Development of Two Avidity-Based Assays to Detect Recent HIV Type 1 Seroconversion Using a Multisubtype gp41 Recombinant Protein
Development of Two Avidity-Based Assays to Detect Recent HIV Type 1 Seroconversion Using a Multisubtype gp41 Recombinant Protein
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DOI：
10.1089/aid.2009.0133
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发表时间：
2010-01-01
影响因子：
1.5
通讯作者：
Parekh, Bharat S.
中科院分区：
文献类型：
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作者：
Wei, Xierong;Liu, Xin;Parekh, Bharat S.
目前检测近期HIV-1感染以估计发病率的实验室方法有各种局限性，包括在不同亚型或人群中的不同性能。因此，需要新的方法来检测具有更高特异性的近期感染。我们开发了一种重组蛋白，rIDR-M，它涵盖了不同的序列从gp 41的免疫显性区域（IDR）的所有主要亚型和重组的HIV-1 M组和大肠杆菌中表达。rIDR-M蛋白与来自不同亚型的血清中的HIV抗体具有高度反应性，并且与来自相应亚型的单个gp 41肽相反，可等效地检测来自泰国的不同亚型B和AE的抗体，这表明其可用于发病率测定。该蛋白以两种不同的测定形式用于测量抗体亲合力：（1）双孔亲合力指数测定（AI-EIA）和（2）新的单孔限制性抗原亲合力测定（LAg-亲合力EIA），两者均使用pH 3.0缓冲液以解离早期感染期间存在的低亲合力抗体。限制抗原的量可以在有或没有解离缓冲液的情况下检测最近的HIV-1感染，但当包括pH 3.0解离缓冲液时，检测效率最高。当使用充分表征的41个成员的血清发病率组（20个近期和21个长期）时，双孔AI-EIA和单孔LAg-亲合力EIA都有效地区分近期和长期感染。使用rIDR-M抗原的新的基于亲合力的检测方法可以提高检测近期HIV-1感染的准确性，并可以更好地估计不同HIV-1亚型的发病率。
Current laboratory methods to detect recent HIV-1 infection for the estimation of incidence have various limitations, including varying performance in different subtypes or populations. Therefore, new methods are needed to detect recent infections with increased specificity. We developed a recombinant protein, rIDR-M, that covered divergent sequences from the immunodominant region (IDR) of gp41 from all major subtypes and recombinants of HIV-1 group M and expressed in Escherichia coli. The rIDR-M protein was highly reactive with HIV antibodies in sera from different subtypes and equivalently detected antibodies to divergent subtypes B and AE from Thailand, in contrast to individual gp41 peptides derived from respective subtypes, suggesting that it can be used for incidence assays. The protein was used in two different assay formats to measure antibody avidity: (1) a two-well avidity index assay (AI-EIA) and (2) a new one-well limiting antigen avidity assay (LAg-avidity EIA), both with a pH 3.0 buffer to dissociate low-avidity antibodies present during early infection. Limiting the amount of antigen allowed detection of recent HIV-1 infection, with or without dissociation buffer, but the detection was most efficient when the pH 3.0 dissociation buffer was included. When a well-characterized 41-member seroincidence panel (20 recent and 21 long-term) was used, both the two-well AI-EIA and one-well LAg-avidity EIA efficiently distinguished recent and long-term infections. The new avidity-based assays using rIDR-M antigen may improve the accuracy of detecting recent HIV-1 infection and allow a better estimation of incidence in diverse HIV-1 subtypes.