Cell-microarray analysis of antigen-specific B-cells: single cell analysis of antigen receptor expression and specificity.
The authors previously developed a cell-microarray system that effectively detects antigen-specific B-cells by monitoring intracellular Ca2+ at single cell levels. Here they present a novel method to detect antigen-specific B-cells using cell-microarray system. To detect antigen-specific B-cells, they arrayed live lymphocytes on a chip, stained cells with fluorescence-labeled nonspecific proteins, and analyzed them with a fluorescence scanner to detect nonspecific protein binding to B-cells. They then stained cells with fluorescence-labeled antigen and analyzed them with the scanner. Cells stained with specific antigen, but not with nonspecific proteins, were determined as antigen-specific B-cells and harvested. Antibody cDNA was amplified from retrieved B-cells by single-cell RT-PCR, inserted into expression vectors, and was examined for its specificity by ELISA. They could detect antigen-specific B-cells at a frequency of 0.01% in a model system using transgenic mice that express antibody to hen-egg lysozyme on the surface of B-cells. They applied this system to directly detect hepatitis B virus surface-antigen (HBs-Ag)-specific B-cells from peripheral blood in HBs-Ag-vaccinated volunteers and succeeded in producing HBs-Ag-specific monoclonal antibody. This novel system allows us to identify human antigen-specific B-cells of very low frequency and is a powerful tool to explore the candidates of antibody therapeutics.
Tajiri
Kazuto
K
Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630, Sugitani, Toyama 930-0194, Japan.
Kishi
Hiroyuki
H
Tokimitsu
Yoshiharu
Y
Kondo
Sachiko
S
Ozawa
Tatsuhiko
T
Kinoshita
Koshi
K
Jin
Aishun
A
Kadowaki
Shinichi
S
Sugiyama
Toshiro
T
Muraguchi
Atsushi
A
D000818:Animals; D000941:Antigens / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002469:Cell Separation / Q000379:methods; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D006801:Humans; D051379:Mice; D046228:Microarray Analysis / Q000379:methods*; D009113:Muramidase / Q000276:immunology; D011947:Receptors, Antigen, B-Cell / Q000032:analysis* / Q000276:immunology; D012680:Sensitivity and Specificity
D000941:Antigens; D006514:Hepatitis B Surface Antigens; D011947:Receptors, Antigen, B-Cell; C421864:hen egg lysozyme; D009113:Muramidase