Abstract
Vascular endothelial growth factor recpetor‐3 (VEGFR‐3) and its ligands, vascular endothelial growth factor‐C (VEGF‐C) and –D (VEGF‐D), are the major molecules involved in developmental and pathological lymphangiogenesis. Here we describe for the first time the development of a specific indirect enzyme‐linked immunosorbent assay (ELISA) for the quantification of VEGFR‐3 in different human cell and tissue lysates. A combination of the goat polyclonal anti‐VEGFR‐3 antibody and the mouse monoclonal anti‐human VEGFR‐3 antibody was used. The assay was highly sensitive and reproducible with a detection range of 0.2–25 ng/ml. The assay was specific for VEGFR‐3, with no cross‐reactivity to VEGFR‐1 or VEGFR‐2. Complex formation with VEGF‐C and VEGF‐D had no effect on the sensitivity of the assay. The VEGFR‐3 concentration in the lysates of cultured human dermal microvascular endothelial cells was 14‐fold higher than in the lysates from human umbilical vein endothelial cells. In human kidney, breast, colon, gastric and lung cancer tissues the protein levels of VEGFR‐3 were in the range of 0.6–16.7 ng/mg protein. Importantly, the level of VEGFR‐3 protein detected in the ELISA correlated significantly with the number of VEGFR‐3 positive vessels observed in histochemical sections, suggesting that the ELISA assay may be a reliable surrogate of measuring VEGFR‐3‐positive vessel density. The protein levels of VEGFR‐3 in 27 renal cell carcinoma samples had a significant correlation with the levels of VEGF‐C (p<0.001), or biological active, free VEGF‐A (p<0.0001), but not with VEGFR‐1 or total VEGF‐A. This assay provides a useful tool for the investigations of the expression levels of VEGFR‐3 in physiological and pathological processes, particular in cancer and in lymphangiogenesis‐related disease. © 2004 Wiley‐Liss, Inc.