Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies.
Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies.
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使用单克隆抗体表征甲状腺微粒体抗原及其与甲状腺过氧化物酶的关系。
DOI：
10.1172/jci113438
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
DeGroot,LJ
中科院分区：
文献类型：
--
作者：
Portmann,L;Fitch,FW;Havran,W;Hamada,N;Franklin,WA;DeGroot,LJ
针对甲状腺微粒体抗原的单克隆抗体已经开发出来。以甲状腺微粒体片段为抗原，Western blot分析均结合101-和107-kD条带。抗微粒体抗体高滴度但没有甲状腺球蛋白或促甲状腺激素受体抗体的患者血清免疫沉淀的微粒体蛋白也与MAb结合。结合模式与还原剂的用量有关。101- kd和107-kD的条带通过添加二硫苏糖醇而增加，而在没有二硫苏糖醇的情况下，存在许多更高分子量的条带，这表明这是一种多聚体蛋白质结构。尽管不能免疫沉淀甲状腺过氧化物酶（TPO）的酶活性，但在Western blot中，单抗与变性纯化的猪TPO和变性免疫纯化的人TPO结合强烈。胰蛋白酶消化101-107-kD抗原产生84-88 kD的双链，对单抗仍有免疫反应。五种多克隆血清中有一种（用鞣制红细胞血凝技术测定微粒体抗体滴度大于1/20,480）也识别出84- 88kd胰蛋白酶片段。添加V8蛋白酶导致107-kD蛋白消失，而101-kD蛋白没有消失，证明该抗原是由两种不同的多肽形成的。MAb与甲状腺上皮结合强烈，而与乳头状癌的结合不存在，滤泡和Hurthle细胞癌的结合程度低或中等。本研究表明，甲状腺微粒体抗原和TPO是相同的，由两种不同的多肽组成。在SDS-PAGE上，抗原显示为两个相邻的带，它们具有共同的表位，但不相同，这可以通过它们的大小和对蛋白水解消化的易感性的差异来证明。该分子的免疫反应性高度依赖于胰蛋白酶敏感位点，这在多克隆血清对抗原的识别中显得很重要，并且可能具有生物学意义。微粒体抗原性的表达在各种甲状腺癌中是不同的。图片
MAb directed to the thyroid microsomal antigen have been developed. All bound to 101- and 107-kD bands in Western blot analysis using thyroid microsomal fraction as antigen. The MAb also bound to microsomal proteins immunoprecipitated by serum from patients having a high titer of anti-microsomal antibody but no antibodies to thyroglobulin or thyrotropin-stimulating hormone receptor. The pattern of binding was related to the amount of reducing agent. The 101- and 107-kD bands were increased by addition of dithiothreitol whereas, in its absence, numerous bands of higher molecular weight were present, suggesting a multimeric protein structure. Despite the inability to immunoprecipitate thyroid peroxidase (TPO) enzymatic activity, the MAb bound intensively in Western blot to denatured purified hog TPO and to denatured immunopurified human TPO. Trypsin digestion of the 101-107-kD antigen produced a doublet of 84-88 kD that was still immunoreactive with MAb. One of five polyclonal sera tested (with a microsomal antibody titer greater than 1/20,480 measured by the tanned red cell hemagglutination technique) also recognized the 84-88 kD trypsin fragments. Addition of V8 protease led to a disappearance of the 107-kD protein, but not the 101-kD protein, proving that this antigen is formed by two different polypeptides. The MAb bound strongly to thyroid epithelium, whereas binding to papillary carcinoma was absent or low and moderate for follicular and Hurthle cell carcinoma. This study indicates that the thyroid microsomal antigen and TPO are identical and are constituted of two different polypeptides. On SDS-PAGE the antigen appears as two contiguous bands which share common epitopes but are not identical, as proven by their size and difference in susceptibility to proteolytic digestion. The immunoreactivity of the molecule is highly dependent on a trypsin-sensitive site, which appears important in the recognition of the antigen by polyclonal sera and may have biological importance. The expression of microsomal antigenicity is variable among various thyroid carcinomas.Images