Hamada N, Grimm C, Mori H, DeGroot L J
J Clin Endocrinol Metab. 1985 Jul;61(1):120-8. doi: 10.1210/jcem-61-1-120.
The molecular identity of the thyroid microsomal antigen was investigated by Western blot and immunoprecipitation using sera from eight patients with autoimmune thyroid disease. All sera had high microsomal antibody titers by hemagglutination test and ELISA; only one had thyroglobulin (Tg) antibody. In an immunoprecipitation study using Triton X-100-solubilized 125I-labeled microsomes, all of the sera precipitated a 107K protein. However, this 107K protein was visualized by only three patients' sera by Western blot analysis under reducing conditions. In Western blots run under nonreducing conditions, four sera, including the three sera mentioned above, recognized poorly defined large mol wt proteins. One serum which also had Tg antibody recognized additional bands by both immunoprecipitation and Western blot. Preincubation of serum with 100 micrograms/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 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. The data indicate that serum 4 can recognize the 107K protein under nondenaturing conditions. These data indicate that the 107K protein described here is the microsomal antigen, and that 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 two antigen epitopes and different patients have different antibodies or different populations of antibodies against these epitopes.
利用8例自身免疫性甲状腺疾病患者的血清，通过蛋白质印迹法和免疫沉淀法研究了甲状腺微粒体抗原的分子特性。通过血凝试验和酶联免疫吸附测定法（ELISA）检测，所有血清的微粒体抗体效价均很高；只有1例含有甲状腺球蛋白（Tg）抗体。在一项使用Triton X - 100溶解的125I标记微粒体的免疫沉淀研究中，所有血清均沉淀出一种107K的蛋白质。然而，在还原条件下通过蛋白质印迹分析，只有3例患者的血清能使这种107K的蛋白质显影。在非还原条件下进行的蛋白质印迹实验中，包括上述3例血清在内的4例血清识别出了界限不清的大分子质量蛋白质。1例同时含有Tg抗体的血清通过免疫沉淀和蛋白质印迹法识别出了其他条带。在蛋白质印迹分析中，将血清与100微克/毫升的Tg或100毫单位/毫升的牛促甲状腺激素（TSH）预温育，并不会改变抗体与107K蛋白质的结合。使用患者血清对肝脏和肾脏微粒体进行蛋白质印迹分析时，未发现107K的蛋白质显影。开展实验以表征在蛋白质印迹中能使107K条带显影的1例血清（1号血清）和不能使其显影的1例血清（4号血清）之间的差异。通过ELISA和蛋白质印迹研究比较了不同稀释度下的反应性。1号血清即使在1:3200的稀释度下仍能识别107K的蛋白质，在ELISA中该稀释度下的光密度值低于4号血清1:200稀释度时的值，而4号血清在任何稀释度下均无法识别107K条带。用与琼脂糖[Reacti-Gel (6X)]偶联的4号血清免疫球蛋白G制备的亲和凝胶，从Triton X - 100溶解的微粒体中去除了1号患者血清在还原条件下通过蛋白质印迹分析所识别的107K蛋白质。数据表明，4号血清在非变性条件下能够识别107K蛋白质。这些数据表明，此处描述的107K蛋白质就是微粒体抗原，并且其可能由于天然状态下存在的二硫键而与另一种蛋白质结合或维持独特的构象。此外，107K蛋白质至少包含两个抗原表位，不同患者针对这些表位具有不同的抗体或不同群体的抗体。
