Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, Texas, USA.
Sci Rep. 2012;2:807. doi: 10.1038/srep00807. Epub 2012 Nov 12.
A bottle-neck in recombinant antibody sandwich immunoassay development is pairing, demanding protein purification and modification to distinguish captor from tracer. We developed a simple pairing scheme using microliter amounts of E. coli osmotic shockates bearing site-specific biotinylated antibodies and demonstrated proof of principle with a single domain antibody (sdAb) that is both captor and tracer for polyvalent Marburgvirus nucleoprotein. The system could also host pairs of different sdAb specific for the 7 botulinum neurotoxin (BoNT) serotypes, enabling recognition of the cognate serotype. Inducible supE co-expression enabled sdAb populations to be propagated as either phage for more panning from repertoires or expressed as soluble sdAb for screening within a single host strain. When combined with streptavidin-g3p fusions, a novel transdisplay system was formulated to retrofit a semi-synthetic sdAb library which was mined for an anti-Ebolavirus sdAb which was immediately immunoassay ready, thereby speeding up the recombinant antibody discovery and utilization processes.
在重组抗体夹心免疫分析的开发中，瓶颈在于配对，需要进行蛋白质纯化和修饰，以区分捕获抗体和示踪抗体。我们开发了一种简单的配对方案，使用带有定点生物素化抗体的大肠杆菌渗透压休克产物，并用单域抗体（sdAb）进行了原理验证，该抗体既是多价马尔堡病毒核蛋白的捕获抗体，也是示踪抗体。该系统还可以容纳针对 7 种肉毒梭菌神经毒素（BoNT）血清型的不同 sdAb 对，从而能够识别同源血清型。诱导型 supE 共表达使 sdAb 群体能够作为噬菌体进行更多的淘选，或者在单个宿主菌株中作为可溶性 sdAb 进行筛选。与链霉亲和素-g3p 融合物结合后，制定了一种新型的转显示系统，用于改造半合成 sdAb 文库，该文库被挖掘出一种抗埃博拉病毒的 sdAb，可立即用于免疫分析，从而加快了重组抗体的发现和利用过程。
