Chapter 2
Isolating Pathogen-Specific Human Monoclonal Antibodies
(hmAbs) Using Bacterial Whole Cells as Molecular Probes
Sara Siris, Camilla A. Gladstone, Yanping Guo, Christopher L. Pinder,
Robin J. Shattock, Paul F. McKay, Paul R. Langford, and Fadil A. Bidmos
Abstract
The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from
individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only
single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the
method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of
plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria
meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here
allowed for a pathogen-specific hmAb cloning efficiency of >80%.
Key words Immunoglobulin capture assay, Pathogen-specific plasmablasts, Bacteria, Whole cells,
Vaccine antigen discovery
1 Introduction
Methods for analysis of the human adaptive immune response to
vaccination or disseminated infection include the cloning and
in vitro expression of human monoclonal antibodies (hmAbs)
from antibody-producing cells (APCs: memory B-cells, plasmablasts and plasma cells). Targeting plasmablasts, especially, for
hmAb cloning is useful as rapid expansion of a plasmablast population occurs immediately following antigen encounter—this expansion is characterized by differentiation leading to increased
specificity for the presenting antigen [1]. Expression cloning of
hmAbs from plasmablasts has been achieved by sorting of individual cells into multiwell plates followed by a sequential PCR that
generates amplicons of the variable regions of heavy (VH) and light
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Sara Siris and Camilla Gladstone contributed equally to this work.
Paul Langford and Fadil Bidmos contributed equally to this work.
9
Isolating Pathogen-Specific Human Monoclonal Antibodies
(hmAbs) Using Bacterial Whole Cells as Molecular Probes
Sara Siris, Camilla A. Gladstone, Yanping Guo, Christopher L. Pinder,
Robin J. Shattock, Paul F. McKay, Paul R. Langford, and Fadil A. Bidmos
Abstract
The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from
individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only
single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the
method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of
plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria
meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here
allowed for a pathogen-specific hmAb cloning efficiency of >80%.
Key words Immunoglobulin capture assay, Pathogen-specific plasmablasts, Bacteria, Whole cells,
Vaccine antigen discovery
1 Introduction
Methods for analysis of the human adaptive immune response to
vaccination or disseminated infection include the cloning and
in vitro expression of human monoclonal antibodies (hmAbs)
from antibody-producing cells (APCs: memory B-cells, plasmablasts and plasma cells). Targeting plasmablasts, especially, for
hmAb cloning is useful as rapid expansion of a plasmablast population occurs immediately following antigen encounter—this expansion is characterized by differentiation leading to increased
specificity for the presenting antigen [1]. Expression cloning of
hmAbs from plasmablasts has been achieved by sorting of individual cells into multiwell plates followed by a sequential PCR that
generates amplicons of the variable regions of heavy (VH) and light
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Sara Siris and Camilla Gladstone contributed equally to this work.
Paul Langford and Fadil Bidmos contributed equally to this work.
9