IMMUNODETECTION OF THE PRESENCE OR ABSENCE OF FULL-LENGTH APC GENE-PRODUCT IN HUMAN COLONIC TISSUES

In order to detect the presence or absence of wild-type adenomatous po lyposis coli (APC) gene protein (APC) in human colonic tissues, we imm unoaffinity purified two polyclonal rabbit antibodies (APC-1 and APC-2 ) directed against defined epitopes in the middle and carboxyl regions of APC. Such antibodies proved useful in western blot analysis of mat ched colonic mucosa and tumor sample pairs. A 300 kDa band correspondi ng to APC was detected in samples from normal colonic mucosa using bot h antibodies. No tumor samples (n=14) showed a detectable 300 kDa band . SW480 colon carcinoma cells, known to express truncated APC lacking the carboxyl half of the protein, were also negative These results ind icate that out antibodies bind to full-length but not truncated APC. T hus, western blot analysis employing APC-1 and APC-2 antibodies may be used to evaluate the absence or presence of wild-type APC. The value of this methodology in detecting APC mutations, which mainly involve p rotein truncation or allelic loss, is based on its ability to demonstr ate negative or reduced level of immunoreactivity toward full-length A PC in tissues that contain such mutations.