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Establishment of a novel assay method for anticardiolipin antibodies.

抗心磷脂抗体新测定方法的建立。

基本信息

批准号：
07557221
负责人：
KOIKE Takao
金额：
$ 0.9万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

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.

抗磷脂综合征（APS）是一种自身免疫性疾病，其表现为抗磷脂抗体，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血功能。心磷脂（CL）包被板的各种aCL免疫测定法（ELISA和RIA）已被用于aCL的检测，然而，我们了解到它们存在一些技术困难和产生假阳性结果。我们还建立了一种新的和准确的测定（ELISA）方法，使用多氧聚苯乙烯板包被β 2-糖蛋白I（β 2-GPI），1）当β 2-GPI与多氧聚苯乙烯板以及由负性-带电荷的磷脂如心磷脂和磷脂酰丝氨酸。2）在常规aCL-ELISA中获得的抗体滴度与使用β 2-GPI包被的多氧板的改进ELISA中获得的抗体滴度之间存在良好的相关性。然而，仅在改进的aCL-ELISA中，与带负电荷的分子非特异性结合的抗体可以忽略不计。改良ELISA中检测到的抗体的出现与血栓形成之间以及与狼疮抗凝剂或梅毒试验的生物学假阳性之间具有良好的相关性。在改进的ELISA中，不仅使用多氧聚苯乙烯板，而且使用未处理的聚苯乙烯板，用缺乏其结构域V的β 2-GPI突变蛋白包被，都可以检测到APS患者的aCL。