Establishment of a novel assay method for anticardiolipin antibodies.

抗磷脂综合征(APS)是一种自身免疫性疾病，出现抗心磷脂抗体(ACL)和狼疮抗凝剂等抗磷脂抗体，被认为是基于这些抗体介导的促凝血特性。目前已有多种使用心磷脂(CL)包被的平板进行ACL免疫检测的方法(ELISA法和RIA法)，但这些方法存在一定的技术难度，且存在假阳性结果。本研究对APS患者ACL的特异性进行了研究。我们还建立了一种新的准确检测acl的方法，用包被β2-糖蛋白I(beta2-gpi)的聚氧聚苯乙烯(beta2-gpi)代替CL包被的板。1)aPS患者的acl在与聚氧聚苯乙烯(beta2-gpi)以及由带负电荷的磷脂(如心磷脂和磷脂酰丝氨酸)组成的脂膜相互作用时，能识别出现在beta2-gpi结构上的隐蔽表位。2)用传统的acl-gpi和改进的包被beta2-gpi的聚氧板得到的抗体效价之间有很好的相关性。非特异性结合负电荷分子的抗体仅在改进的ACL-ELISA中可以忽略。3)在改进的ELISA中检测到的抗体的出现与血栓形成以及与狼疮抗凝剂或梅毒试验的生物假阳性之间有很好的相关性。4)在改进的ELISA中，不仅可以使用多氧包被的APS患者的ACL，而且可以检测到包被缺乏其结构域V的β2-GPI突变蛋白的未经处理的聚苯乙烯平板。

An autoimmune disease, "antiphospholipid syndrome (APS) ", with appearance of antiphospholipid antibodies such as anticardiolipin antibodies (aCL) and lupus anticoagulants is thought to be based on these antibodies mediated-procoagulant features. Various aCL-immunoassays (ELISA and RIA) using cardiolipin (CL) -coated plates have been used for aCL detection, however, we learned that they have some technical difficulties and produce false-positive results.In the present study, we investigated the specificity of aCL from patients with APS and obtained following results. We also established a novel and accurate assay (ELISA) method for aCL using polyoxygenated polystirene plates coated with beta2-glycoprotein I (beta2-GPI), instead of CL-coated plates.1) aCL from patients with APS recognized an cryptic epitope appearing on the beta2-GPI structure when beta2-GPI interacts with polyoxygenated polystirene plates as well as with lipid membranes composed of negatively-charged phospholipids such as cardiolipin and phosphatidylserine.2) There was a good correlation between the antibody titers obtained in the conventional aCL-ELISA and in the improved ELISA using polyoxygenated plates coated with beta2-GPI.However, antibodies non-specifically bound to negatively charged molecules were negligible only in the improved aCL-ELISA.3) There was good correlation between the appearance of antibodies detected in the improved ELISA and thrombosis and also between the appearance and lupus anticoagulants or biological false positive for the test of syphilis.4) aCL from APS-patients could be detected in the improved ELISA using not only the polyoxygenated but also the non-treated polystirene plates coated with the beta2-GPI mutant protein lacking its domain V.