Human EGFR Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human EGF R immunoassay is a 4.5 hour solid phase ELISA designed to measure EGF R in cell culture supernates, serum, plasma, and human milk. It contains NS0-expressed recombinant human EGF R and has been shown to accurately quantitate the recombinant factor. Results obtained using natural EGF R showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human EGFR.

Preparation and Storage

Background: EGFR

The EGF R subfamily of receptor tyrosine kinases comprises four members: EGF R (also known as HER-1, ErbB1, or ErbB), ErbB2 (Neu, HER-2), ErbB3 (HER-3), and ErbB4 (HER-4). All family members are type I transmembrane glycoproteins with an extracellular ligand binding domain containing two cysteine-rich domains separated by a spacer region and a cytoplasmic domain containing a membrane-proximal tyrosine kinase domain followed by multiple tyrosine autophosphorylation sites. The human EGF R cDNA encodes a 1210 amino acid (aa) precursor with a 24 aa signal peptide, a 621 aa extracellular domain (ECD), a 23 aa transmembrane segment, and a 542 aa cytoplasmic domain. Soluble receptors consisting of the extracellular ligand binding domain are generated by alternate splicing in human and mouse. Within the ECD, human EGF R shares 88% aa sequence identity with mouse and rat EGF R. It shares 43% - 44% aa sequence identity with the ECD of human ErbB2, ErbB3, and ErbB4. EGF R binds a subset of the EGF family ligands, including EGF, amphiregulin, TGF-alpha, betacellulin, epiregulin, HB-EGF, and epigen. Ligand binding induces EGF R homodimerization as well as heterodimerization with ErbB2, resulting in kinase activation, heterodimerization tyrosine phosphorylation and cell signaling. EGF R can also be recruited to form heterodimers with the ligand-activated ErbB3 or ErbB4. EGF R signaling regulates multiple biological functions including cell proliferation, differentiation, motility, and apoptosis. EGF R is overexpressed in a wide variety of tumors and is the target of several anti-cancer drugs.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.    Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.    Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.    Add 50 μL of Assay Diluent to each well.

4.    Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.    Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.    Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.    Aspirate and wash 5 times.

8.    Add 100 μL Substrate Solution to each well.

9.    Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.