Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot

J Immunol Methods. 2020 Mar:478:112715. doi: 10.1016/j.jim.2019.112715. Epub 2019 Dec 3.

Abstract

Analysis of B-cell specificities at the single cell level provides important information on how the B-cell compartment responds when challenged by infection or vaccination. We recently developed a reversed B-cell FluoroSpot assay and showed that it could be used to detect B cells specific for different antigens simultaneously in a mouse model. The aim of this study was to further develop the method to detect and quantify antigen-specific memory B cells (MBCs) in humans where circulating antigen-specific cells are less frequent. We show that MBCs specific for three antigens, tetanus toxoid, hepatitis B surface antigen and cytomegalovirus pp65, could be detected simultaneously in one well. In addition to enumerating antigen-specific MBCs, we also assessed the spot volume to estimate the intensity of the response in individual cells and found this to be a new and sensitive approach to study MBC responses after vaccination. This unique B-cell FluoroSpot approach provides a simple and sensitive multiplex analysis of MBCs and can be adapted to most antigens and host species.

Keywords: Antibody; Antigen; B cell ELISpot; FluoroSpot; Vaccine.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Viral / immunology*
  • B-Lymphocytes / immunology*
  • Blood Buffy Coat / cytology
  • Cell Separation / instrumentation
  • Cell Separation / methods*
  • Cytomegalovirus / immunology
  • Feasibility Studies
  • Fluorescent Antibody Technique / instrumentation
  • Fluorescent Antibody Technique / methods*
  • Fluorescent Dyes / chemistry
  • Hepatitis B Vaccines / administration & dosage
  • Hepatitis B Vaccines / immunology
  • Hepatitis B virus / immunology
  • High-Throughput Screening Assays / instrumentation
  • High-Throughput Screening Assays / methods*
  • Humans
  • Hybridomas
  • Immunogenicity, Vaccine
  • Immunologic Memory*
  • Mice
  • Recombinant Proteins / immunology
  • Reproducibility of Results
  • Staining and Labeling
  • Tetanus Toxoid / immunology
  • Vaccination
  • Workflow

Substances

  • Antigens, Viral
  • Fluorescent Dyes
  • Hepatitis B Vaccines
  • Recombinant Proteins
  • Tetanus Toxoid