Holzinger A, Dingle S, Bejarano P A, Miller M A, Weaver T E, DiLauro R, Whitsett J A
Division of Pulmonary Biology, University of Cincinnati College of Medicine, Ohio 45229-3039, USA.
Hybridoma. 1996 Feb;15(1):49-53. doi: 10.1089/hyb.1996.15.49.
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.
甲状腺转录因子-1（TTF-1）是同源结构域转录因子NKx2家族的成员，在甲状腺和肺的上皮细胞中表达。为了制备对TTF-1具有特异性的单克隆抗体，该多肽在大肠杆菌中表达，并利用带有多组氨酸标签的TTF-1融合蛋白的亲和色谱法进行纯化。用重组TTF-1免疫的BALB/c小鼠的脾细胞与P3x/63Ag8.653骨髓瘤细胞融合以产生杂交瘤。采用重组TTF-1作为抗原，通过ELISA筛选组织培养上清液中的抗TTF-1活性。产生高亲和力抗体的杂交瘤通过有限稀释法进行亚克隆。从IgG1克隆（8G7G3/1）的组织培养液中获得的抗体，该抗体可对石蜡包埋的人甲状腺组织细胞核进行染色，经沉淀纯化后进一步鉴定。在已知表达TTF-1 mRNA的细胞系的核提取物或裂解物的免疫印迹中，该抗体可识别一条单一的40 kDa多肽。单克隆抗体8G7G3/1也以高度特异性的方式对组织细胞核进行染色，这与使用已有的多克隆TTF-1抗体和原位杂交获得的表达模式一致。单克隆抗体8G7G3/1用于人肺腺癌、结肠癌和乳腺癌以及肺小细胞癌的TTF-1免疫组织化学检测。在原发性肺腺癌和小细胞癌中检测到TTF-1，而在结肠癌和乳腺癌中未检测到。这些发现表明，抗TTF-1单克隆抗体8G7G3/1能在细胞提取物和组织中特异性结合TTF-1，可用于区分肿瘤的肺源性和非肺源性。
