Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin
AuthorAguilar, OA; Tanaka, M; Balaji, GR; Berry, R; Rossjohn, J; Lanier, LL; Carlyle, JR
Source TitleJournal of Immunology
PublisherAMER ASSOC IMMUNOLOGISTS
University of Melbourne Author/sRossjohn, Jamie
AffiliationMicrobiology and Immunology
Document TypeJournal Article
CitationsAguilar, O. A., Tanaka, M., Balaji, G. R., Berry, R., Rossjohn, J., Lanier, L. L. & Carlyle, J. R. (2020). Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin. JOURNAL OF IMMUNOLOGY, 205 (6), pp.1709-1717. https://doi.org/10.4049/jimmunol.2000687.
Access StatusAccess this item via the Open Access location
The generation of reliable mAb of unique and desired specificities serves as a valuable technology to study protein expression and function. However, standard approaches to mAb generation usually involve large-scale protein purification and intensive screening. In this study, we describe an optimized high-throughput proof-of-principle method for the expanded generation, enrichment, and screening of mouse hybridomas secreting mAb specific for a protein of interest. Briefly, we demonstrate that small amounts of a biotinylated protein of interest can be used to generate tetramers for use as prime-boost immunogens, followed by selective enrichment of Ag-specific B cells by magnetic sorting using the same tetramers prior to hybridoma generation. This serves two purposes: 1) to effectively expand both low- and high-affinity B cells specific for the antigenic bait during immunization and 2) to minimize subsequent laborious hybridoma efforts by positive selection of Ag-specific, Ab-secreting cells prior to hybridoma fusion and validation screening. Finally, we employ a rapid and inexpensive screening technology, CELLISA, a high-throughput validation method that uses a chimeric Ag fused to the CD3ζ signaling domain expressed on enzyme-generating reporter cells; these reporters can detect specific mAb in hybridoma supernatants via plate-bound Ab-capture arrays, thereby easing screening. Using this strategy, we generated and characterized novel mouse mAb specific for a viral immunoevasin, the mouse CMV m12 protein, and suggest that these mAb may protect mice from CMV infection via passive immunity.
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