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Immunoassays | Prototype

Erenna® EGFR Immunoassay Prototype Assay (Cat# 03-0037-AA)

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Introduction

The Erenna® EGFR Immunoassay Evaluation Reagent Kit uses a quantitative fluorescent sandwich immunoassay technique to measure EGFR in human serum samples. A capture antibody specific for EGFR has been pre-coated onto paramagnetic microparticles (MP). The user pipettes MP, standards, and samples into uncoated microplate wells. During incubation, the EGFR present in the sample binds to the capture antibody on the coated MP. Unbound molecules are washed away during the subsequent buffer exchange and wash steps. Fluor-labeled detection antibody is added to each well and incubated. This detection antibody recognizes and binds to EGFR that has been captured onto the MP. During the following wash step the MPs are transferred to a clean plate. Elution buffer is then added and incubated. The elution buffer dissociates the bound protein sandwich from the MP surface, releasing the labeled antibodies. These antibodies are separated during transfer to a final microplate. The plate is loaded into the Erenna System where the labeled molecules are detected and counted. The number of fluor-labeled detection antibodies counted is directly proportional to the amount of EGFR present in the sample when captured. The amount of EGFR in unknown samples is interpolated from a standard curve.

Assay Performance


TABLE 1. Analytical sensitivity of the Erenna® EGFR Immunoassay Prototype Assay1
Lower Limit of Detection 207 pg/mL
Lower Limit of Quantification2 3125 pg/mL
Upper Limit of Quantification 100,000 pg/mL
Low-end CV% Range 1 - 16%
Low-end CV% Average 8%
Recommended Sample Volume 100 μL
Minimum Sample Volume Required3 15 μL

1 See product insert for updated values
2 LLoQ = 20% CV and ± 20% recovery
3 based upon median [EGFR] in a healthy reference


FIGURE 1. [EGFR] in EDTA plasma from 14 healthy donors, with median and interquartile range. The Erenna® EGFR Immunoassay Evaluation Reagent Kit reliably quantifi es EGFR in healthy subjects, who have a median [EGFR] of 27 ng/mL that is well above the detection limit of 207 pg/mL.

FIGURE 2. Erenna® EGFR Immunoassay Evaluation Reagent Kit assay low-end standard curve signal (left) and curve fit (right).

Representative data shown for demonstration purposes only. Individual results may vary depending upon samples tested and protocol used.

Biology and Disease

The Epidermal Growth Factor Receptor (EGFR) is a receptor tyrosine kinase that is activated by binding to the epidermal growth factor family. Ligand binding invokes receptor dimerization and auto-trans-phosphorylation of tyrosine residues in the intracellular C-terminal domain. This subsequently activates several intracellular signaling pathways that control cell proliferation and DNA synthesis downstream, including the PI3-Kinase and MAP-Kinase signaling cascades. Over-activity of signal transduction pathways spurred by EGFR signaling have been shown to be associated with the development of lung, breast, colorectal, and ovarian cancer. Many recently developed therapeutic approaches involve inhibition of EGFR activity, and some cancers can be typifi ed by their EGFR status as being responsive or non-responsive to such therapies.


TABLE 2. Additional UniProtKB/Swiss-Prot Information*
Protein Name: Epidermal growth factor receptor
UniProtKB/Swiss-Prot ID: P00533
Protein-protein interaction database: P00533
Alternative Names: Proto-oncogene c-ErbB-1, Receptor tyrosine-protein kinase erbB-1
Gene Names: EGFR
Synonym Gene Names: ERBB1
Function: Receptor for EGF, but also for other members of the EGF family, as TGF-alpha, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, GP30 and vaccinia virus growth factor. Is involved in the control of cell growth and differentiation. Phosphorylates MUC1 in breast cancer cells and increases the interaction of MUC1 with SRC and CTNNB1/beta-catenin.
Biological Processes: positive regulation of MAP kinase activity, positive regulation of catenin protein nuclear translocation
Molecular Function: MAP/ERK kinase kinase activity, epidermal growth factor receptor activity

* Source Info from www.uniprot.org.


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