用体外表达的血小板膜蛋白GPⅢa偶联Luminex微球检测HPA-1a抗体

摘要

Objective To generate recombinant GP Ⅲ a as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.Methods The full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1.The recombinant plasmid was transfected into CHO cells,and those with stable expression were screened with G418.Expressed protein was identified and coupled with Luminex xMAP beads,which were then reacted with sera samples.Subsequently,phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.Results DNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa.The recombinant GP Ⅲ a was coupled with Luminex xMAP beads.The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL).The Luminex beads assay could specifically identify the HPA 1a antibody from the test sera,and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology.Cross-reactivity was not observed with the samples containing HLA,ABO and other HPA antibodies (HPA-3a and HPA-5b).The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.Conclusion Recombinant GPⅢ a was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.%目的 拟利用体外克隆表达的GPⅢa蛋白作为HPA-1a抗原与Luminex xMAP微球相连,建立和评估用Luminex微球技术检测血清中抗HPA-1a的效果.方法 扩增并克隆GPⅢa蛋白的编码基因ITGB3至真核表达载体,转染中国仓鼠卵巢癌细胞株.利用G418筛选稳定表达细胞株,体外获取并鉴定相应的特异性膜糖蛋白GPⅢa.将重组蛋白通过表面游离氨基和Luminex xMAP微球表面激活的羧基相偶联,再利用偶联后的微球与待测血清反应.利用PE-抗人IgG进行显色,在Luminex-100仪上读取数据并判断结果.结果 ITGB3基因测序为HPA-1aa,将克隆后的GPⅢa重组蛋白偶联Luminex xMAP微球,Luminex微球法检测HPA 1a抗体的灵敏度为滴度1/32(抗体浓度3.125 U/mL).微球技术能特异性鉴别出待测样本的HPA-1a抗体,与MAIPA结果一致,而且与HLA抗体、ABO抗体以及其他HPA血小板系统抗体(HPA-3a、HPA-5b)均不发生交叉反应,说明建用立的Luminex微球技术来检测血小板抗体是可行的.结论 成功获取了重组表达的血小板膜蛋白GPⅢa,并初步建立了Luminex微球检测HPA-1a抗体方法.