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Novel VEGF-A ELISA allows sensitive quantification of human total bioactive VEGF-A
25 February 2020 (online)
Introduction Vascular endothelial growth factor A (VEGF-A), a prominent member of growth factors that regulate angiogenesis and development of normal vasculature, plays an important role in bone development and remodeling. Studies have shown that ossification requires vascularization a priori and that most VEGF in the bone comes from osteoblastic cells. Upon secretion from osteoblasts, VEGF activates endothelial cell migration/proliferation and vessel permeability. Moreover, it regulates osteoclastic differentiation and migration in bone repair. Thus, VEGF represents a relevant therapeutic target. The sensitive measurement of low amounts of circulating VEGF-A found in control cohorts of apparently healthy individuals proves to be difficult. Hence, there is a need for a high-sensitivity assay that reliably measures low VEGF-A concentrations.
Methods We developed a high-sensitivity sandwich ELISA for the detection of human total bioactive VEGF-A using high quality, well-characterized recombinant monoclonal and polyclonal anti-human VEGF-A antibodies. The linear epitopes of the polyclonal detection antibody were mapped with microarray technology. Analyte stability was determined and in accordance with ICH and EMEA guidelines, assay parameters like specificity, dilution linearity, and spike recovery were assessed.
Results We demonstrate that bioactive human VEGF-A can reliably be measured in plasma preparations. In contrast, serum VEGF levels are clearly increased in some samples. This indicates that serum preparation might have an influence on the VEGF amount measured as VEGF can be released from platelets during sample manipulation. Most importantly, we show that samples of apparently healthy individuals are measurable over background. The assay covers a calibration range between 0 and 2000 pg/ml and assay characteristics as well as analyte stability meet the international standards of acceptance. The recombinant capture antibody recognizes a structural epitope in the conserved receptor binding-site of VEGF-A, and thus, specifically binds to all bioactive isoforms of VEGF-A. The polyclonal detection antibody recognizes linear epitopes in the first 120 amino acids of the VEGF-A molecule.
Discussion Our novel VEGF-A ELISA provides a reliable and accurate tool for the quantitative determination of all biologically active VEGF-A isoforms with high sensitivity.
Keywords Angiogenesis, Bone Formation, Growth Factors, Bone Repair, Vascular Endothelial Growth Factor
Korrespondenzadresse Gabriela Berg, Biomedica Medizinprodukte, Divischgasse 4, 1210 Wien, Österreich,