High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses.
High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses.
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HIV-1和SIV特异性ADCC抗体反应的高通量定量分析。
DOI：
10.1002/cyto.a.21084
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发表时间：
2011-08
期刊：
影响因子：
3.7
通讯作者：
Ferrari, Guido
中科院分区：
文献类型：
--
作者：
Pollara, Justin;Hart, Lydia;Brewer, Faraha;Pickeral, Joy;Packard, Beverly Z.;Hoxie, James A.;Komoriya, Akira;Ochsenbauer, Christina;Kappes, John C.;Roederer, Mario;Huang, Ying;Weinhold, Kent J.;Tomaras, Georgia D.;Haynes, Barton F.;Montefiori, David C.;Ferrari, Guido
我们已经开发了一个高通量平台来检测HIV-1和SIV特异性ADCC介导的抗体应答的存在。该测定基于细胞可渗透荧光肽底物的水解，该底物含有丝氨酸蛋白酶粒酶B（Gz B）识别的序列。由于抗原（Ag）特异性Ab-Fcγ受体相互作用，GzB通过细胞毒性效应细胞递送至靶细胞。在靶细胞内，效应细胞衍生的GzB水解底物，产生荧光信号，允许通过流式细胞术鉴定已接受致死性打击的单个靶细胞。结果报告为具有GzB活性的靶细胞的百分比（%GzB）。新鲜分离或冷冻保存的PBMC和/或NK细胞可用作效应细胞。在（i）用重组包膜糖蛋白包被，（ii）用表达原代和实验室适应病毒的Env抗原的感染性分子克隆感染，或（iii）用HIV-1/IIIB变体（称为A1953）慢性感染后，将表达CCR 5共受体的NKR细胞用作靶细胞。此外，体外感染HIV-1的原代CD 4 + T细胞也可用作靶点。该测定具有高度重现性，变异系数小于25%。在不存在血清/血浆的情况下，使用靶细胞和效应细胞群计算背景（8.6±2.3%）。我们确定，检测人和非人灵长类动物样本分别需要1：50和1：100的初始稀释度。该检测允许快速定量HIV-1或SIV特异性ADCC介导的抗体，这些抗体是在接种疫苗后或感染的自然过程中产生的，从而为研究人员提供了一种新的方法，用于研究ADCC介导的抗体作为HIV-1和SIV感染的控制或预防相关物的作用。
We have developed a high throughput platform to detect the presence of HIV-1 and SIV-specific ADCC-mediating antibody responses. The assay is based on the hydrolysis of a cell-permeable fluorogenic peptide substrate containing a sequence recognized by the serine protease, Granzyme B (GzB). GzB is delivered into target cells by cytotoxic effector cells as a result of antigen (Ag)-specific Ab-Fcγ receptor interactions. Within the target cells, effector cell-derived GzB hydrolyzes the substrate, generating a fluorescent signal that allows individual target cells that have received a lethal hit to be identified by flow cytometry. Results are reported as the percentage of target cells with GzB activity (%GzB). Freshly isolated or cryopreserved PBMC and/or NK cells can be used as effector cells. CEM.NKR cells expressing the CCR5 co-receptor are used as a target cells following (i) coating with recombinant envelope glycoprotein, (ii) infection with infectious molecular clones expressing the Env antigens of primary and lab adapted viruses, or (iii) chronic infection with a variant of HIV-1/IIIB, termed A1953. In addition, primary CD4+ T cells infected with HIV-1 in vitro can also be used as targets. The assay is highly reproducible with a coefficient of variation of less than 25%. Target and effector cell populations, in the absence of serum/plasma, were used to calculate background (8.6±2.3%). We determined that an initial dilution of 1:50 and 1:100 is required for testing of human and non-human primate samples, respectively. This assay allows for rapid quantification of HIV-1 or SIV-specific ADCC-mediating antibodies that develop in response to vaccination, or in the natural course of infection, thus providing researchers with a new methodology for investigating the role of ADCC-mediating antibodies as correlates of control or prevention of HIV-1 and SIV infection.
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影响因子：
3.7
作者：
Edmonds TG;Ding H;Yuan X;Wei Q;Smith KS;Conway JA;Wieczorek L;Brown B;Polonis V;West JT;Montefiori DC;Kappes JC;Ochsenbauer C
通讯作者：
Ochsenbauer C
影响因子：
5.4
作者：
Hessell, Ann J.;Rakasz, Eva G.;Burton, Dennis R.
通讯作者：
Burton, Dennis R.
影响因子：
4.4
作者：
Packard, Beverly Z.;Telford, William G.;Henkart, Pierre A.
通讯作者：
Henkart, Pierre A.
影响因子：
6.4
作者：
LJUNGGREN, K;MOSCHESE, V;JONDAL, M
通讯作者：
JONDAL, M
影响因子：
6.4
作者：
Kohl, S;Charlebois, ED;Burke, RL
通讯作者：
Burke, RL