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Mol Cell Proteomics. 2012 Jun;11(6):O111.016253. doi: 10.1074/mcp.O111.016253. Epub 2012 Feb 3.

Rapid identification of monospecific monoclonal antibodies using a human proteome microarray.

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1
Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Abstract

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.

PMID:
22307071
PMCID:
PMC3433917
DOI:
10.1074/mcp.O111.016253
[Indexed for MEDLINE]
Free PMC Article
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