Moteki S, Leung P S, Coppel R L, Dickson E R, Kaplan M M, Munoz S, Gershwin M E
Division of Rheumatology, Allergy and Clinical Immunology, School of Medicine, University of California Davis, CA 95616, USA.
Hepatology. 1996 Jul;24(1):97-103. doi: 10.1002/hep.510240117.
The detection of antimitochondrial autoantibodies (AMAs) is critical in the diagnosis of primary biliary cirrhosis (PBC). However, conventional laboratory assays to detect AMA are dependent on the time-consuming method of immunofluorescence microscopy, a method often plagued by problems of nonspecificity. AMAs react against mitochondrial autoantigens including the E2 components of the pyruvate dehydrogenase complex (PDC-E2), the branched-chain 2-oxo-acid dehydrogenase complex (BCOADC-E2), and the 2-oxo-glutarate dehydrogenase complex (OGDC-E2). Interestingly, the immunodominant epitopes of PDC-E2, BCOADC-E2, and OGDC-E2 are all conformational lipoate binding sites, but antibodies against them do not cross-react. Although 80% to 90% of sera from patients with PBC react to PDC-E2, approximately 10% patients with PBC react only to BCOADC-E2 and/or OGDC-E2. We have taken advantage of our epitope-mapping studies of the E2 components of PDC, BCOADC, and OGDC, and constructed a "designer" hybrid clone, designated as pML-MIT3, that coexpresses the immunodominant epitopes within the three distinct lipoyl domains. We examined a total of 321 sera, including 186 sera from patients with PBC, to test the immunoreactivity of pMIT3. Of 186 sera from patients with PBC, 152 sera (81.7%) reacted with recombinant fusion protein of PDC-E2, whereas 171 sera (91.9%) showed positive reactivities when probed by immunoblotting against the recombinant fusion protein expressed from the pML-MIT3 clone. Of 34 PBC sera that did not react with recombinant PDC-E2, 18 sera contained BCOADC-E2-specific AMA and 1 serum possessed only OGDC-E2-specific AMA. We also developed an enzyme-linked immunosorbent assay (ELISA), using affinity-purified recombinant fusion protein of pML-MIT3 clone as the antigen source, to quantify specific AMAs in patients with PBC. None of the 135 control sera from patients with primary sclerosing cholangitis (PSC), chronic autoimmune hepatitis (CAH), systemic lupus erythematosus (SLE), or healthy volunteers showed significant reactivity against pML-MIT3 recombinant fusion protein in the ELISA assay. Our results indicate that an ELISA using recombinant, cloned autoantigen of pML-MIT3 is a powerful and very specific method for the detection of AMA.
抗线粒体自身抗体（AMA）的检测对原发性胆汁性肝硬化（PBC）的诊断至关重要。然而，传统实验室检测AMA的方法依赖耗时的免疫荧光显微镜法，该方法常受非特异性问题困扰。AMA可与包括丙酮酸脱氢酶复合物（PDC-E2）、支链2-氧代酸脱氢酶复合物（BCOADC-E2）及2-氧代戊二酸脱氢酶复合物（OGDC-E2）的E2成分在内的线粒体自身抗原发生反应。有趣的是，PDC-E2、BCOADC-E2和OGDC-E2的免疫显性表位均为构象性硫辛酸结合位点，但针对它们的抗体不会发生交叉反应。虽然80%至90%的PBC患者血清与PDC-E2反应，但约10%的PBC患者仅与BCOADC-E2和/或OGDC-E2反应。我们利用对PDC、BCOADC和OGDC的E2成分进行的表位作图研究，构建了一个“设计型”杂交克隆，命名为pML-MIT3，它在三个不同的硫辛酰结构域中共表达免疫显性表位。我们共检测了321份血清，包括186份PBC患者血清，以检测pMIT3的免疫反应性。在186份PBC患者血清中，152份血清（81.7%）与PDC-E2重组融合蛋白反应，而当用针对pML-MIT3克隆表达的重组融合蛋白进行免疫印迹检测时，171份血清（91.9%）显示出阳性反应。在34份不与重组PDC-E2反应的PBC血清中，18份血清含有BCOADC-E2特异性AMA，1份血清仅含有OGDC-E2特异性AMA。我们还开发了一种酶联免疫吸附测定（ELISA），使用pML-MIT3克隆的亲和纯化重组融合蛋白作为抗原来源，以定量PBC患者中的特异性AMA。在ELISA测定中，来自原发性硬化性胆管炎（PSC）、慢性自身免疫性肝炎（CAH）、系统性红斑狼疮（SLE）患者的135份对照血清或健康志愿者的血清均未显示出对pML-MIT3重组融合蛋白的显著反应。我们的结果表明，使用pML-MIT3重组克隆自身抗原的ELISA是一种检测AMA的强大且非常特异的方法。
