MAC-CCD system: a novel lymphocyte microwell-array chip system equipped with CCD scanner to generate human monoclonal antibodies against influenza virus.

Abstract

We previously developed a lymphocyte microwell-array system, which effectively detects antigen-specific B-cells by monitoring intracellular Ca(2+) mobilization at the single-cell level with a fluorescent Ca(2+) indicator, fluo-4. However, it is difficult for the system to perform time-lapse monitoring. Here, we developed a novel method, a lymphocyte microwell-array chip system equipped with a charge-coupled device (CCD) time-lapse scanner (MAC-CCD system), for monitoring intracellular Ca(2+) mobilization. The MAC-CCD system is able to monitor intracellular Ca(2+) mobilization of more than 15,000-20,000 individual live B-cells every 10 s. In addition, we adopted a correlation method in a MAC-CCD system, which enabled us to detect B-cells with a frequency of as few as 0.046%. Furthermore, we succeeded in obtaining six influenza nucleoprotein-specific human monoclonal antibodies from the peripheral blood of influenza-vaccinated volunteers. These results demonstrate that the MAC-CCD system with a correlation method could detect very rare antigen-specific B-cells.