高通量血清学 HPA-1a 分型的快速、低成本直接 ELISA 方法

Fast and low-cost direct ELISA for high-throughput serological HPA-1a typing.

机构信息

Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands.

Department of Experimental Immunohematology, Sanquin, Amsterdam, The Netherlands.

出版信息

Transfusion. 2019 Sep;59(9):2989-2996. doi: 10.1111/trf.15454. Epub 2019 Jul 22.

DOI:10.1111/trf.15454

PMID:31329320

Abstract

BACKGROUND

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay.

STUDY DESIGN AND METHODS

A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500.

RESULTS

The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03.

CONCLUSION

A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions.

摘要

背景

胎儿和新生儿同种免疫性血小板减少症（FNAIT）是由母体针对胎儿人类血小板抗原（HPAs）的同种抗体引起的，主要由抗-HPA-1a 引起。基于人群的 FNAIT 筛查仍然是一个有争议的话题。在后勤和财务方面，实施的主要挑战是对孕妇进行分型，以识别 2%的 HPA-1a 阴性女性。因此，需要一种高通量且低成本的 HPA-1a 分型检测方法。

研究设计和方法

开发了一种夹心 ELISA，使用单克隆抗-GPIIIa 作为包被抗体，辣根过氧化物酶标记的重组抗-HPA-1a 作为检测抗体。将 ELISA 结果与等位基因鉴别 PCR 检测方法进行比较。在第一阶段，使用两种检测方法对未经选择的连续孕妇样本进行了测试。第二阶段是妊娠前瞻性筛查研究的一部分，仅对任意 OD 值<0.500 的样本进行基因分型。

结果

ELISA 进行了优化，无需对储存管进行额外的处理（搅拌或旋转）。在第一阶段，测试了 506 个样本。在第二阶段，对另外 62171 个连续样本进行了表型分析，并在 1902 个样本中进行了支持性基因分型。总共对 1585 名 HPA-1a 阴性和 823 名 HPA-1a 阳性女性进行了基因分型。该检测方法的截止 OD 值为 0.160 时，灵敏度达到 100%，特异性为 99.9%，假 HPA-1a 阴性率为 0.03。