Optimization of antibodies for detection of the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 using a biotin-free visualization system.
Testing for microsatellite instability (MSI) has become an important step in the planning of therapeutic and follow-up procedures for patients with colorectal cancer, both as a prognostic marker and as a screening tool for hereditary non-polyposis colorectal cancer. Today the gold standard for MSI testing is based on the polymerase chain reaction. Immunohistochemistry may represent an alternative or complement to molecular MSI testing. Antibodies against the protein products of the most commonly affected mismatch repair genes (hMLH1, hMSH2, hMSH6, and hPMS2) have been available for some time now. However, the quality of the primary antibody and optimization of the antigen retrieval methods are essential to get reproducible results. The aim of the present study was to test and optimize a panel of antibodies against the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 using biotin-free, polymer-based visualization systems. The antibodies were tested on multitissue blocks containing normal tissue and tumor tissue from patients with known microsatellite-stable and microsatellite-instable tumors. For all four antibody groups, the chosen clones gave specific and reproducible staining. Furthermore, with the PowerVision+ detection system, the influence of endogenous biotin was eliminated, the incubation time with the primary antibody was significantly reduced, and the primary antibody could be further diluted. The authors found that immunohistochemistry may provide a cost-effective and time-saving complement to the molecular MSI analysis, and using the PowerVision+ detection system has greatly decreased the turnaround time as well as reduced the cost of immunohistochemistry in the authors' laboratory.
Department of Pathology, Odense University Hospital, Winslowparken 15, DK-5000, Odense, Denmark. tineplato@hotmail.com
Tine Plato
TP
Hansen
Ole
O
Nielsen
Claus
C
Fenger
D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000276:immunology; D000906:Antibodies / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D001710:Biotin; D002352:Carrier Proteins / Q000032:analysis* / Q000276:immunology; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000175:diagnosis / Q000473:pathology*; D045643:DNA Repair Enzymes / Q000032:analysis* / Q000276:immunology; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000032:analysis* / Q000276:immunology; D009687:Nuclear Proteins / Q000032:analysis* / Q000276:immunology
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D001710:Biotin; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; C497172:MSH2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D045643:DNA Repair Enzymes