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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.

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


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.


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

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