HLA Antibody Specification Using Single-Antigen Beads-A Technical Solution for the Prozone Effect
HLA Antibody Specification Using Single-Antigen Beads-A Technical Solution for the Prozone Effect
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DOI：
10.1097/tp.0b013e31822872dd
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发表时间：
2011-09-01
期刊：
影响因子：
6.2
通讯作者：
Wernet, Dorothee
中科院分区：
文献类型：
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作者：
Schnaidt, Martina;Weinstock, Christof;Wernet, Dorothee
背景。 Luminex 单抗原珠 (SAB) 检测的引入在人类白细胞抗原抗体规范方面取得了实质性进展。当事实证明这种方法容易产生前带效应，导致在高抗体滴度的情况下出现假阴性结果时，这一进展受到了损害。平行检测一名患者的血清和乙二胺四乙酸（EDTA）血浆，仅用血清样本观察前带效应。这促使我们研究补体成分 1 (C1) 作为 SAB 测试中前带的原因。我们还找到了一种克服前带效应的简单方法。在SAB测定中测试具有前带效应的血清，应用不同的血清预处理方法来探索导致前带效应的参数。结果。检测EDTA血浆或添加EDTA的血清时，未观察到前带。此外，添加二硫苏糖醇、添加C1抑制剂或血清热灭活消除了前带效应。将新鲜的非免疫血清添加到热灭活的血清中恢复了前带效应。仅发现显示前带的珠子被 C1q 覆盖。结论。我们的观察结果与补体 C1 解离或破坏消除前带效应的假设一致。在高度免疫患者的血清中添加 EDTA 是避免因前带效应引起的 SAB 检测假阴性结果的最简单方法。
Background. Substantial progress in human leukocyte antigen antibody specification has been made by the introduction of Luminex single-antigen bead (SAB) assays. This progress was impaired when it turned out that this method is prone to a prozone effect leading to false-negative results in the case of high antibody titers. Testing serum and ethylenediaminetetraacetic acid (EDTA) plasma of one patient in parallel, we observed the prozone effect with the serum sample only. This led us to investigate complement component 1 (C1) as the cause of the prozone in SAB testing. We also found an easy way to overcome the prozone effect.Methods. Sera with a prozone effect were tested in the SAB assay, applying different methods of serum pretreatment to explore the parameters leading to the prozone.Results. The prozone was not observed when EDTA plasma or serum with EDTA added were tested. Further, addition of dithiothreitol, addition of C1 inhibitor, or heat inactivation of the sera abolished the prozone effect. Adding fresh nonimmune serum to heat-inactivated sera restored the prozone effect. Only beads showing a prozone were found to be covered with C1q.Conclusion. Our observations are consistent with the hypothesis that dissociation or destruction of complement C1 eliminates the prozone effect. Addition of EDTA to serum of highly immunized patients is the easiest way to avoid false-negative results in SAB testing caused by a prozone effect.