Monoclonal antibody to thyroid transcription factor-1: Production, characterization, and usefulness in tumor diagnosis

Thyroid transcription factor-1 (TTF-1), a member of the NKx2 family of homeodomain transcription factors, is expressed in epithelial cells of the thyroid gland and the lung, To produce monoclonal antibodies specific for TTF-1, the polypeptide was expressed in E, coli and purified utilizing affinity chromatography of a polyhistidine-tagged TTF-1 fusion protein. Splenocytes from BALB/c mice immunized with recombinant TTF-1 were fused with P3x/63Ag8.653 myeloma cells to produce hybridomas. Tissue culture supernatant was screened for anti TTF-1 activity by ELISA employing recombinant TTF-1 as antigen, Hybridomas producing high-affinity antibodies were subcloned by limiting dilution, Antibodies from tissue culture fluid from an IgG(1) clone (8G7G3/1) that stained the nuclei of paraffin-embedded human thyroid tissues were precipitation-purified and further characterized, The antibody stained a single 40-kDa polypeptide in immunoblots of nuclear extracts or lystates of cell lines known to express TTF-1 mRNA. MAb 8G7G3/1 also stained nuclei of tissue in a highly specific manner consistent with the pattern of expression obtained with an established polyclonal TTF-1 antibody and by in situ hybridization, MAb 8G7G3/1 was used for TTF-1 immunohistochemistry of human adenocarcinomas of the lung, colon, and breast as well as small cell carcinomas of the lung, TTF-1 was detected in primary lung adenocarcinomas and small cell carcinomas and was absent in colon and breast carcinomas, These findings demonstrate that anti-TTF-l MAb 8G7G3/1 specifically binds TTF-1 in cell extracts and tissues and can be used to distinguish between lung and nonlung origin of a tumor.