Vascular endothelial growth factor (VEGF or VEGF-A), also known as vascular permeability factor (VPF), is a potent mediator of both angiogenesis and vasculogenesis in the fetus and adults (1-3). It is a member of the plateletderived growth factor family that is characterized by a cysteine knot structure formed by eight conserved cysteine residues (4). Mouse embryos expressing only the VEGF120 isoform do not survive to term and show defects in skeletogenesis (5). Mouse VEGF120 shares 98% aa sequence identity with corresponding regions of rat, 89% with canine, feline, equine and porcine, and 87% with human, ovine and bovine VEGF, respectively. VEGF binds the type I transmembrane receptor tyrosine kinases VEGF R1 (also called Flt 1) and VEGF R2 (Flk1/KDR) on endothelial cells (4). VEGF is required during embryogenesis to regulate the proliferation, migration, and survival of endothelial cells (3, 4). In adults, VEGF functions mainly in wound healing and the female reproductive cycle (3). Pathologically, it is involved in tumor angiogenesis and vascular leakage (6, 7). Circulating VEGF levels correlate with disease activity in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus (8).VEGF is induced by hypoxia and cytokines such as IL-1, IL-6, IL-8, oncostatin M and TNF-α (3, 4, 9).

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.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with antibody to mouse VEGF (8 x 12)

2. 2 vials mouse VEGF Standard lyophilized, 2000 pg/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-mouse VEGF antibody

4. 2 vials Streptavidin-HRP solution

5. 1 bottle Standard /sample Diluent

6. 1 bottle Biotin-Conjugate antibody Diluent

7. 1 bottle Streptavidin-HRP Diluent

8. 1 bottle Wash Buffer Concentrate 20x (PBS with 1% Tween-20)

9. 1 vial Substrate Solution

10. 1 vial Stop Solution

11. 4 pieces Adhesive Films

12. package insert

NOTE: [96 Tests]

Unopened Kit：Store at 2 -8° C. Do not use past kit expiration date. opened/ReconstitutedReagents：Please refer to the datasheets for detail information.

1. Ferrara, N. et al. (2003) Nat. Med. 9:669.

2. Shima, D.T. et al. (1996) J. Biol. Chem. 271:3877.

3. Breier, G. et al. (1992) Development 114:521.

4. Claffey, K.P. et al. (1992) J. Biol. Chem. 267:16317.

5. Conn, G. et al. (1990) Proc. Natl. Acad. Sci. USA 87:2628.

6. Scheidegger, P. et al. (1999) Biol. Chem. 380:1449.

7. Sharma, H.S. et al. (1995) Biochim. Biophys. Acta 1260:235.

8. Berse, B. (2002) GenBank Accession # P26617.

9. Keck, P.J. et al. (1989) Science 246:1309.