) is produced by endothelial cells by differential splicing of the f lt-1 gene, and it is a secreted gene product. In order to develop a specifi c 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 th e seven Ig-like domains of the extracellular receptor protein but have no n eutralizing activity in ligand binding assays. Together with a polyclonal a ntiserum, 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 sol uble receptors. Using this assay, human sVEGFR-1 was measured in the supern atant of different VEGFR-1 expressing cell types. No sVEGFR-1 protein was d etectable after heparin Sepharose treatment or size-exclusion filtration(<3 0 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 amni ocentesis 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 powerfu l tool for investigations concerning the physiological function of the solu ble receptor under normal and pathophysiological conditions.