IDENTIFICATION OF A THYROID MICROSOMAL ANTIGEN BY WESTERN BLOT AND IMMUNOPRECIPITATION
IDENTIFICATION OF A THYROID MICROSOMAL ANTIGEN BY WESTERN BLOT AND IMMUNOPRECIPITATION
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DOI：
10.1210/jcem-61-1-120
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发表时间：
1985-01-01
影响因子：
5.8
通讯作者：
DEGROOT, LJ
中科院分区：
文献类型：
--
作者：
HAMADA, N;GRIMM, C;DEGROOT, LJ
用免疫印迹和免疫沉淀法对8例自身免疫性甲状腺疾病患者的血清进行了微粒子抗原的分子鉴定。血凝试验和酶联免疫吸附试验均为高滴度的微粒子抗体，仅1例血清有甲状腺球蛋白(TG)抗体。在用Triton X-100溶解的125I标记的微粒进行的免疫沉淀研究中，所有的血清都沉淀了一个107K的蛋白质。在还原条件下，只有3例患者的血清可见这一107K蛋白。在非还原条件下运行的Western blotts中，包括上述3种血清在内的4种血清识别出定义不清的大分子蛋白质。一份同时含有TG抗体的血清通过免疫沉淀和免疫印迹识别出额外的条带。用100mU/mlTG或100mU/ml牛TSH预先孵育血清，在Western印迹分析中不改变抗体与107K蛋白的结合。在使用患者血清的肝和肾微粒体的蛋白质印迹图中没有检测到107K蛋白。通过实验研究了一种血清(1号血清)和一种不能显示107K条带的血清(4号血清)之间的差异。用ELISA法和Western印迹法比较不同稀释度下的反应性。血清1在1：3200稀释度时仍能识别107K蛋白，其吸光度值低于血清4的1：200稀释度，而血清4在任何稀释度下都不能识别107K条带。从Triton X-100溶解的微粒子中去除病人1血清在还原条件下经Western印迹分析识别的107K蛋白，用琼脂糖凝胶[Reacti-Gel(6 X)]连接血清4免疫球蛋白G制备的亲和凝胶。显然，血清4在非变性条件下可以识别107K蛋白。显然，这里描述的107K蛋白是微粒体抗原；它可能与另一种蛋白结合，或由于天然状态下存在的二硫键而保持独特的构象。此外，107K蛋白包括至少2个抗原表位，不同的患者针对这些表位有不同的抗体或不同的抗体群。
The molecular identify of the thyroid microsomal antigen was investigated by Western blot and immunoprecipitation using sera from 8 patients with autoimmune thyroid disease. All sera had high microsomal antibody titers by hemagglutination test and ELISA [enzyme linked immunosorbent assay] only 1 had thyroglobulin (Tg) antibody. In an immunoprecipitation study using Triton X-100-solubilized 125I-labeled microsomes, all of the sera precipitated a 107K protein. This 107K protein was visualized by only 3 patients'' sera by Western blot analysis under reducing conditions. In Western blots run under nonreducing conditions, 4 sera, including the 3 sera mentioned above, recognized poorly defined large MW proteins. One serum which also had Tg antibody recognized additional bands by both immunoprecipitation and Western blot. Preincubation of serum with 100 .mu.g/ml Tg or 100 mU/ml bovine TSH did not alter the binding of antibody to 107K protein in Western blot analysis. The 107K protein was not visualized in Western blots of liver and kidney microsomes using patients'' sera. Experiments were performed to characterize the difference between one serum (no. 1) which visualized the 107K [kdalton] band in Western blots and a serum (no. 4) which did not. Reactivity at different dilutions was compared by ELISA and Western blot studies. Serum 1 recognized the 107K protein even at a 1:3200 dilution, which gave a lower optical density value than a 1:200 dilution of serum 4 in ELISA, while serum 4 failed to recognize the 107K band at any dilution. An affinity gel prepared from serum 4 immunoglobulin G linked to agarose [Reacti-Gel (6 X)] removed from Triton X-100-solubilized microsomes the 107K protein which patient 1 serum recognized by Western blot analysis under reducing conditions. Apparently, serum 4 can recognize the 107K protein under nondenaturing conditions. Evidently, the 107K protein described here is the microsomal antigen; it may associate with another protein or maintain a unique conformation due to disulfide bonds present in the native state. In addition, the 107K protein includes at least 2 antigen epitopes and different patients have different antibodies or different populations of antibodies against these epitopes.