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.
Visual Abstract
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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