Abcam Epitomics’ new L858R specific antibody verified by Femtopath EGFR mutation kit

Abcam Epitomics’ new L858R specific antibody verified by Femtopath EGFR mutation kit. In past 2 years, Femtopath help Abcam-Epitomics to develop a new EGFR L858R specific antibody. Epitomics’ Dr. Aihua Li had posted the latest data in 2015 CAP policy meeting.



New EGFR L858R Specific Rabbit Monoclonal Antibody for Immunohistochemical Application



Aihua Li, Yuekai Zhang, Hongyang Pan, Jackie Chan, Shyh-Chang Chen, John Wang, Chi-Kuan Chen, Johan Chien, Daniel Lin, Taiying Chen. Epitomics – an Abcam Company, Burlingame, CA; Taichung Veterans General Hospital, Taichung, Taiwan; Mackay Memorial Hospital, Taipei, Taiwan; Hong Jing Biotechnology Inc (FemtoPath), Taipei, Taiwan



Background: Determining EGFR mutation status is valuable for lung cancer patients in selecting treatment regimens. However, genetic testing remains costly and time-consuming. Due to the challenging nature of developing mutation specific antibodies, only a few EGFR L858R monoclonal antibodies exist for immunohistochemical (IHC) testing. Here we report a new EGFR L858R RabMAb developed by utilizing the RabMAb® technology, designated as clone EP344. The concordance of the IHC staining results with EGFR L858R mutation status was analyzed.
Design: Rabbits were immunized with EGFR L858R peptide. Sera collected from immunized rabbits were screened by ELISA, WB and IHC. Hybridoma was generated by fusing splenocytes with the fusion partner cell line (240E-W2). Antibody from final hybridoma cell line was further characterized by extensive IHC testing using formalin fixed paraffin embedded (FFPE) human normal, tumor and EGFR L858R mutant tumor tissues. EGFR L858R gene mutation was detected by FemtoPath (Patent No. P2708-TW) or Sanger dideoxy sequencing (ABI 3730) mutation analysis. The concordance of EP344 IHC staining results with mutation status was analyzed and compared against EGFR L858R rabbit monoclones SP125 and 43B2.
Results: A 175 KDa EGFR L858R protein was detected by WB in EGFR L858R mutant H1975 cells, but not in wild type cells. IHC analysis of EP344, SP125 and 43B2 on human tissue microarray (TMA) comprising 16 types of normal tissues showed that all 3 antibodies stained negatively in all normal cells. In two tumor TMAs comprise of 16 types of tumors showed that 3 in 16 lung adenocarcinomas and 2 in 21 lung squamous carcinomas are positive with all 3 antibodies. EP344 is negative in all other tumors, while SP125 and 43B2 positive staining was found in breast carcinoma with EGFR wild type. Furthermore, a separate group of 13 lung carcinomas with EGFR L858R mutation was stained with all antibodies. We found that the concordance of EP344, SP125 and 43B2 staining results with mutation status was 92.3%, 77% and 23% respectively.
Conclusions: RabMAbs anti-EGFR L858R, clone EP344 is specific and sensitive in the detection of target mutant proteins by IHC in FFPE tissues. EP344 is highly concordant with EGFR L858R mutation status. Comparing to current EGFR L858R antibodies, EP344 may be a potentially better tool for predicting EGFR L858R mutation status by IHC testing.
Category: Pulmonary Pathology (including Mediastinal)