Antigen‐specific memory B cell (MBC) populations mediate the rapid, strong, and high‐affinity secondary antibody responses that play a key role in combating infection and generating protective responses to vaccination. Recently, cell staining with fluorochrome‐labeled antigens together with sequencing methods such as Drop‐seq and CITE‐seq have provided information on the specificity, phenotype, and transcriptome of single MBCs. However, characterization of MBCs at the level of antigen‐reactive populations remains an important tool for assessing an individual's B cell immunity and responses to antigen exposure. This is readily performed using a long‐established method based on in vitro polyclonal stimulation of MBCs to induce division and differentiation into antibody‐secreting cells (ASCs). Post‐stimulation antigen‐specific measurement of the MBC‐derived ASCs (or the secreted antibodies) indicates the size of precursor MBC populations. Additional information about the character of antigen‐reactive MBC populations is provided by analysis of MBC‐derived antibodies of particular specificities for binding avidity and functionality. This article outlines a simple and reliable strategy for efficient in vitro MBC stimulation and use of the ELISpot assay as a post‐stimulation readout to determine the size of antigen‐specific MBC populations. Other applications of the in vitro stimulation technique for MBC analysis are discussed. The following protocols are included. © 2020 Wiley Periodicals LLC
While immunoglobulins find ubiquitous use in biotechnology as static binders, recent developments have created proantibodies that enable orthogonal switch‐like behavior to antibody function. Previously, peptides with low binding affinity have been genetically fused to antibodies, to proteolytically control binding function by blocking the antigen‐binding site. However, development of these artificial blockers requires panning for peptide sequences that reversibly affect antigen affinity for each antibody. Instead, a more general strategy to achieve dynamic control over antibody affinity may be feasible using protein M (ProtM) from
- NSF-PAR ID:
- 10116807
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 116
- Issue:
- 11
- ISSN:
- 0006-3592
- Page Range / eLocation ID:
- p. 2834-2842
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Basic Protocol 1 : Polyclonal stimulation of memory B cells using unfractionated PBMCsAlternate Protocol : Stimulation of small PBMC numbers using 96‐well plates with U‐bottom wellsBasic Protocol 2 : ELISpot assay for enumeration of memory B cell−derived antibody‐secreting cells -
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