title: 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.
authors.affiliation: Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ozawa
authors.forename: K
authors.initials: K
authors.lastname: Kinoshita
authors.forename: S
authors.initials: S
authors.lastname: Kadowaki
authors.forename: K
authors.initials: K
authors.lastname: Tajiri
authors.forename: S
authors.initials: S
authors.lastname: Kondo
authors.forename: R
authors.initials: R
authors.lastname: Honda
authors.forename: M
authors.initials: M
authors.lastname: Ikemoto
authors.forename: L
authors.initials: L
authors.lastname: Piao
authors.forename: A
authors.initials: A
authors.lastname: Morisato
authors.forename: K
authors.initials: K
authors.lastname: Fukurotani
authors.forename: H
authors.initials: H
authors.lastname: Kishi
authors.forename: A
authors.initials: A
authors.lastname: Muraguchi
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D005456:Fluorescent Dyes; D006801:Humans; D008214:Lymphocytes / Q000276:immunology*; D044085:Microfluidics; D008856:Microscopy, Fluorescence; D009975:Orthomyxoviridae / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes