Human EGF ELISA Assay
The Human EGF ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 15 pg/mL
Dynamic Range: 62.5 – 2000 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µL
Alternative Names: Epidermal Growth Factor
Epidermal growth factor (EGF) is the founding member of the EGF family that also includes TGFα, amphiregulin (AR), betacellulin (BTC), epiregulin (EPR), heparin binding EGF-like growth factor (HB-EGF), epigen, and the neuregulins (NRG)1 through-6. 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. 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. EGF is present in various body fluids, including blood, milk, urine, saliva, seminal fluid, pancreatic juice, cerebrospinal fluid, and amniotic fluid. Four ErbB (HER) family receptor tyrosine kinases including EGFR/ErbB1, ErbB2, ErbB3 and ErbB4, mediate responses to EGF family members. These receptors undergo a complex pattern of ligand induced homo or heterodimerization to transducer EGF family signals. 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. Biological activities ascribed to EGF include epithelial development, angiogenesis, and inhibition of gastric acid secretion, fibroblast proliferation, and colony formation of epidermal cells in culture.
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The Eagle Biosciences Human Epidermal Growth Factor (EGF) ELISA Assay Kit 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 colored 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.
- Prepare all reagents and working standards as directed in the previous sections.
- Determine the number of microwell strips required to test the desired number of samples plus an appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
- Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided.
- Incubate for 1.5 hours at 37°C.
- Aspirate each well and wash, repeating the process three times for a total of four washes.
- Wash by filling each well with Wash Buffer (350 µL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance.
- After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
- Add 100 µL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C
- Repeat the aspiration/wash.
- Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C Avoid placing the plate in direct light.
- Repeat the aspiration/wash.
- Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
- Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
Typical Standard Curve
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