Yokoyama N, Taurog A, Dorris M L, Klee G G
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
J Clin Endocrinol Metab. 1990 Mar;70(3):758-65. doi: 10.1210/jcem-70-3-758.
We have isolated highly purified thyroid peroxidase (TPO) from human thyroid tissue to study further the relationship between TPO and the thyroid microsomal antigen that elicits the production of microsomal autoantibodies in patients with autoimmune thyroid disease. Serum samples were obtained from 24 patients with suspected autoimmune thyroid disease, and from 7 normal subjects. Microsomal autoantibodies in the patient sera, as determined by the microsomal hemagglutination assay (MCHA), varied between 1:100 and 1:102,400. Antithyroglobulin antibodies, however, were very low (less than 1:100). Binding of serum autoantibodies to purified human TPO, as determined by enzyme-linked immunosorbent assay, correlated fairly well with MCHA titers (r = 0.72; P less than 0.001). An immunoblot procedure was developed to study the binding of serum antibodies to the major active fragment of TPO (93 kDa), after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Binding under both conditions correlated very well with MCHA titers (r = 0.80-0.84; P less than 0.001). Studies were performed to determine the inhibitory effect of patient serum on the enzymatic activity of purified human TPO. A marked inhibitory effect on guaiacol activity was observed when TPO was preincubated with as little as 10 microL high titer serum. There was a significant correlation (r = 0.47; P less than 0.01) between MCHA titer and inhibitory effect. The addition of 2 micrograms purified human TPO completely or almost completely inhibited the binding of serum antibodies to thyroid microsomes (enzyme-linked immunosorbent assay) in 10 of 11 patient sera with high MCHA titers (1:25,600 or greater).
我们从人甲状腺组织中分离出了高度纯化的甲状腺过氧化物酶（TPO），以进一步研究TPO与甲状腺微粒体抗原之间的关系，该抗原可在自身免疫性甲状腺疾病患者体内引发微粒体自身抗体的产生。血清样本取自24例疑似自身免疫性甲状腺疾病患者以及7名正常受试者。通过微粒体血凝试验（MCHA）测定，患者血清中的微粒体自身抗体在1:100至1:102,400之间变化。然而，抗甲状腺球蛋白抗体水平非常低（低于1:100）。通过酶联免疫吸附测定法确定，血清自身抗体与纯化的人TPO的结合与MCHA滴度具有较好的相关性（r = 0.72；P < 0.001）。开发了一种免疫印迹程序，用于研究在还原和非还原条件下进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后，血清抗体与TPO主要活性片段（93 kDa）的结合。两种条件下的结合与MCHA滴度均具有很好的相关性（r = 0.80 - 0.84；P < 0.001）。进行了相关研究以确定患者血清对纯化的人TPO酶活性的抑制作用。当TPO与低至10微升的高滴度血清预孵育时，观察到对愈创木酚活性有明显的抑制作用。MCHA滴度与抑制作用之间存在显著相关性（r = 0.47；P < 0.01）。在11例MCHA滴度较高（1:25,600或更高）的患者血清中，添加2微克纯化的人TPO可完全或几乎完全抑制血清抗体与甲状腺微粒体的结合（酶联免疫吸附测定法）。
