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[ MSD 应用案例] 病毒学顶级杂志Journal of Virology :艾滋病疫苗开发,多因子技术检测抗体特异性
您是第位阅读本文  发布时间:2011-5-6 15:40:42

中和性抗体是由血液中获得性免疫细胞暴露于病毒后产生的一类可溶性蛋白。在血液中,它们能与病毒结合,阻止其进一步感染人体细胞,并可导致病毒颗粒裂解, 引起“中和”反应。由于中和性抗体可以在病毒感染人体细胞之前将其“消灭”,因此,如果此类抗体在人体暴露于HIV之前就存在,将可以预防感染发生。只有 少数HIV阳性病人在感染HIV病毒数年后能产生强效广谱中和性抗体。动物实验显示,如果一种预防性疫苗能引发这些抗体,就能阻止HIV感染。PG9和 PG16是科学家首次发现的强效HIV广谱性抗体,可以与HIV-1中的一个糖蛋白(gp120)特定表位结合,阻止HIV-1感染人体细胞。由于传统 ELISA和病毒中和实验并不能解决接种疫苗动物产生的抗体特异性。为鉴定表位特异性,Hoffenberg等在MSD的多因子微孔板内点了PG9, PGT126, PGV04, b6, 17b, and 2F5等抗体。这是基于Meso Scale Discovery (MSD)技术开发了一种竞争结合(competition-binding assay)方法,用以检测接种疫苗后的动物血清是否含有识别表位的抗体。在Journal of Virology ,有多篇文章应用和开发Meso Scale Discovery (MSD)电化学发光技术。

MSD多因子竞争抗体结合:
Multiplexed competition antibody binding assay.To determine epitope specificity, we developed a competition-binding assay (CBA) based on Meso Scale Discovery (MSD) Sector Imager technology. MSD uses electrochemiluminescence (ECL) to detect binding events in a unique and sensitive manner with a _4-log dynamic range. The goal of the CBA is to determine whether sera from vaccinated animals contain Abs recognizing epitopes similar to those bound by well-characterized Env-specific Abs. The Abs PG9, PGT126, PGV04, b6, 17b, and 2F5 were each spotted onto MSD multiarrays. Dilutions of sera were preincubated for 1 h at 37°C with an excess of soluble biotinylated BG505 L111A gp120 or with a clade C gp120 chimera based on isolate 16096 with a V1 to V3 loop substitution from subtype 16055, a clade C isolate that we term 16936-V55. This mixture was then transferred to an MSD plate blocked with PBS containing 3% BSA and incubated for 1 h at room temperature. Plates were washed with PBS containing 0.02% Tween 20 before incubation with a streptavidin-Sulfo tag reporter reagent. Plates were read using the Sector Imager 2400. A reduction in signal reflects the presence of Abs in serum that competed with spotted Abs for binding to gp120. An advantage of this assay configuration is that it detects binding to soluble antigen, which may possess a more native structure compared with plate-bound antigen.

实验结果:


原文:Hoffenberg S, Powell R, Carpov A, Wagner D, Wilson A, Kosakovsky Pond S, Lindsay R, Arendt H, Destefano J, Phogat S, Poignard P, Fling SP, Simek M, Labranche C, Montefiori D, Wrin T, Phung P, Burton D, Koff W, King CR, Parks CL, Caulfield MJ. Identification of an HIV-1 clade A envelope that exhibits broad antigenicity and neutralization sensitivity and elicits antibodies targeting three distinct epitopes.  J Virol. 2013 May;87(10):5372-83. doi: 10.1128/JVI.02827-12. Epub 2013 Mar 6.
有关Meso Scale Discovery (MSD)公司:Amgen的创始人之一Sam Wohlstadter投资了MSD。美国MSD成立于1995年,在2009年全球首次检测到人携带甲型H1N1病毒,在2013年被评为“全球前五的 免疫分析品牌”。其研发的基于微孔板的电化学发光检测技术(简称:微孔板ECL )是全面升级/替代ELISA, Western Blot,多蛋白因子检测的第三代免疫分析技术。已经广泛被医药研发中心、药物安全性评价中心、GLP实验室、合同外包企业(CRO)、诊断、生物技术企 业应用于药物筛选,动物模型,药物代谢,血药浓度,免疫原性,中和实验,Biomarker筛选,抗体亲和力,疫苗评估,宿主细胞残留,诊断研究。

>>>查询Journal of Virology 上发表的“Meso Scale Discovery”(点击此处)<<<。



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