Immunohistochemistry of carcinomas using monoclonal antibody, JAA-F11
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Thomsen Friedenreich antigen (TF-Ag) is a carbohydrate antigen that is exposed on various carcinomas including those of the breast, colon, bladder and prostate . The ubiquitous nature of this antigen indicates that it may have potential as an antigen to assist in pathologic diagnosis. JAA-F11, mouse monoclonal antibody shows high binding and specificity to Thomsen Friedenreich antigen. Preliminary analysis to determine if JAA-F11 could be used for immunohistochemistry to aid in immunopathologic analysis of human tissue was promising, but repeat analysis by another group was not as promising. In order to understand these differences and optimize the conditions of JAA-F11 use for immunohistochemical staining, the mouse 4T1 tumor cell line was used. This tumor cell line was used because of experimental evidence that indicated cell surface reactivity with JAA-F11 including the fact that JAA-F11 decreased 4T1 tumor metastasis and increased survival . In addition, enzyme immunoassays, flow cytometry and radiolabeled antibody in vivo tumor localization experiments all indicate the presence of TF-Ag which is reactive with the JAA-F11 on the surface of 4T1 cells. The radiolocalization experiments were particularly important in showing the lack of reaction with normal mouse tissue and the reaction with tumor tissue. Thus, in optimization of the immunohistochemical analysis 4T1 tumors were used as a positive control and normal mouse tissues as a negative control. To optimize staining, all of the following were analyzed: (1) tissue fixation methods, (2) tissue hydration and dehydration methods and timing, (3) Antibody concentrations and incubation times, and (4) blocking agents and (5) use of secondary antibody or biotinylated antibody.