Identification of antigen-specific B cells by concurrent monitoring of intracellular Ca²⁺ mobilization and antigen binding with microwell array chip system equipped with a CCD imager

B cells are very heterogeneous, consisting of more than 10⁹ B-cell clones with distinct specificities for antigens in each individual. To identify single B cells with antigen specificity, we have been developing cell microarray technology using microwell array chips whose microwells each capture a single B cell. Using microwell array chips, we detected antigen-specific B cells by monitoring antigen-induced intracellular Ca²⁺ mobilization with a CCD scanner (MAC-CCD system) or the binding of fluorescence-labeled antigen to cells with a confocal laser scanner. We retrieved target cells from the chip, cloned immunoglobulin genes, and produced antigen-specific antibodies. However, these methods present some difficulties: the former technique could not detect cells whose frequency was less than 0.05% and the latter one took a long time to identify the objective cells although it could detect cells at a frequency of 0.01%. Here, we have combined the advantages of these two methods. Monitoring antigen-induced intracellular Ca²⁺ mobilizations and the binding of fluorescence-labeled antigens simultaneously with a MAC-CCD system enabled us to detect rapidly, antigen-specific B cells whose frequency was less than 0.01% with high efficiency. Our system provides a superior screening system for antigen-specific B cells and extends the horizons of multiparameter singlecell analysis in heterogeneous cell populations.