Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany.
J Immunol Methods. 2013 May 31;391(1-2):72-80. doi: 10.1016/j.jim.2013.02.011. Epub 2013 Feb 27.
Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) β3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsβ3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems.
及时、准确地检测人类血小板抗原 1a（HPA-1a）同种异体抗体对新生儿同种免疫性血小板减少症（NAIT）的临床诊断至关重要。目前用于检测 HPA-1 同种异体抗体的抗原特异性检测方法在技术上对大多数诊断实验室来说非常复杂和繁琐。因此，我们设计并应用了展示 HPA-1a 或 HPA-1b 表位的重组可溶性（rs）β3 整合素来开发单抗原磁珠检测（SAMBA）。重组可溶性 HPA-1a 和 HPA-1b 在人胚肾 293（HEK293）细胞中以重组方式产生，并进行了差异标记。然后将重组可溶性蛋白固定在顺磁珠上，并通过酶联免疫吸附试验（ELISA）用于分析 HPA-1 同种异体抗体。我们使用来自 NAIT 患者的 7 份 HPA-1a 血清样本（n=7）、惰性血清和含有非 HPA-1a 抗体的血清来评估 SAMBA 的灵敏度和特异性。将 V5-His 或 GS-SBP-His 标签融合到 rsβ3 整合素上导致高产量表达。SAMBA 能够检测到 MAIPA 特异性固定血小板抗原试验（MAIPA）识别的所有 HPA-1a 和 -1b 同种异体抗体。没有观察到血清之间的交叉反应。在 MAIPA 中，7 份 HPA-1a 同种异体抗体阳性血清中有 2 份显示出弱到中度反应性，但在 SAMBA 中显示出强信号。SAMBA 提供了一种非常可靠的方法，用于检测 HPA-1 抗体，具有高特异性和灵敏度。这种简单、快速的检测方法可以适用于任何常规实验室，并可能适用于自动化系统。
