EMab-300 Detects Mouse Epidermal Growth Factor Receptor-Expressing Cancer Cell Lines in Flow Cytometry

Abstract

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 IgG1, 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 KD of EMab-300 for CHO/mEGFR and NMuMG was 4.3 × 10−8 M and 1.9 × 10−8 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.

Development of Highly Sensitive Anti-Mouse HER2 Monoclonal Antibodies for Flow Cytometry

“…We have developed the Cell-Based Immunization and Screening (CBIS) method which includes antigen-overexpressing cell immunization and high-throughput screening of hybridoma supernatants using flow cytometry. Using the CBIS method, we have developed mAbs against HER families, including human EGFR [17], HER2 [18], HER3 [19], and mouse EGFR [20]. In this study, we developed novel anti-mHER2 mAbs using the CBIS method and evaluated its application to flow cytometry.…”

Establishment of a Novel Anti-mouse CCR1 Monoclonal Antibody C₁Mab-6

“…[14] Until now, we have developed mAbs for mouse CCR3 [15], CCR8 [16], and CXCR4 [17] using the Cell-Based Immunization and Screening (CBIS) method. [18][19][20][21][22][23][24][25] In this study, we established a novel anti-mCCR1 mAb using the CBIS method and evaluated its applications.…”

Establishment of a Highly-sensitive Anti-EphB2 Monoclonal Antibody Eb2Mab-3 for Flow Cytometry

“…To evaluate the expression of EphB2 and targeting the EphB2-positive cancer cells, the development of monoclonal antibodies (mAbs) against EphB2 is essential. We have developed antireceptor tyrosine kinase mAbs against human epidermal growth factor (EGFR) (clone EMab-17) [17], mouse EGFR (EMab-300) [18], HER2 (H2Mab-19) [19], mouse HER2 (H2Mab-304) [20], and HER3 (H3Mab-17) [21] using the Cell-Based Immunization and Screening (CBIS) method. The CBIS method includes immunizing antigen-overexpressed cells and high-throughput hybridoma screening using flow cytometry.…”