Rapid identification of monospecific monoclonal antibodies using a human proteome microarray.
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.
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Related Subject Headings
- Recombinant Fusion Proteins
- Proteome
- Protein Array Analysis
- Mice, Inbred BALB C
- Mice
- Immobilized Proteins
- Hybridomas
- Humans
- Cell Line, Tumor
- Biochemistry & Molecular Biology
Citation
Published In
DOI
EISSN
ISSN
Publication Date
Volume
Issue
Start / End Page
Related Subject Headings
- Recombinant Fusion Proteins
- Proteome
- Protein Array Analysis
- Mice, Inbred BALB C
- Mice
- Immobilized Proteins
- Hybridomas
- Humans
- Cell Line, Tumor
- Biochemistry & Molecular Biology