| Summary: | Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates the sustaining proliferative signaling, which is an important hallmark of cancer. Human EGFR-targeting monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patients with colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mouse model is essential to further develop the mAb therapy against EGFR. Therefore, sensitive mAbs against mouse EGFR (mEGFR) should be established. In this study, we developed a specific and sensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mEGFR mAb, EMab-300 (rat IgG<sub>1</sub>, kappa), reacted with mEGFR-overexpressed Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells, using flow cytometry. The kinetic analysis using flow cytometry indicated that the <i>K</i><sub>D</sub> of EMab-300 for CHO/mEGFR and NMuMG was 4.3 × 10<sup>−8</sup> M and 1.9 × 10<sup>−8</sup> M, respectively. These results indicated that EMab-300 applies to the detection of mEGFR using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.
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