Monoclonal antibody to thyroid transcription factor-1: Production, characterization, and usefulness in tumor diagnosis
Monoclonal antibody to thyroid transcription factor-1: Production, characterization, and usefulness in tumor diagnosis
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DOI：
10.1089/hyb.1996.15.49
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发表时间：
1996-02-01
期刊：
影响因子：
--
通讯作者：
Whitsett, JA
中科院分区：
文献类型：
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作者：
Holzinger, A;Dingle, S;Whitsett, JA
甲状腺转录因子-1（TTF-1）是同源结构域转录因子NKx 2家族的成员，在甲状腺和肺的上皮细胞中表达。为了产生对TTF-1特异的单克隆抗体，在大肠杆菌中表达多肽，并利用多组氨酸标记的TTF-1融合蛋白的亲和层析纯化。用重组TTF-1免疫BALB/c小鼠，取脾细胞与P3 x/63Ag8.653骨髓瘤细胞融合，制备杂交瘤细胞株。用重组TTF-1作为抗原，通过ELISA筛选组织培养上清液的抗TTF-1活性，通过有限稀释法亚克隆产生高亲和力抗体的杂交瘤，沉淀纯化来自IgG（1）克隆（8 G7 G3/1）的组织培养液的抗体，所述IgG（1）克隆（8 G7 G3/1）染色石蜡包埋的人甲状腺组织的核，该抗体在已知表达TTF-1 mRNA的细胞系的核提取物或裂解物的免疫印迹中染色单个40-kDa多肽。MAb 8 G7 G3/1还以与用已建立的多克隆TTF-1抗体和通过原位杂交获得的表达模式一致的高度特异性方式染色组织的细胞核。MAb 8 G7 G3/1用于肺、结肠和乳腺的人腺癌以及肺的小细胞癌的TTF-1免疫组织化学，TTF-1在原发性肺腺癌和小细胞癌中检测到，而在结肠癌和乳腺癌中不存在，这些发现证明抗TTF-1 MAb 8 G7 G3/1特异性结合TTF-1。1，并且可用于区分肿瘤的肺和非肺起源。
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.