重组血小板糖蛋白Ⅲa联合Luminex微球在人血小板同种抗原-1a抗体检测中的应用
[Application of recombinant GPⅢa combined Luminex beads for the detection of HPA-1a antibody].
作者信息
Tao Sudan, Liu Ying, He Yanming, Ying Yanling, He Ji, Zhu Faming
机构信息
Blood Center of Zhejiang Province, Key Laboratory of Blood Safety Research of the Ministry of Health, Key Laboratory of Blood Safety Research of Zhejiang Province, Hangzhou, Zhejiang 310006, China.
出版信息
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2017 Feb 10;34(1):40-44. doi: 10.3760/cma.j.issn.1003-9406.2017.01.009.
OBJECTIVE
To generate recombinant GPⅢa 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 GPⅢa 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 GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.
目的
制备重组糖蛋白Ⅲa（GPⅢa）作为人血小板同种抗原-1a（HPA-1a）抗原的替代来源，并将其与Luminex xMAP微球结合用于检测HPA-1a特异性同种抗体。
方法
扩增整合素β3（ITGB3）基因的完整编码区并与pcDNA3.1连接。将重组质粒转染至中国仓鼠卵巢（CHO）细胞，用G418筛选稳定表达的细胞。对表达的蛋白进行鉴定并与Luminex xMAP微球偶联，然后与血清样本反应。随后，向反应孔中加入藻红蛋白标记的抗种属IgG抗体，并在Luminex-100分析仪上测定中位荧光强度。
结果
DNA测序证实克隆的ITGB3基因为HPA-1aa。重组GPⅢa与Luminex xMAP微球偶联。Luminex微球法检测HPA-1a抗体的灵敏度为1/32稀释度（3.125 U/mL）。Luminex微球法可从检测血清中特异性鉴定HPA-1a抗体，结果与血小板抗原单克隆抗体特异性固定技术（MAIPA）一致。未观察到与含有人类白细胞抗原（HLA）、ABO及其他HPA抗体（HPA-3a和HPA-5b）的样本发生交叉反应。结果表明，用Luminex xMAP微球技术检测HPA抗体是可行的。
结论
成功获得重组GPⅢa，并用于建立基于Luminex技术的HPA抗体检测方法。
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