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Front Immunol. 2018 Mar 23;9:493. doi: 10.3389/fimmu.2018.00493. eCollection 2018.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

Author information

1
St. John's Institute of Dermatology, School of Basic & Medical Biosciences, King's College London, Guy's Hospital, London, United Kingdom.
2
NIHR Biomedical Research Centre at Guy's and St. Thomas's Hospitals and King's College London, King's College London, London, United Kingdom.
3
Breast Cancer Now Research Unit, School of Cancer & Pharmaceutical Sciences, King's College London, Guy's Cancer Centre, London, United Kingdom.
4
Department of Applied Research and Technology Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
5
School of Cancer & Pharmaceutical Sciences, King's College London, Guy's Hospital, London, United Kingdom.
6
Breast Cancer Now Toby Robins Research Centre, Institute of Cancer Research, London, United Kingdom.
7
Immunology and Inflammation Therapeutic Research Area, Sanofi US, Cambridge, MA, United States.
8
Department of Oncology, Haematology and Stem Cell Transplantation, University Hospital of Hamburg Eppendorf, Hamburg, Germany.

Abstract

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

KEYWORDS:

B cell; antibody expression; antigen; fluorescent bead; human antibodies; humoral immune response; single cell sorting

PMID:
29628923
PMCID:
PMC5876289
DOI:
10.3389/fimmu.2018.00493
[Indexed for MEDLINE]
Free PMC Article

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