Establishment of a novel assay method for anticardiolipin antibodies.

一种自身免疫性疾病，“抗磷脂综合征（APS）”，出现抗磷脂抗体，如抗心磷脂抗体（aCL）和狼疮抗凝剂，被认为是基于这些抗体介导的促凝特征。使用心磷脂（CL）包被板的各种aCL免疫测定法（ELISA和RIA）已用于aCL检测，然而，我们了解到它们存在一些技术困难并产生假阳性结果。在本研究中，我们研究了APS患者aCL的特异性，得到了以下结果。我们还建立了一种新的、准确的测定aCL的方法（ELISA），使用包被β 2-糖蛋白I （β 2- gpi）的多氧聚苯乙烯板代替cl包被板。1) APS患者的aCL识别出β - gpi与多氧聚苯乙烯板以及与带负电荷的磷脂（如心磷脂和磷脂酰丝氨酸）组成的脂膜相互作用时，在β - gpi结构上出现的一个隐表位。2)采用β 2- gpi包被多氧板改进的ELISA法与常规的aCL-ELISA法所得抗体滴度具有良好的相关性。然而，非特异性结合负电荷分子的抗体仅在改进的aCL-ELISA中可以忽略不计。3)改良ELISA检测抗体的出现与血栓形成有良好的相关性，与狼疮抗凝剂或梅毒生物学假阳性有良好的相关性。4)改良后的酶联免疫吸附法不仅可以检测到aps患者的aCL，还可以检测到包被缺失V结构域的β 2- gpi突变蛋白的多氧和未处理的聚苯乙烯板。

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.