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Rapid Identification of Monospecific Monoclonal Antibodies Using a Human Proteome Microarray

2012; Elsevier BV; Volume: 11; Issue: 6 Linguagem: Inglês

10.1074/mcp.o111.016253

1535-9484

Jun Seop Jeong, Lizhi Jiang, Edisa Albino, Josean Marrero, Hee Sool Rho, Jianfei Hu, Shaohui Hu, Carlos Vera, Diane Bayron-Poueymiroy, Zully Ann Rivera-Pacheco, Leonardo Ramos, Cecil Torres-Castro, Jiang Qian, Joseph Bonaventura, Jef D. Boeke, W Y Yap, Ignacio Pino, Daniel Eichinger, Heng Zhu, Seth Blackshaw,

Glycosylation and Glycoproteins Research

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-His 6 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.