Quantification of vascular endothelial growth factor-C (VEGF-C) by a novel ELISA.
Lymphangiogenesis plays an important role in several normal and pathological conditions such as wound healing, inflammation or metastasis formation in several malignancies. VEGF-C and VEGF-D are important and specific regulatory factors for lymphatic endothelial proliferation and lymphangiogenesis. In order to develop a highly sensitive and specific detection system for VEGF-C, we produced soluble binding proteins and antibodies for a microtiterplate-based assay. Here we describe a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of human, rat and murine VEGF-C. The different antibodies developed against human and rat VEGF-C could be combined to detect processed and partially processed VEGF-C in a specific way. The ELISA was able to detect human and rat VEGF-C with a minimum detection limit of 100 pg/ml. The assay did not show any cross-reactivity with the related protein VEGF-D. Furthermore, complex formation with its soluble receptors VEGFR-2 and VEGFR-3 did not restricted the sensitivity of the assay. Using this assay, VEGF-C was measured in supernatants and lysates of different cell types and in tumour tissue samples of murine, rat and human origin. Cell lines secrete VEGF-C in very low amounts (<1 ng/ml) whereas VEGF-C transfected cells can secrete up to 50 ng/ml VEGF-C into the supernatant. In human tumour tissue samples VEGF-C was detected in some carcinomas in the low protein range. This ELISA will be a useful tool for investigations concerning the physiological function of VEGF-C in lymphangiogenesis under normal and pathophysiological conditions.
Department of Gene Regulation and Differentiation National Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany. weich@gbf.de
Herbert A
HA
Weich
Hiroko
H
Bando
Maren
M
Brokelmann
Petra
P
Baumann
Masakazu
M
Toi
Bernhard
B
Barleon
Kari
K
Alitalo
Bence
B
Sipos
Jonathan
J
Sleeman
D000818:Animals; D000906:Antibodies / Q000032:analysis; D015152:Blotting, Northern; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D009369:Neoplasms / Q000737:chemistry; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D042582:Vascular Endothelial Growth Factor C / Q000032:analysis*
D000906:Antibodies; D042582:Vascular Endothelial Growth Factor C