Recombinational biopanning method for multiplex antibody:antigen screening

摘要 DNA测序技术已经取得了巨大的进步，以降低成本， 增加能力，但蛋白质组学技术在过去的30至40年中并没有太大的进步， 年虽然存在许多用于产生重组亲和试剂的方法，但是成本昂贵。 和吞吐量的当前技术的发展是重大的障碍， 可再生亲和试剂的全面和广泛可用的资源。我们建议 开发一种技术，使我们能够筛选M13噬菌体文库，展示> 1010种不同的 针对在E. coli细胞表面。F菌毛是M13感染E.杆菌拟议 在该技术中，细胞的噬菌体感染依赖于M13展示的抗体和 细菌细胞表面展示的瞬时F-减E的抗原。油包水型大肠杆菌宿主 乳剂下一代DNA测序和多重条形码方案将用于 鉴定抗体：抗原对。我们将使用一组高价值的免疫肿瘤学靶点， 开发该方法并与用更传统的噬菌体展示获得的抗体进行比较 生物淘选技术该技术本身应该适用于任何生物体，包括 病毒、细菌和人类蛋白质组，以及单一和混合群体的蛋白质组。

ABSTRACT There has been tremendous progress in DNA sequencing technology to decrease cost and increase capacity, yet technologies for proteomics have not advanced much in the last 30 to 40 years. While a number of approaches for generating recombinant affinity reagents exist, the cost and throughput of current technologies represent significant roadblocks to the development of a comprehensive and broadly available resource of renewable affinity reagents. We propose to develop a technology that will allow us to screen a library of M13 phage displaying >1010 different antibodies against a library of >104 different protein (and/or peptide) antigens displayed on an E. coli cell surface. The F-pilus is the attachment site for M13 infection of E. coli. In the proposed technology, phage infection of the cells relies on the interaction of an M13-displayed antibody and the bacterial cell surface-displayed antigen of a transiently-F-minus E. coli host in water-in-oil emulsions. NextGeneration DNA sequencing and a multiplex barcoding scheme will be used to identify antibody:antigen pairs. We will use a set of high-value, immuno-oncology targets to develop the method and compare to antibodies obtained with more traditional phage display biopanning techniques. The technology itself should be applicable to any organism, including viral, bacterial and human proteomes, as well as single- and mixed-populations of proteomes.