%0 Generic %A Correa, Isabel %A Ilieva, Kristina M. %A Crescioli, Silvia %A Lombardi, Sara %A Figini, Mariangela %A Cheung, Anthony %A Spicer, James F. %A Tutt, Andrew N. J. %A O. Nestle, Frank %A Karagiannis, Panagiotis %A Lacy, Katie E. %A N. Karagiannis, Sophia %D 2018 %T table_2.docx %U https://frontiersin.figshare.com/articles/dataset/table_2_docx/6020225 %R 10.3389/fimmu.2018.00493.s003 %2 https://frontiersin.figshare.com/ndownloader/files/10832828 %K B cell %K humoral immune response %K single cell sorting %K fluorescent bead %K human antibodies %K antibody expression %K antigen %X

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.

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