Bernard N F, Nygen T N, Tyutyunikov A, Stolarski C, Scalise D, Genovese C, Hayes M B, Ludgate M, Wall J R
Thyroid Study Center, Montreal General Hospital Research Institute, Quebec, Canada.
Clin Immunol Immunopathol. 1994 Mar;70(3):225-33. doi: 10.1006/clin.1994.1033.
We have tested for serum antibodies reactive with 1D, a recombinant 65-kDa human thyroid protein which is also expressed in eye muscle, in patients with thyroid autoimmunity and ophthalmopathy by immunofluorescence and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. We also measured antibodies to a 64-kDa pig eye muscle membrane protein which is identified by SDS-PAGE and Western blotting, correlating the two reactivities. While antibodies to 1D, expressed in Chinese hamster ovary (CHO) cell membrane, were detected in approximately 40% of patients with ophthalmopathy, in both tests the greatest prevalence, by immunofluorescence, 73%, was demonstrated in patients with Graves' hyperthyroidism without clinically evident eye disease, although only 50% of these patients were positive in immunoblotting. When the two tests for anti-1D antibodies were compared, immunofluorescence appeared to be the more specific and immunoblotting appeared to be the more sensitive. The greatest prevalence of antibodies reactive with a 64-kDa pig eye muscle protein, 71%, was in patients with TAO of less than 1 year duration; tests were positive in 49% of patients with more chronic ophthalmopathy and in 50% of patients with Graves' hyperthyroidism without evident eye disease. Antibodies reactive with 1D were detected in 17% of normals by immunofluorescence and 24% by immunoblots, while antibodies reactive with the 64-kDa pig eye muscle protein were detected in only 10% of the normal subjects tested. Lesser prevalences of antibodies to the two 64-kDa proteins in patients with established eye disease suggest that such antibodies may be an early abnormality in patients with Graves' hyperthyroidism who are predisposed to develop ophthalmopathy. Although the association was not close, reactivity against 1D by immunoblotting, but not immunofluorescence, was significantly correlated with reactivity to a 64-kDa eye muscle membrane protein by immunoblotting. On the other hand, when sera containing antibodies reactive with both 1D and the 64-kDa eye muscle protein were incubated with CHO (1D) cell membrane, reactivity against 1D was absorbed while that against the eye muscle protein was not. The precise relationship between the two 64-kDa proteins can only be clarified by cloning the 64-kDa protein from an eye muscle expression library and comparing the sequences with those of 1D.
我们通过免疫荧光、SDS - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和蛋白质印迹法，检测了甲状腺自身免疫和眼病患者血清中与1D（一种重组的65 kDa人甲状腺蛋白，也在眼肌中表达）反应的抗体。我们还检测了针对一种64 kDa猪眼肌膜蛋白的抗体，该蛋白通过SDS - PAGE和蛋白质印迹法鉴定，并关联了这两种反应性。虽然在大约40%的眼病患者中检测到了在中国仓鼠卵巢（CHO）细胞膜上表达的1D抗体，但在两项检测中，通过免疫荧光法，Graves甲亢且无明显眼病的患者中抗体阳性率最高，为73%，不过这些患者中只有50%在免疫印迹法中呈阳性。当比较两种抗1D抗体检测方法时，免疫荧光法似乎更具特异性，而免疫印迹法似乎更敏感。与64 kDa猪眼肌蛋白反应的抗体阳性率最高为71%，出现在病程小于1年的甲状腺相关眼病（TAO）患者中；在病程较长的眼病患者中，49%检测呈阳性，在Graves甲亢且无明显眼病的患者中，50%检测呈阳性。通过免疫荧光法在17%的正常人中检测到与1D反应的抗体，通过免疫印迹法在24%的正常人中检测到；而在仅10%的受试正常受试者中检测到与64 kDa猪眼肌蛋白反应的抗体。在已确诊眼病的患者中，针对这两种64 kDa蛋白的抗体阳性率较低，这表明此类抗体可能是Graves甲亢且易患眼病患者的早期异常表现。虽然这种关联并不紧密，但通过免疫印迹法而非免疫荧光法检测到的与1D的反应性，与通过免疫印迹法检测到的与64 kDa眼肌膜蛋白的反应性显著相关。另一方面，当将含有与1D和64 kDa眼肌蛋白都反应的抗体的血清与CHO（1D）细胞膜孵育时，与1D的反应性被吸收，而与眼肌蛋白的反应性未被吸收。只有通过从眼肌表达文库中克隆64 kDa蛋白并将其序列与1D的序列进行比较，才能阐明这两种64 kDa蛋白之间的确切关系。
