Immunodetection and quantification of vascular endothelial growth factor receptor-3 in human malignant tumor tissues.
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.
Bando
Hiroko
H
Department of Gene Regulation and Differentiation, National Research Centre for Biotechnology (GBF), Braunschweig, Germany.
Brokelmann
Maren
M
Toi
Masakazu
M
Alitalo
Kari
K
Sleeman
Jonathan P
JP
Sipos
Bence
B
Gröne
Hermann-Josef
HJ
Weich
Herbert A
HA
D000818:Animals; D000911:Antibodies, Monoclonal; D042783:Endothelial Cells; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006041:Goats; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000737:chemistry* / Q000473:pathology*; D009389:Neovascularization, Pathologic; D012680:Sensitivity and Specificity; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D040321:Vascular Endothelial Growth Factor Receptor-3