Development of rapid production of human monoclonal antibodies against emerging infectious pathogens using lymphocyte chip

We developed a single-cell microarray system to analyze cellular response of individual lymphocytes using microwell array chips, with over 230,000 microwells that can accommodate only single-lymphocytes. We also developed new methods for(1)detection of Ag-specific B cells with Ca^2+ response and Ag-binding, and (2) detection of Ab-producing B cells by Fluorescence-linked immuno-spot (FLTSPOT) assay. Since the address of each lymphocyte on the chip is fixed, we could easily recover them using a micromanipulator. Antibody cDNA could be augmented from each single lymphocyte by RT-PCR and could be expressed in cells. By applying this system, we successfully obtained Abs for type B hepatitis virus surface antigen (HBs-Ag) as well as influenza virus from peripheral blood lymphocytes of HBs-Ag-and influenza-HA immunized volunteers. The new methods for detection of Ag-specific B cells is following; (1) Simultanous Ca^2+ response and Ag-binding assay in which B-cells were applied on the chip and stained with non-specific protein-Cy5, followed by Antigen-Cy5, and fluorescence of cells was monitored with the Cell Scanner. This method enabled us to efficiently detect Ag-specific B cells with low frequency of false-positive Ag-specific B-cells.(B) FLTSPOT Assay in which anti-Ig-Ab was coated on the surface of microwell array chip and cells containing Ab-secreting cells were added to the chip, and Ag-specific Ab-secreting cells were detected using fluorescence-labeled Ag. Then Ag-specific Ab-secreting cells were retrieved from the chip, Ab cDNA was amplified with RT-PCR and recombinant Abs were produced. In the case of HBs antigen/Influenza-HA, enriched CD138^+ human lymphocytes were cultured shortly on chips and Ab-secreting cells were detected using biotinylated-antigens, followed by streptavidine-Cy3. V_H and V-L cDNAs cloned from single antibody-secreting cells were transfected into 293T cells and recombinant Abs were generated. Ag-specificity of antibodies were examined with ELISA in the presence of soluble antigens.