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，以降低成本和 增加能力，但蛋白质组学技术在过去30到40年里并没有太大进步 好几年了。虽然存在许多生产重组亲和试剂的方法，但成本 和当前技术的吞吐量是开发 可再生亲和试剂的广泛来源。我们建议 开发一种技术，使我们能够筛选不同的M13噬菌体展示文库 抗&gt；104不同蛋白(和/或肽)抗原库的抗体显示在E. Coli细胞表面。F菌毛是大肠埃希菌M13感染的附着部位。在建议的 技术，细胞的噬菌体感染依赖于M13显示的抗体和 细菌细胞表面展示瞬时F-阴性大肠杆菌宿主在油包水中的抗原 乳剂。下一代DNA测序和多路条形码方案将用于 识别抗体：抗原对。我们将使用一组高价值的免疫肿瘤学靶点来 发展该方法，并与更传统的噬菌体展示获得的抗体进行比较 生物扫描技术。这项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.