RADIOIMMUNOASSAY OF THE APC GENE-PRODUCT USING ANTIBODIES AGAINST ITSMIDDLE AND CARBOXYL REGIONS

A radioimmunoassay (RIA) has been developed for the adenomatous polypo sis coli protein (APC). High-avidity rabbit polyclonal antibodies were produced against synthetic peptides corresponding to amino acids 1865 -1881 (APC-1) and to amino acids 1336-1350 (APC-2) in APC's 2844 amino acid sequence. Both antibodies were utilized in RIA to evaluate full- length APC that is present in the insoluble particulate fraction of ce ll lysates. High salt extraction, often employed for coiled-coil type protein preparations, was found to be useful for extraction of APC fro m lysates of normal colonic epithelium. Proteolytic digestion of high salt extracts increased antibody reactivity toward both epitopes, sugg esting that APC-1 and APC-2 antigenic sites are partially concealed du e to APC's involvement in multiprotein complexes. Thus, RIA using our antibodies will provide a valuable tool for APC protein purification a nd in studies for elucidating APC's biologic function. (C) 1995 Academ ic Press, Inc.