Vascular endothelial growth factor receptor-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.