An in vitro enzyme immunoassay (EIA) kit for quantitative determination of human Phosphorylated EGF receptor in serum, cell lysate and other biological fluids

Description

EGF Receptor
The epidermal growth factor (EGF) receptor is a glycoprotein that consists of 130 kDa protein (1186 amino acid residues) and 40 kDa sugar chain. EGF receptor molecule is made up of four domains; glycosylated extracellular domain that is the binding site of EGF and TGFα, a transmembrane domain, tyrosin kinase domain and a to-phosphorylation domain. Tyrosin kinase domain is an intracellular domain which is homologous with the oncogene v-erbB product¹⁾. The binding of EGF to the receptor results in DNA reproduction and cell proliferation. During this step, ruffuring of cell membrane, phosphorylation of EGF receptor ⁶⁾, internalization, pH change, enzyme activation, reorganization of actin filament, and oncogene protein induction are occured. Although the mechanism of the EGF signal transduction is not clear, it is assumed that EGF binding stimulation should initiate a series of signal transduction through tyrosine auto-phosphorylation and phosphorylation of interacting proteins²⁾. The half-life of EGF receptor on the cell surface is approximately 20 hours. When the receptor accepts the ligand, the degradation of the receptor is accelerated (degraded approximately in 5 hours)³⁾. The human squamous epithelium carcinoma cell line, A431, possess many EGF receptor, 5-10 times of standard cell line's (2-3 × 10⁶/cell). This kit enables to measure the quantity of phosphorylated EGF receptor by ELISA. It is useful for analysis of receptor phosphorylation and for in vitro screening of specific inhibitors to EGF receptor kinase.

Storage

4°C

Kit components

1.	Antibody Coated Microtiterplate	1 plate (8 well × 12 strips)
2.	Antibody-POD Conjugate	1 vial (for 11 ml × 1)
3.	Standard	1 vial (for 1 ml × 1)
4.	Sample Diluent	2 vials (11 ml × 2)
5.	Substrate Solution	1 vial (12 ml × 1)
8.	Receptor Extraction Buffer	1 vial (11 ml × 1)

Performance

Range of assay:	0.1-5.0 fmol/ml
Sensitivity:	0.1 fmol/ml
Assay duration:	2.5 hours for assay protocol
Specificity:	This kit specifically measures human phosphorylated EGF receptor. Inactive EGF receptor that is dephosphorylated is not detected with this kit.
Test specimen type:	Human serum or plasma, cell extracts; If each test sample is run in duplicate, about 210 µl (100 µl per assay well plus approx. 10 µl for waste per transfer) is required.

Monoclonal antibodies

This kit uses Anti-Human EGF Receptor Antibody (B4G7) (Cat.# M059) as solid antibody and Anti-Phosphotyrosin Antibody (PY20)-HRP as labeled antibody. B4G7 recognizes EGF binding domain peptide on the human EGF receptor⁴⁾.
PY20 is specific to phosphotyrosin without reacting phosphoserine, phosphothreonin nor dephosphorylated tyrosin⁵⁾.

Typical standard curve (Do not use to calculate unknowns)