Improved Detection of HIV Gag p24 Protein Using a Combined Immunoprecipitation and Digital ELISA Method.
Improved Detection of HIV Gag p24 Protein Using a Combined Immunoprecipitation and Digital ELISA Method.
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DOI：
10.3389/fmicb.2021.636703
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发表时间：
2021
影响因子：
5.2
通讯作者：
Zuck P
中科院分区：
文献类型：
--
作者：
Wu G;Cheney C;Huang Q;Hazuda DJ;Howell BJ;Zuck P
超过90%的HIV-1前病毒被认为是有缺陷的，不能进行病毒复制。虽然有复制能力的前病毒是疾病进展或传播的主要关注点，但研究表明，即使是有缺陷的前病毒也不是沉默的，并且可以产生病毒蛋白，这可能有助于炎症和免疫应答。病毒蛋白表达也对基于免疫的HIV-1清除策略有影响，该策略依赖于抗原识别。因此，需要旨在定量复制能力的前病毒和有缺陷的但具有复制能力的前病毒的灵敏测定，以了解病毒蛋白对HIV-1发病机制的贡献，并确定HIV-1治愈干预措施的有效性。以前，我们报告了一种改进的HIV-1 gag p24数字酶联免疫吸附试验与单分子阵列（Simoa）检测细胞相关的病毒蛋白。在这里，我们报告了一种新的p24蛋白富集方法与数字免疫分析相结合，以进一步扩展病毒蛋白检测的灵敏度和特异性。免疫捕获HIV gag p24，然后以Simoa兼容的格式洗脱，导致更高的蛋白质回收率和更低的背景，从各种生物基质和样品体积。从ART抑制的HIV参与者的外周血或组织中分离的细胞的细胞裂解物中，以及猿猴-人免疫缺陷病毒感染的非人灵长类动物（NHP）中，低至1 fg的p24蛋白的定量，具有高回收率和重现性。将这些增强的方法应用于患者来源的样品有可能进一步研究持续的HIV状态，并检查对治疗的体外反应，以及来自各种供体的免疫活性细胞的离体研究。
Greater than 90% of HIV-1 proviruses are thought to be defective and incapable of viral replication. While replication competent proviruses are of primary concern with respect to disease progression or transmission, studies have shown that even defective proviruses are not silent and can produce viral proteins, which may contribute to inflammation and immune responses. Viral protein expression also has implications for immune-based HIV-1 clearance strategies, which rely on antigen recognition. Thus, sensitive assays aimed at quantifying both replication-competent proviruses and defective, yet translationally competent proviruses are needed to understand the contribution of viral protein to HIV-1 pathogenesis and determine the effectiveness of HIV-1 cure interventions. Previously, we reported a modified HIV-1 gag p24 digital enzyme-linked immunosorbent assay with single molecule array (Simoa) detection of cell-associated viral protein. Here we report a novel p24 protein enrichment method coupled with the digital immunoassay to further extend the sensitivity and specificity of viral protein detection. Immunocapture of HIV gag p24 followed by elution in a Simoa-compatible format resulted in higher protein recovery and lower background from various biological matrices and sample volumes. Quantification of as little as 1 fg of p24 protein from cell lysates from cells isolated from peripheral blood or tissues from ART-suppressed HIV participants, as well as simian–human immunodeficiency virus–infected non-human primates (NHPs), with high recovery and reproducibility is demonstrated here. The application of these enhanced methods to patient-derived samples has potential to further the study of the persistent HIV state and examine in vitro response to therapies, as well as ex vivo study of translationally competent cells from a variety of donors.
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影响因子：
4.1
作者：
Evans DT;Silvestri G
通讯作者：
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