Research Use Only

EGFr ELISA

Intended Use
The EGFr ELISA is an enzyme-linked immunoassay used to quantitate human epidermal growth factor receptor (EGFr) in human serum, plasma, and cell culture fluids. This assay is for research use only and is not to be used for diagnostic purposes.

Background
The activation and overexpression of cellular oncogenes is an important factor in the development of human cancer. One important member of the oncogene family is the epidermal growth factor receptor (EGFr), which is structurally and functionally related to the oncogenic protein encoded by the v-erb B retrovirus. Retroviral oncogenes have been shown to convert normal cells to cancer cells [1]. The EGFr protein, also known as HER-1, is a transmembrane tyrosine kinase cell surface growth receptor that is expressed on normal epithelial cells and on a variety of malignant cell types. The full length EGFr oncoprotein has a molecular weight of 170,000 Daltons and is composed of three domains: the internal tyrosine kinase portion, responsible for signal transduction; a short transmembrane portion; and an external extracellular domain (ECD), which is the portion that binds growth factors such as EGF and which is shed from the cell surface [2].

The other members of the v-erb B family include HER-2/neu, HER-3, and HER-4, which are also cell surface receptors. Studies have shown that the ECDs of receptor tyrosine kinases interact with polypeptide ligands. Ligand binding to the ECD results in the formation of receptor homodimers or heterodimers, stimulates intrinsic kinase activity, and leads to phosphorylation of tyrosine residues in the intracellular domain. This, in turn, stimulates signal transduction [3].

Studies have shown that the EGFr ECD is shed into culture fluids of A431 cancer cells and has a molecular weight of 110,000 Daltons [4,5]. Circulating ECD is produced either by proteolytic cleavage [6] of the receptor, or by alternative transcription of primary RNAs [7]. Numerous immunohistochemistry studies [8,9] have shown that EGFr is overexpressed in tumor cells of a variety of cancer types.

However, there are reports that the EGFr ECD can be either elevated or decreased in cancer patients when compared to a normal population. For instance, some immunoassay studies showed that soluble EGFr ECD levels are increased in the serum of patients with asbestosis-induced lung cancer [10] and in the urine of patients with squamous cell carcinomas of the head, neck, and lung [11]. In a 1999 report by Baron, et al., it was shown that serum EGFr ECD levels were significantly lower in epithelial ovarian cancer patients with stage III or IV disease compared to controls. The investigators also reported that serum EGFr ECD levels changed temporally during the course of a patient’s disease [12]. In addition, a follow-up study by Baron, et al., in 2003 observed that soluble EGFr concentrations are useful in detecting stage I/II and stage III/IV epithelial ovarian cancer in young, premenopausal women [13]. Other investigators have concluded that the EGF/ErbB family of receptors may have clinical utility as serum biomarkers of disease activity and could be ideal for the development of novel therapeutics in the treatment of ovarian cancer patients [14].

In a study presented at American Association for Cancer Research (AACR) in 2002, it was reported that, compared to the normal EGFr ECD range (45–78 ng/mL), patients with cancer had decreased levels of serum EGFr ECD. In fact, it was reported that 42% of the patients with lung cancer, 44% of patients with late stage prostate cancer, 48% of patients with ovarian cancer, 62%of patients with colon cancer, 44% of women with stage III breast cancer, and 32% of women with metastatic breast cancer had significantly decreased serum EGFr ECD levels compared to normal subjects [15].

In studies reported at American Society of Clinical Oncology (ASCO) in 2002, it was shown
that pretreatment serum EGFr levels were significantly decreased in metastatic breast cancer patients compared to healthy controls. The breast cancer patients who had decreased serum EGFr levels had reduced clinical benefit, shortened time to progression, and a shortened overall survival compared to patients with normal serum EGFr levels [16].

Recent work has shown that tumors that overexpress EGFr may be amenable to treatment
with a variety of therapies which target EGFr. Among these are small molecule inhibitors of the
kinase activity or antibody-based therapies that are directed to the surface EGFr proteins. In addition, these novel anti-EGFr therapies are being combined with traditional therapies to increase therapeutic efficacy [17–20].

The EGFr ELISA is designed to provide the investigator with a convenient, accurate, and reproducible method to determine EGFr levels in human serum or plasma. Comparison of results between investigators, using a common method such as the EGFr ELISA, will contribute to consistency in observations and will aid in more clearly defining the role of EGFr in the development and progression of cancer. In addition, the EGFr ELISA may be used in conjunction with other immunoassays.

Assay Principle
The EGFr ELISA is a sandwich type immunoassay which uses a mouse monoclonal capture antibody and a alkaline phosphatase labeled mouse monoclonal as detector. Both capture and detector reagents specifically recognize the extracellular domain of EGFr. The capture antibody recognizes a protein domain on the extracellular portion of EGFr, does not inhibit EGF binding, and does not cross react with erbB-2 oncoprotein or human blood group A antigen. The capture antibody has been immobilized on the interior surface of the plate wells. To perform the test, an appropriate volume of specimen is incubated in the wells to allow binding of the antigen by the capture antibody. The immobilized antigen is then exposed to the alkaline phosphatase labeled detector antibody. Addition of substrate to the wells allows the catalysis of a chromogen into a colored product, the intensity of which is proportional to the amount of EGFr which has been bound to the plate.

Standards are provided in the kit that allow accurate, quantitative determinations of EGFr in suitable samples. Using a microtiter plate reader, one can measure simultaneously the absorbance of the colored product in the Standards and sample wells. Correlating the absorbance values of samples with the Standards allows the investigator to determine the levels of EGFr in a sample. Samples may be assigned a quantitative value of EGFr in nanograms per mL (ng/mL) of serum, plasma, or culture fluid.

Selected References

1. Coussens L, Yang-Feng TL, Liao YC, et al. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science 1985 Dec 6;230(4730):1132–1139.
2. Brandt-Rauf PW, Pincus MR, Carney WP. The c-erbB-2 protein in oncogenesis: Molecular structure to molecular epidemiology. Crit Rev Oncog 1994;5(2–3):313–329.
3. Kaptain S, Tan LK, Chen B. HER-2/neu and breast cancer. Diagn Mol Pathol 2001 Sep;10(3):139–152.
4. Merlino GT, Xu YH, Ishii S, et al. Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells. Science 1984 Apr 27;224(4647):417–419.
5. Weber W, Gill GN, Spiess J. Production of an epidermal growth factor receptor-related protein. Science 1984 Apr 20;224(4646):294–297.
6. Zabrecky JR, Lam T, McKenzie SJ, et al. The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3. J Biol Chem 1991 Jan 25;266(3):1716–1720.
7. Xu YH, Richert N, Ito S, et al. Characterization of epidermal growth factor receptor gene expression in malignant and normal human cell lines. Proc Natl Acad Sci USA 1984 Dec;81(23):7308–7312.
8. Gullick WJ, Marsden JJ, Whittle N, et al. Expression of epidermal growth factor receptors on human cervical, ovarian, and vulval carcinomas. Cancer Res 1986 Jan;46(1):285–292.
9. Wright C, Nicholson S, Angus B, et al. Relationship between c-erbB-2 protein product expression and response to endocrine therapy in advanced breast cancer. Br J Cancer 1992 Jan;65(1):118–121.
10. Partanen R, Hemminki K, Koskinen H, et al. The detection of increased amounts of the extracellular domain of the epidermal growth factor receptor in serum during carcinogenesis in asbestosis patients. J Occup Med 1994 Dec;36(12):1324–1328.
11. Witters LM, Curley EM, Kumar R, et al. Epidermal growth factor receptor ectodomain in the urine of patients with squamous cell carcinoma. Clin Cancer Res 1995 May;1(5):551–557.
12. Baron AT, Lafky JM, Boardman CH, et al. Serum sErbB1 and epidermal growth factor levels as tumor biomarkers in women with stage III or IV epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev 1999 Feb;8(2):129–137.
13. Baron AT, Cora EM, Lafky JM, et al. Soluble epidermal growth factor receptor (sEGFR/sErbB1) as a potential risk, screening, and diagnostic serum biomarker of epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev 2003 Feb;12(2):103–113.
14. Maihle NJ, Baron AT, Barrette BA, et al. EGF/ErbB receptor family in ovarian cancer. Cancer Treat Res 2002 107:247–258.
15. Carney W, Burrell M, Morris L, et al. Normal levels of serum EGFR and decreases in several cancers. Proceedings AACR 2002. Vol 43, Abstract #240.
16. Marx III JH, Leitzel K, Ali SM, et al. Serum EGFR in metastatic breast cancer patients. Proceedings ASCO 2002; Abstract #1743.
17. Dullea RG, Barbacci EG, Miller PE, et al. Induction of apoptosis by CP 358, 774, an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, in combination with cisplatin (CDDP). Proceedings AACR 2000. Vol 41, Abstract #2550.
18. Nelson JM and Fry DW. Inhibition of ErbB family receptors by CI-1033 enhances the cytotoxicity of gemcitabine via modulation of AKT and MAP kinases. Proceedings AACR 2000. Vol 41, Abstract #1533.
19. O’Reilly T, Cozens R, Traxler P. In vivo antitumor activity of the epidermal growth factor receptor (EGFR) inhibitor PKI166. Proceedings AACR 2000. Vol 41, Abstract #3069.
20. Kelly HC, et al. ZD1839 (“IRESSA”), an oral epidermal growth factor receptor tyrosine kinase inhibitor (EGFRTKI): Pharmacokinetics in a phase I study of patients with advanced cancer. Proceedings AACR 2000. Vol 41, Abstract #3896.