EGFR (L858R Mutant Specific), Cell Signaling Technology, 3197

We generated a top 1000 list of the most studied and most published proteins and analyzed the literature validation data for the commercially available research antibody tools. EGFR is the third most studied gene on the list. Cell Signaling 3197 was validated in the original peer-reviewed paper from Cell Signaling Technology by Yu et al., 2009, Clinical Cancer Research entitled Mutation-Specific Antibodies for the Detection of EGFR Mutations in Non–Small-Cell Lung Cancer The authors tested the mutation-specific antibody by Western blot, immunofluorescence, and immunohistochemistry. The antibody was validated for Western blotting using a genotype-defined mutant/wild-type cell line panel, including L858R-positive, exon19-deletion-positive, and negative lung cancer cell lines. The L858R antibody detected only cells expressing L858R and did not detect exon19-deletion models (Supplementary data). Cells carrying alternate EGFR mutation or lacking target mutation, and total EGFR control antibody were used for controls. This shows strong mutation specificity of the antibody, although not a gene knockout/knockdown experiment. The Cell Signaling antibody was further validated for IF using a genotype-defined mutant/wild-type cell line and xenograft panel: H3255/H1975 L858R vs H1650/HCC827 exon19 deletion and negative cells IF staining occurred only in L858R-expressing cells and correlated spatially with total EGFR staining (Fig. 1). This provides high-quality application-specific mutation validation using multiple genetically characterized positives and negatives. Further validation in IHC using genotype-defined xenografts, followed by IHC using 40 genotyped tumors as samples, and then followed by IHC using 340 tumors of unknown genotype at the time of staining. Samples were FFPE xenografts and human NSCLC, respectively. Controls constituted known L858R, exon19 deletion, and mutation-negative samples; DNA sequencing provided information on genotype. Mutation-specific IHC showed high agreement with DNA genotype; combined assay reported 92% sensitivity and 99% specificity in the larger panel. Data shown in Figures 2-3 and tables 1-3. These data provide very strong IHC validation for mutation detection because the readout is compared against independent DNA sequencing. Dilution is not disclosed in the publication. We rated the Cell Signaling antibody based on the available information and according to the pAbmAbs criteria.