MONOCLONAL-ANTIBODY TO THYROID TRANSCRIPTION FACTOR-I - 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 o f the thyroid gland and the lung, To produce monoclonal antibodies spe cific for TTF-1, the polypeptide was expressed in E, coli and purified utilizing affinity chromatography of a polyhistidine-tagged TTF-1 fus ion protein. Splenocytes from BALB/c mice immunized with recombinant T TF-1 were fused with P3x/63Ag8.653 myeloma cells to produce hybridomas . Tissue culture supernatant was screened for anti TTF-1 activity by E LISA employing recombinant TTF-1 as antigen, Hybridomas producing high -affinity antibodies were subcloned by limiting dilution, Antibodies f rom tissue culture fluid from an IgG(1) clone (8G7G3/1) that stained t he nuclei of paraffin-embedded human thyroid tissues were precipitatio n-purified and further characterized, The antibody stained a single 40 -kDa polypeptide in immunoblots of nuclear extracts or lystates of cel l lines known to express TTF-1 mRNA. MAb 8G7G3/1 also stained nuclei o f tissue in a highly specific manner consistent with the pattern of ex pression obtained with an established polyclonal TTF-1 antibody and by in situ hybridization, MAb 8G7G3/1 was used for TTF-1 immunohistochem istry of human adenocarcinomas of the lung, colon, and breast as well as small cell carcinomas of the lung, TTF-1 was detected in primary lu ng adenocarcinomas and small cell carcinomas and was absent in colon a nd breast carcinomas, These findings demonstrate that anti-TTF-l MAb 8 G7G3/1 specifically binds TTF-1 in cell extracts and tissues and can b e used to distinguish between lung and nonlung origin of a tumor.