Development of the method for detection of antigen-specific lymphocytes and antigen receptors using cell-microarray

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Lymphocytes express antigen-specific receptors and binding of antigen to the receptors initiates lymphocyte-activation as well as differentiation. Lymphocyte-activation is usually monitored by the proliferation or the protein-secretion of bulk culture of lymphocytes in 96 well plates. At the clonal level, each lymphocyte expresses antigen-receptors with distinct specificity and responds to distinct epitopes in an antigenic molecule. When lymphocyte-activation could be monitored at the clonal level, more precise information on lymphocyte-activation will be obtained. To that end, we have been developing the system for detecting antigen-specific lymphocytes at the clonal level. Lymphocytes are loaded with fluorescent probe of calcium indicator, fluo-4, and added to a silicon chip (2 cm x 2 cm) containing array of about 2 x 10^5 microwells of which size is just fit for a single lymphocyte. After removing the residual cells that do not enter microwells, 70 to 90% of wells contain single lymphocytes. Fluorescence of lymphocytes before activation is monitored by a scanner. Then lymphocytes are activated with stimulants such as antigens and fluorescence of lymphocytes is monitored by the scanner. By comparing the fluorescence before and after activation, activated lymphocytes are detected and recovered from the chip using micromanipulator, and cDNA of antigen-receptors is recovered from a single lymphocyte. By using this system, we detected antigen-responding human B-lymphocytes and obtained antigen-specific antibodies. This system is simple and useful to develop antibody medicine. It will be also used to detect antigen-specific T-lymphocytes and useful to study lymphocyte-activation in basic research as well as clinical application