Abstract Background: MLH1, a DNA mismatch repair gene, plays a critical role in colorectal cancer (CRC) development. Loss of nuclear MLH1 is attributed to hypermethylation of its promoter, occurring in 78% of microsatellite instability (MSI) which composes 15% of all CRC. Immunohistochemical (IHC) analysis of MLH1 in CRC is routinely performed in pathology laboratories, but remains challenging with the current selection of available MLH1 antibodies. Here we report a new MLH1 rabbit monoclonal antibody (RabMAb) developed with RabMAb® technology, designated as clone EP481. Design: Sera collected from rabbits immunized with recombinant MLH1 protein were screened by ELISA, WB, and IHC. After fusion, antibody from a final hybridoma cell line was further characterized by extensive IHC testing using formalin-fixed paraffin-embedded human normal and tumor tissue microarrays (TMAs) comprised of 99 CRCs. EP481 performance was compared against MLH1 mouse monoclonal antibodies, G168-15, G168-728, and the current benchmark, ES05. Cases without tumor cell staining were considered negative; those also lacking stroma staining were excluded, as it could not be determined if there was germline mutation or poor tissue quality. Agreement was evaluated with Cohen's kappa. Results: An 85 kDa MLH1 protein was detected by WB in SW480 cell lysate, but absent in the MLH1-negative cell line, HCT116. IHC analysis of EP481 was positive in normal epithelium and surrounding stroma, but negative in 10% of tumors. G168-728 showed nonspecific cytoplasmic staining and deviated from its counterparts. Concordance data (Table 1) demonstrates that EP481 is equivalent with ES05 and incongruous with G168-15. Conclusion: The RabMAb MLH1 antibody, clone EP481, specifically labels normal cells and is absent in 10% of CRCs. This finding is consistent with both literature and the gold-standard anti-mouse clone, ES05. EP481 is a new alternative for MLH1 IHC and a promising tool to identify MSI in CRC. Table 1. Concordance staining of MLH1 clone EP481 with clones ES05 and 168-15 on CRC TMA.ES05 +ES05 -Total168-15 +168-15 -TotalEP481 +7417566975EP481 -178268Total75883681583Cohen's kappaRatersCoefficientAgreementEP481/ES0586%ExcellentEP481/168-1545%Moderate Citation Format: Sean Cobb, Hongyang Pan, Qiong Yang, Jackie Chan, Julia Wu, Jason Law, Aihua Li. A new MLH1 rabbit monoclonal antibody for immunohistochemical application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4516.