Cucnik S, Kveder T, Rozman B, Bozic B
Department of Rheumatology, University Medical Centre, Vodnikova 62, 1000 Ljubljana, Slovenia.
Rheumatology (Oxford). 2004 Nov;43(11):1353-6. doi: 10.1093/rheumatology/keh349. Epub 2004 Jul 27.
We studied the influence of different binding conditions on the interaction between high- or low-avidity IgG anti-beta2-glycoprotein I antibodies (anti-beta2-GPI) and beta2-GPI, which was either free in solution or bound to microtitre plates or nitrocellulose membranes.
Sera from 30 patients with antiphospholipid syndrome and/or systemic lupus erythematosus were selected on the basis of anti-beta2-GPI positivity. Avidity of IgG anti-beta2-GPI was determined by chaotropic ELISA, using increased NaCl concentration during the antibody binding. Immunodetection on nitrocellulose membrane followed reducing or non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE) of beta2-GPI. In converted, non-reducing PAGE, the preparation with high-affinity Fab fragments (obtained by papain digestion) was subjected to electrophoresis, while purified beta2-GPI was used as the sample in immunodetection.
Anti-beta2-GPI antibodies of high, low or heterogeneous (low and high) avidity were found in 5/30, 9/30 and 16/30 sera, respectively. The density of beta2-GPI, which was 20-30 times higher on the nitrocellulose membrane than on the surface of ELISA plates, was not sufficient for the recognition of the antigen by anti-beta2-GPI: 2/5 high-avidity samples reacted only with non-reduced beta2-GPI, 3/9 low-avidity samples recognized only denatured and reduced beta2-GPI, and 1/16 samples with heterogeneous-avidity antibodies reacted with reduced and non-reduced beta2-GPI.
Our results suggest that neither high density of the antigen nor high avidity of the antibodies (or Fab fragments) alone was sufficient for the binding of anti-beta2-GPI to beta2-GPI. Some conformational modifications and, consequently, exposed neo-epitopes are required for the recognition of beta2-GPI by polyclonal anti-beta2-GPI antibodies.
我们研究了不同结合条件对高亲和力或低亲和力IgG抗β2-糖蛋白I抗体（抗β2-GPI）与β2-GPI之间相互作用的影响，β2-GPI在溶液中游离、结合于微量滴定板或硝酸纤维素膜上。
基于抗β2-GPI阳性，从30例抗磷脂综合征和/或系统性红斑狼疮患者中选取血清。通过离液剂ELISA测定IgG抗β2-GPI的亲和力，在抗体结合过程中增加NaCl浓度。β2-GPI经还原或非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（PAGE）后在硝酸纤维素膜上进行免疫检测。在转化的非还原PAGE中，用高亲和力Fab片段（通过木瓜蛋白酶消化获得）制备物进行电泳，而纯化的β2-GPI用作免疫检测的样品。
分别在5/30、9/30和16/30份血清中发现了高、低或异质（低和高）亲和力的抗β2-GPI抗体。β2-GPI在硝酸纤维素膜上的密度比ELISA板表面高20 - 30倍，但不足以被抗β2-GPI识别抗原：2/5高亲和力样品仅与非还原β2-GPI反应，3/9低亲和力样品仅识别变性和还原的β2-GPI，1/16份具有异质亲和力抗体的样品与还原和非还原β2-GPI反应。
我们的结果表明，单独的高抗原密度或高抗体（或Fab片段）亲和力都不足以使抗β2-GPI与β2-GPI结合。多克隆抗β2-GPI抗体识别β2-GPI需要一些构象修饰以及由此暴露的新表位。
