Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay
Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay
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DOI：
10.1016/j.omtm.2018.11.007
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发表时间：
2019-06-14
影响因子：
4.7
通讯作者：
Xiao, Weidong
中科院分区：
文献类型：
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作者：
Guo, Ping;Zhang, Junping;Xiao, Weidong
重组腺相关病毒(RAAV)已成为基础研究和人类基因治疗的成功载体。然而，抗甲型肝炎病毒衣壳蛋白的中和抗体可以消除甲型肝炎病毒对靶细胞的感染性，从而降低转导效率。AAV NAB缺失已成为参加基因治疗试验的患者的先决条件。然而，对AAV NAB的准确评估仍然是一项具有挑战性的任务。在此，我们建立了一种基于以下观察结果的快速检测方法：AAV NAB抑制rAAV与宿主细胞表面的结合，并且NAB滴度与AAV基因组与靶细胞结合的数量呈负相关。通过在有抗血清存在的情况下对靶细胞上的AAV基因组进行定量，可以准确地确定AAVNAB滴度。用这种方法测定的效价与经典的基于转导的分析方法有很好的相关性。这种方法的一个主要优点是，它可以用30分钟的结合分析来进行，而不需要漫长的等待转导结果。该检测与AAV血清型在靶细胞中的转导性能无关。因此，AAV细胞结合法检测NAB为体内NAB检测提供了一种新的方法。
Recombinant adeno-associated virus (rAAV) has been developed as a successful vector for both basic research and human gene therapy. However, neutralizing antibodies (NAbs) against AAV capsids can abolish AAV infectivity on target cells, reducing the transduction efficacy. Absence of AAV NAb has become a prerequisite qualification for patients enrolled in gene therapy trials. Nevertheless, accurate assessment of AAV NAb has remained a challenging task. Here we developed a rapid assay based on the observations that AAV NAb inhibits rAAV binding to the host cell surface and NAb titers are negatively related to the amount of AAV genomes binding to the target cells. By quantifying the AAV genome on the target cells in the presence of anti-sera, AAV NAb titers can be accurately determined. The titer determined by this assay correlates well with the classical transduction-based assays. A major advantage of this method is that it can be carried out with a 30-min binding assay without the lengthy wait for a transduction outcome. This assay is independent of transduction performance of AAV serotype in the target cells. Therefore, the AAV cell-binding assay for NAb determination offers an alternative method for in vivo NAb assay.