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Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin

Oscar A. Aguilar, Miho Tanaka, Gautham R. Balaji, Richard Berry, Jamie Rossjohn, Lewis L. Lanier and James R. Carlyle
J Immunol September 15, 2020, 205 (6) 1709-1717; DOI: https://doi.org/10.4049/jimmunol.2000687
Oscar A. Aguilar
*Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada;
†Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143;
‡Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94143;
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Miho Tanaka
*Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada;
†Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143;
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Gautham R. Balaji
§Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia;
¶Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, Australia; and
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Richard Berry
§Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia;
¶Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, Australia; and
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Jamie Rossjohn
§Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia;
¶Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, Australia; and
‖Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff CF14 4XN, United Kingdom
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Lewis L. Lanier
†Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143;
‡Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94143;
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James R. Carlyle
*Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada;
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Key Points

  • A novel method to generate mAb using tetramers and reporter cells is described.

  • Ab against the MCMV immunoevasin m12 are generated and characterized.

  • An mAb was identified that is capable of blocking m12-mediated NK inhibition.

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Abstract

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.

Footnotes

  • This work was supported by Canadian Institutes of Health Research Operating Grants MOP 106491 and PJT 159450 (to J.R.C.) and by Burroughs Wellcome Fund (BWF) Investigator in the Pathogenesis of Infectious Disease Award 1007761 (to J.R.C.) and a BWF Postdoctoral Enrichment Program award (to O.A.A). O.A.A. is a Cancer Research Institute Irvington Fellow supported by the Cancer Research Institute. O.A.A. and L.L.L. are supported by the Parker Institute for Cancer Immunotherapy. J.R. is a recipient of Australian Research Council Laureate Fellowship FL160100049. R.B. is a recipient of National Health and Medical Research Council of Australia Career Development Award APP1109901.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ADCC
    Ab-dependent cellular cytotoxicity
    CPRG
    chlorophenol-red-β-d-galactopyranoside
    CTV
    CellTrace Violet
    DMEM-HG
    high-glucose DMEM
    HAT
    hypoxanthine–aminopterin–thymidine
    HT
    hypoxanthine–thymidine
    LAK
    lymphokine-activated killer
    MCMV
    mouse CMV
    P3
    P3XAg8.653.1
    rhIL-2
    recombinant human IL-2
    SA
    streptavidin.

  • Received June 8, 2020.
  • Accepted July 19, 2020.
  • Copyright © 2020 by The American Association of Immunologists, Inc.
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The Journal of Immunology: 205 (6)
The Journal of Immunology
Vol. 205, Issue 6
15 Sep 2020
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Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin
Oscar A. Aguilar, Miho Tanaka, Gautham R. Balaji, Richard Berry, Jamie Rossjohn, Lewis L. Lanier, James R. Carlyle
The Journal of Immunology September 15, 2020, 205 (6) 1709-1717; DOI: 10.4049/jimmunol.2000687

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Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin
Oscar A. Aguilar, Miho Tanaka, Gautham R. Balaji, Richard Berry, Jamie Rossjohn, Lewis L. Lanier, James R. Carlyle
The Journal of Immunology September 15, 2020, 205 (6) 1709-1717; DOI: 10.4049/jimmunol.2000687
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