MSD應(yīng)用案例：艾滋病疫苗開(kāi)發(fā)，多因子技術(shù)檢測(cè)抗體特異性

 

中和性抗體是由血液中獲得性免疫細(xì)胞暴露于病毒后產(chǎn)生的一類(lèi)可溶性蛋白。在血液中，它們能與病毒結(jié)合，阻止其進(jìn)一步感染人體細(xì)胞，并可導(dǎo)致病毒顆粒裂解，引起“中和”反應(yīng)。由于中和性抗體可以在病毒感染人體細(xì)胞之前將其“消滅”，因此，如果此類(lèi)抗體在人體暴露于HIV之前就存在，將可以預(yù)防感染發(fā)生。只有少數(shù)HIV陽(yáng)性病人在感染HIV病毒數(shù)年后能產(chǎn)生強(qiáng)效廣譜中和性抗體。動(dòng)物實(shí)驗(yàn)顯示，如果一種預(yù)防性疫苗能引發(fā)這些抗體，就能阻止HIV感染。PG9和PG16是科學(xué)家首次發(fā)現(xiàn)的強(qiáng)效HIV廣譜性抗體，可以與HIV-1中的一個(gè)糖蛋白(gp120)特定表位結(jié)合，阻止HIV-1感染人體細(xì)胞。由于傳統(tǒng)ELISA和病毒中和實(shí)驗(yàn)并不能解決接種疫苗動(dòng)物產(chǎn)生的抗體特異性。為鑒定表位特異性，Hoffenberg等在MSD的多因子微孔板內(nèi)點(diǎn)了PG9, PGT126, PGV04, b6, 17b, and 2F5等抗體。這是基于Meso Scale Discovery (MSD)技術(shù)開(kāi)發(fā)了一種競(jìng)爭(zhēng)結(jié)合（competition-binding assay）方法，用以檢測(cè)接種疫苗后的動(dòng)物血清是否含有識(shí)別表位的抗體。在Journal of Virology ，有多篇文章應(yīng)用和開(kāi)發(fā)Meso Scale Discovery (MSD)電化學(xué)發(fā)光技術(shù)。

 

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.

 

 

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.  J Virol. 2013 May;87(10):5372-83. doi: 10.1128/JVI.02827-12. Epub 2013 Mar 6.

 

有關(guān)Meso Scale Discovery （MSD）公司：Amgen的創(chuàng)始人之一Sam Wohlstadter投資了MSD。美國(guó)MSD成立于1995年，在2009年全球首次檢測(cè)到人攜帶甲型H1N1病毒，在2013年被評(píng)為“全球前五的免疫分析品牌”。其研發(fā)的基于微孔板的電化學(xué)發(fā)光檢測(cè)技術(shù)（簡(jiǎn)稱(chēng)：微孔板ECL ）是全面升級(jí)/替代ELISA， Western Blot，多蛋白因子檢測(cè)的第三代免疫分析技術(shù)。已經(jīng)廣泛被醫(yī)藥研發(fā)中心、藥物安全性評(píng)價(jià)中心、GLP實(shí)驗(yàn)室、合同外包企業(yè)（CRO）、診斷、生物技術(shù)企業(yè)應(yīng)用于藥物篩選，動(dòng)物模型，藥物代謝，血藥濃度，免疫原性，中和實(shí)驗(yàn)，Biomarker篩選，抗體親和力，疫苗評(píng)估，宿主細(xì)胞殘留，診斷研究。