| A pilot project for generation of rabbit monoclonal antibodies and an immunohistochemistry database exploring the proteome. Poster at Human Protein Organization (HUPO) Congress 2006
Douglas T. Ross 1, Rodney A. Beck 2, Harry Au 3, Maria Frolkis 3, Stephanie DeFoe 2, Brian Z. Ring 1, Yanling Wang 1, Weimin Zhu 3, Robert S. Seitz 2, Guo-Liang Yu 3
1Applied Genomics Inc, Burlingame, CA; 2Applied Genomics Inc, Huntsville, AL; 3Epitomics Inc, Burlingame, CA
A significant barrier to the characterization and cataloging of the expression patterns of proteins and protein variants across the proteome is the availability of affinity capture reagents selected specifically for their particular end-use. We have endeavored to pilot the proteomic scale production of monoclonal antibodies screened specifically for utility as immunohistochemistry (IHC) reagents. We selected twenty human target proteins and immunized rabbits with two distinct synthesized peptides and one bacterial produced recombinant protein fragment for each of these twenty targets. Polyclonal antisera from immunized rabbits were screened for anti-antigen titer by ELISA and Western blotting and rabbits selected for hybridoma production. Hybridoma fusions from successful immunizations were screened by ELISA and positive wells further screened by both Western blotting and direct testing of culture supernatents on tissue arrays by immunohistochemistry. Over 1600 tissue arrays were used to screen candidate clones as well as resultant subclones from positive wells which were then used to stain tissue arrays containing 100 normal and tumor tissue samples. Clones positive by Western blot were frequently not useful for immunohistochemistry, while clones selected by subjective immunohistochemical staining patterns were often not able to be confirmed by Western blotting. To date, of the 20 proteins targeted, 14 have successfully yielded at least one high quality IHC monoclonal antibody. For several proteins, multiple independent monoclonal antibodies to the same protein target were isolated with different yet striking tissue specific staining patterns. This may relate to alternate splice forms or other protein variants. Data documenting success rates for these sixty hybridoma projects and dramatic staining patterns revealed by different clones targeting protein kinase and cyclin gene families will be shown.
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