title: Immunodetection and molecular forms of plasma vascular endothelial growth factor-C.
abstract: Vascular endothelial growth factor (VEGF)-C is a member of the VEGF family. VEGF-C is involved in developmental lymphangiogenesis and may be important in pathological lymphangiogenesis, lymphatic invasion and metastasis in carcinoma. We describe the development of an indirect enzyme-linked immunosorbent (ELISA) assay for the quantification of VEGF-C in plasma. Capture of VEGF-C was achieved using goat anti-human VEGF-C antibody, followed by detection with rabbit anti-human VEGF-C antibody. The sensitivity of the assay was amplified using the biotin-avidin and enhanced chemiluminescence (ECL) systems. The assay was highly sensitive and reproducible with a detection range of 0.4-100 U/ml and the intra- and inter-assay variations were less than 8%. Substitutional tests demonstrated that the assay was specific for VEGF-C with no cross-reaction with VEGF-A or VEGF-D. Practical application of the assay was evaluated in 41 colorectal cancer patients and 31 controls. Median plasma levels of VEGF-C were 35.0 U/ml (range: 17.4-75.9 U/ml) in colorectal cancer patients in contrast to 11.5 U/ml (range: 5.4-21.5 U/ml) in controls (p<0.001). Moreover, VEGF-C levels tended to be elevated in patients with advanced disease compared to early disease, but this was not statistically significant owing to a relatively small number of patients in each group. Immunoprecipitation and immunoblotting confirmed detection of VEGF-C in plasma and revealed that two forms of VEGF-C were present in the plasma corresponding to approximately 40 and approximately 80 kDa. The measurement of plasma VEGF-C offers opportunities to explore clinical applications in the management of malignancy, in particular in the prediction of lymphatic spread and in other lymphangiogenesis-related diseases.
authors.affiliation: Laboratory Medicine Academic Group, Department of Experimental Pathology, Stopford Building, University of Manchester, Manchester M13 9PT, UK. sarah.duff@man.ac.uk
authors.forename: S E
authors.initials: SE
authors.lastname: Duff
authors.forename: C
authors.initials: C
authors.lastname: Li
authors.forename: A
authors.initials: A
authors.lastname: Renehan
authors.forename: S T
authors.initials: ST
authors.lastname: O'Dwyer
authors.forename: S
authors.initials: S
authors.lastname: Kumar
mesh_terms: D000230:Adenocarcinoma / Q000097:blood; D000818:Animals; D001360:Avidin; D014408:Biomarkers, Tumor / Q000097:blood; D001710:Biotin; D015179:Colorectal Neoplasms / Q000097:blood; D003429:Cross Reactions; D016228:Endothelial Growth Factors / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006041:Goats; D006801:Humans; D008163:Luminescent Measurements; D009363:Neoplasm Proteins / Q000097:blood; D020033:Protein Isoforms / Q000097:blood / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D042582:Vascular Endothelial Growth Factor C
chemical_list: D014408:Biomarkers, Tumor; D016228:Endothelial Growth Factors; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D042582:Vascular Endothelial Growth Factor C; D001360:Avidin; D001710:Biotin