Recombinational biopanning method for multiplex antibody:antigen screening

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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.