Epidermal growth factor (EGF) is the founding member of the EGF family that also includes TGFα, amphiregulin (AR), betacellulin (BTC), epiregulin(EPR), heparinbinding EGF-like growth factor (HB-EGF), epigen, and the neuregulins (NRG)1 through-6(1). Members of the EGF family share a structural motif, the EGF-like domain, which is characterized by three intramolecular disulfide bonds that are formed by six similarly spaced conserved cysteine residues (2). All EGF family members are synthesized as type I transmembrane precursor proteins that may contain several EGF domains in the extracellular region. The 1207 amino acid (aa) human EGF precursor contains nine EGF domains and nine LDLR class B repeats. The mature protein consists of 53 aa and is generated by proteolytic excision of the EGF domain proximal to the transmembrane region (3). EGF is present in various body fluids, including blood, milk, urine, saliva, seminal fluid, pancreatic juice, cerebrospinal fluid, and amniotic fluid (4). Four ErbB (HER) family receptor tyrosine kinases including EGFR/ErbB1, ErbB2, ErbB3 and ErbB4, mediate responses to EGF family members (5). These receptors undergo a complex pattern of ligand induced homoor heterodimerization to transducer EGF family signals (6, 7). EGF binds ErbB1 and depending on the context, induces the formation of homodimers or heterodimers containing ErbB2. Dimerization results in autophosphorylation of the receptor at specific tyrosine residues to create docking sites for a variety of signaling molecules (5, 8). Biological activities ascribed to EGF include epithelial development, angiogenesis, inhibition of gastric acid secretion, fibroblast proliferation, and colony formation of epidermal cells in culture.

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for EGF has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any EGF present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for EGF is added to the wells and binds to the combination of capture antibody- EGF 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 EGF 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 EGF standard dilutions and EGF sample concentration determined.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with antibody to human EGF (812)

2. 2 vials human EGF Standard lyophilized, 20 ng/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-human EGF 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. Harris, R.C. et al. (2003) Exp. Cell Res. 284:2.

2. Carpenter, G. and S. Cohen, (1990) J. Biol. Chem. 265:7709.

3. Bell, G.I. et al. (1986) Nucl. Acids Res. 14:8427.

4. Carpenter, G. and Zendegui, J.G. (1986) Exp. Cell Res.164:1.

5. Jorissen, R.N. et al. (2003) Exp. Cell Res. 284:31.

6. Gamett, D.C. et al. (1997) J. Biol. Chem. 272:12052.

7. Qian, X. et al. (1994) Proc. Natl. Acad. Sci. 91:1500.

8. Qian, X. et al. (1999) J. Biol. Chem. 274:574.