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Rapid Identification of Monospecific Monoclonal Antibodies Using a Human Proteome Microarray*

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To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His6 fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.

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This work was supported by National Institutes of Health Grants R41MH088008 (to S. B. and I. P.) and 1R01EY017015 (to S. B.), by National Institutes of Health Common Fund Grant U54RR020839 (to H. Z. and J. D. B.), and by a grant from the Puerto Rico Science and Technology Trust. Additional support was obtained from the Institute for Cell Engineering and the Fund for Medical Discovery, Johns Hopkins University School of Medicine, and the Puerto Rico Science and Technology Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.