Hussack Greg, Baral Toya Nath, Baardsnes Jason, van Faassen Henk, Raphael Shalini, Henry Kevin A, Zhang Jianbing, MacKenzie C Roger
Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada.
Human Health Therapeutics Research Centre, National Research Council Canada, Montréal, QC, Canada.
Front Immunol. 2017 Oct 30;8:1406. doi: 10.3389/fimmu.2017.01406. eCollection 2017.
ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.
ABTAG是一种骆驼科单域抗体(sdAb),它以低皮摩尔亲和力与牛血清白蛋白(BSA)结合。在使用BSA表面的表面等离子体共振(SPR)分析中,结合的ABTAG在低pH下可在多个循环中从BSA表面完全解离,而BSA表面结合ABTAG的能力不会有任何降低。本文描述了一种基于SPR的中等通量抗体筛选测定法,该方法利用了ABTAG的独特特性。从用癌胚抗原相关细胞粘附分子6(CEACAM6)免疫的羊驼重链抗体库衍生的噬菌体展示sdAb文库中分离出抗CEACAM6 sdAb。经过一轮或两轮淘选后,富集的克隆在微量滴定板培养物中表达为ABTAG融合蛋白。培养上清液中的sdAb-ABTAG融合蛋白被捕获在BSA表面,然后将CEACAM6抗原与捕获的分子结合。SPR筛选方法可读出融合蛋白的相对表达水平以及捕获分子与CEACAM6结合的动力学和亲和力常数。该文库也通过传统方法进行淘选和筛选,阳性克隆被亚克隆并表达用于SPR分析。与传统的淘选和筛选相比,基于SPR的ABTAG方法产生的结合物多样性要高得多,其中一些结合物的亲和力比通过传统淘选鉴定的结合物高三个数量级。