Description
Principle of the assay: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for VEGF has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any VEGF present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for VEGF is added to the wells and binds to the combination of capture antibody- VEGF in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A coloured product is formed in proportion to the amount of VEGF present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven VEGF standard dilutions and VEGF sample concentration determined.
Background: Vascular endothelial growth factor (VEGF or VEGF-A), also known as vascular permeability factor (VPF), is well known for its important roles in regulating both physiological and pathological blood vessel growth (1). It is a member of the VEGF family that also includes VEGF-B, -C, -D, -E, and PlGF (placental growth factor) (2). Family members exhibit a cysteine knot motif formed by eight characteristically spaced cysteine residues (3 - 5). Various cells and tissues express VEGF including skeletal and cardiac muscle, hepatocytes, osteoblasts, neutrophils, macrophages, keratinocytes, platelets, brown adipose tissue, CD34+ cells, astrocytes, neurons, and endothelial cells (6-17). VEGF can be detected in both human plasma and serum samples, with higher levels found in serum due to its release from platelets (17). VEGF transcription is potentiated in response to hypoxia, oncogenic transformation, and growth factors. VEGF has several physiological roles both during development and in the adult. It has a well-documented and critical role in embryonic vasculogenesis (1). Targeted deletion of a single VEGF allele results in significant defects in vascular development and is embryonic lethal (18). VEGF acts as an endothelial cell survival factor, mitogen, and inducer of migration, functions that potentially involve an array of signaling molecules including G proteins, PI3 kinase, MAP kinases, FAK, PKC, and NO (3, 19-25). VEGF affects bone formation by regulating blood vessel growth and cartilage remodeling, activities important for growth plate morphogenesis (26). In the adult, VEGF has potential roles in cyclic angiogenesis in the female reproductive system. Other diverse functions include the regulation of hematopoietic development and roles as a neurotrophic factor. VEGF has received much attention for its involvement in tumor-associated angiogenesis and its potential as a target for cancer therapy. Numerous studies have demonstrated that VEGF or its receptors are upregulated in several forms of human cancer. Neutralizing VEGF antibodies are shown to reduce tumor growth in cancer models