Rapid Identification of Monospecific Monoclonal Antibodies Using a Human Proteome Microarray

• Published in: Molecular & cellular proteomics Vol. 11; no. 6; p. O111.016253
• Main Authors: Jeong, Jun Seop, Jiang, Lizhi, Albino, Edisa, Marrero, Josean, Rho, Hee Sool, Hu, Jianfei, Hu, Shaohui, Vera, Carlos, Bayron-Poueymiroy, Diane, Rivera-Pacheco, Zully Ann, Ramos, Leonardo, Torres-Castro, Cecil, Qian, Jiang, Bonaventura, Joseph, Boeke, Jef D., Yap, Wendy Y., Pino, Ignacio, Eichinger, Daniel J., Zhu, Heng, Blackshaw, Seth
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-06-2012; The American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Antibodies, Monoclonal, Murine-Derived - immunology; Antibody Specificity; Antigens - chemistry; Antigens - immunology; Cell Line, Tumor; Humans; Hybridomas; Immobilized Proteins - chemistry; Immobilized Proteins - immunology; Mice; Mice, Inbred BALB C; Protein Array Analysis; Proteome - chemistry; Proteome - immunology; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - immunology
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.O111.016253

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.