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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 CD3\u03b6 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.<\/jats:p>","DOI":"10.4049\/jimmunol.2000687","type":"journal-article","created":{"date-parts":[[2020,8,19]],"date-time":"2020-08-19T16:05:13Z","timestamp":1597853113000},"page":"1709-1717","source":"Crossref","is-referenced-by-count":3,"title":["Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin"],"prefix":"10.1093","volume":"205","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7990-8745","authenticated-orcid":false,"given":"Oscar A","family":"Aguilar","sequence":"first","affiliation":[{"name":"Department of Immunology, University of Toronto , Toronto, Ontario M5S 1A8 ,","place":["Canada"]},{"name":"Department of Microbiology and Immunology, University of California , San Francisco, San Francisco, CA 94143"},{"name":"Parker Institute for Cancer Immunotherapy, University of California , San Francisco, San Francisco, CA 94143"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miho","family":"Tanaka","sequence":"additional","affiliation":[{"name":"Department of Immunology, University of Toronto , Toronto, Ontario M5S 1A8 ,","place":["Canada"]},{"name":"Department of Microbiology and Immunology, University of California , San Francisco, San Francisco, CA 94143"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7436-4689","authenticated-orcid":false,"given":"Gautham R","family":"Balaji","sequence":"additional","affiliation":[{"name":"Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University , Clayton, Victoria 3800 ,","place":["Australia"]},{"name":"Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University , Clayton, Victoria 3800 ,","place":["Australia"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard","family":"Berry","sequence":"additional","affiliation":[{"name":"Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University , Clayton, Victoria 3800 ,","place":["Australia"]},{"name":"Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University , Clayton, Victoria 3800 ,","place":["Australia"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2020-7522","authenticated-orcid":false,"given":"Jamie","family":"Rossjohn","sequence":"additional","affiliation":[{"name":"Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University , Clayton, Victoria 3800 ,","place":["Australia"]},{"name":"Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University , Clayton, Victoria 3800 ,","place":["Australia"]},{"name":"Institute of Infection and Immunity, Cardiff University School of Medicine , Cardiff CF14 4XN ,","place":["United Kingdom"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1308-3952","authenticated-orcid":false,"given":"Lewis L","family":"Lanier","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of California , San Francisco, San Francisco, CA 94143"},{"name":"Parker Institute for Cancer Immunotherapy, University of California , San Francisco, San Francisco, CA 94143"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James R","family":"Carlyle","sequence":"additional","affiliation":[{"name":"Department of Immunology, University of Toronto , Toronto, Ontario M5S 1A8 ,","place":["Canada"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2020,9,15]]},"reference":[{"key":"2026041420283424800_r1","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1038\/nri2744","article-title":"Monoclonal antibodies: versatile platforms for cancer immunotherapy","volume":"10","author":"Weiner","year":"2010","journal-title":"Nat. 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