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 IgG1 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 lysates 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-1 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.
Holzinger
A
A
Division of Pulmonary Biology, University of Cincinnati College of Medicine, Ohio 45229-3039, USA.
Dingle
S
S
Bejarano
P A
PA
Miller
M A
MA
Weaver
T E
TE
DiLauro
R
R
Whitsett
J A
JA
D000230:Adenocarcinoma / Q000175:diagnosis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry*; D014408:Biomarkers, Tumor / Q000276:immunology*; D001943:Breast Neoplasms / Q000175:diagnosis; D003110:Colonic Neoplasms / Q000175:diagnosis; D005260:Female; D006801:Humans; D007120:Immunochemistry; D008175:Lung Neoplasms / Q000175:diagnosis; D051379:Mice; D008807:Mice, Inbred BALB C; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013194:Staining and Labeling; D013964:Thyroid Neoplasms / Q000175:diagnosis; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; C000614412:NKX2-1 protein, human; C000614414:Nkx2-1 protein, mouse; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors