The Use of Antibodies in the Study of EGFR Signaling
Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that has been associated with cancers. The receptor depends on the binding of specific ligands to induce autophosphorylation and receptor dimerization of the intracellular domain of the tyrosine Kinase. EGFR has been identified as an oncogene opening channels for its targeting in cancer therapy. Recently, there has been a development of EGFR molecular testing whose use in the clinical setting has become prevalent. As mentioned above, molecular testing has become prevalent in both clinical and research settings. One of the techniques used in molecular testing exploits antibodies. It has been critical in the personalization of treatment in cancer. This paper seeks to explore antibodies use in the study of EGFR.
Whirl-Carrillo et al. (2012) explain that EGFR has three major signaling pathways. These pathways include that of RAS-RAF-MAPK pathway. In this pathway, phosphorylation of EGFR causes recruitment of guanine-nucleotide exchange factor through Shcadapter proteins and GRB2 causing RAS activation that stimulates MAP and RAF kinase pathways. These kinase pathways are associated with such as tumor invasion and cell proliferation. P13K/AKT is another pathway that is concerned with the activation of anti-apoptosis and major cellular survival signals by nuclear transcription factors activation. On the other hand, JAK/STAT pathway is concerned with activation of cell survival transcription genes. EGFR can be targeted using antibodies to evaluate the signaling pathways such as by using C225 (Scaltriti & Baselga, 2006). Another antibody that can be used is ABX-EGF (Nahta, Hortobagyi & Esteva, 2003).
Often, characterization of EGF receptor tyrosine kinase is required. In such cases, EGF receptor purification is required to ensure relevant inferences. Several methods of EGF receptor purification are available such as the use of sequential ion exchange and wheat germ agglutinin-sepharose. The use of antibodies has revolutionized the process of EGF receptor purification. Antibodies allow EGF receptor purification through a single step in the process of immunoaffinity chromatography (Weber, Bertics & Gill, 1984). Purification of EGF receptor requires the use of a monoclonal anti-EGF receptor antibody. This antibody is competitive to the binding sites of EGF receptor. Moreover, this process has allowed the characterization of the self-phosphorylation kinetics and EGF receptor binding stoichiometry.
Antibodies are of pivotal role in studying phosphorylation activity of EGFR. EGFR has an intrinsic tyrosine kinase activity (Cohen, 2000). According to Park, Schaefer and Nathans (1996), the tyrosine kinase in EGFR is activated by the binding of a ligand. Antibodies can be used to evaluate the process through which the tyrosine kinase is activated to cause phosphorylation to other biomolecules. According to Mandell (2003), phosphorylation state-specific antibodies (PSSA) development has allowed the study of the process of phosphorylation in situ. Such study is of critical importance due to the association of EGF receptor phosphorylation with poor clinical outcomes in cancer treatment. PSSA has enabled determination of the degree of efficiency of drugs that are targeting to a given intracellular signaling pathways. PSSA has been used to determine the efficacy of ZD1839, an EGF receptor inhibitor, to prevent phosphorylation.
According to Sheffield and Gavinski (2003), the interaction of molecules in the body is pertinent to understanding signal transduction. The interaction of EGF receptor is pertinent especially for its association with cancer. Understanding the affinity of EGF binding is pertinent in such cases. EGF receptors have been revealed to have low and high affinity that results in differences in their abundance. The low-affinity EGF receptors are more abundant compared to those with high affinity and are rarer. Den Hartigh et al. (1992) explain that the understanding of differences in affinity can help to evaluate the interaction of signal transduction. Antibodies are of critical importance in such cases of characterization. Antibodies can be used to induce selective inhibition to characterize protein-protein interactions. For instance, Den Hartigh et al. (1992) explain that the competition of the antibody and the EGF receptor binding site can be determined to evaluate EGF receptor actin-binding domain. Moreover, antibodies can also be used in the evaluation of EGF receptor tyrosine kinase role in the progression of cells in G1 phase and their entry into S phase of the cell cycle (Voldborg et al., 1997). Monoclonal antibodies can be used to block EGF receptor ligand binding to determine their effects on such as the activities of cyclin-dependent kinases of the G1 phase. The signaling by EGF receptor can be assessed using techniques such as western blot (Sheffield & Gavinski, 2003).
EGFR has been associated with cancer. Two antibodies have been developed to the two most prevalent EGFR mutations that are used as to characterize EGFR biomarker. These antibodies are mutation-specific and are used to characterize EGFR that acts as a biomarker in immunohistochemistry assays (Hutchinson et al., 2015; Wen et al., 2013). They include clone 43B2 and rabbit monoclonal antibody clone 6B6. These antibodies are used in the assessment of EGFR protein expression (Hutchinson et al., 2015). The two antibodies have been shown to have high predictive value and sensitivity in the detection of EGFR in lung carcinoma. Use on antibodies in the study of EGCR through immunohistochemistry allows diagnosis of mutation (Wen et al., 2013). Antibodies have been used to evaluate the relationship between EGFR phosphorylation, downstream signaling, and intracellular trafficking. Such evaluation of this relationship requires tracing EGFR and its adaptor protein in all the organelles involved. According to Oksvold et al. (2000), antibodies allow undertaking of this tracing using techniques such as immunoelectron and immunofluorescent microscopy that are used to localize EGFR phosphorylation species.
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