Cell‐microarray analysis of antigen‐specific B‐cells: Single cell analysis of antigen receptor expression and specificity

Abstract

The authors previously developed a cell-microarray system that effectively detects antigen-specific B-cells by monitoring intracellular Ca 21 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 singlecell 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. To date, many investigators have developed various methods to detect antigenspecific B-cells using fluorescence-labeled antigen. However, nonspecific binding of fluorescence-labeled antigen (0.1%-1% in B-cells) makes it difficult to detect antigen-specific cells by hiding specific signals in background noise. In this study, we have developed a cell-microarray system that consists of microwell-array chips and a cell scanner. Microwell-array chips with a regular array of 45,000 or 234,000 wells that can accommodate only single lymphocytes enable us to analyze cells on a single cell basis (13). Since the position of each cell is fixed on the chip, it allows us to analyze the same single cell repeatedly. Here, we show that we can identify antigen-specific B-cells using this cell-microarray system by distinguishing signals from background

Identification of antigen‐specific B cells by concurrent monitoring of intracellular Ca2+ mobilization and antigen binding with microwell array chip system equipped with a CCD imager

“…Compared with that protocol, the protocol in this study showed much higher detection efficiency because we could amplify HyHEL10 cDNA from 95% and 67% of retrieved cells out of 0.05% and 0.01% of the MD4 B cells, respectively. We also reported that we could amplify HyHEL10 cDNA from 50% of the retrieved cells when analyzing the 0.01% MD4 B-cell preparation by detecting the binding of the fluorescence-labeled antigen (17). In that protocol, it took about 1 h to detect antigen-specific B cells.…”

“…Cytomics is becoming increasingly important for analyzing cell conditions and cellular responses to stimuli from extracellular milieu, which will contribute to drug discovery as well as predictive medicine. Cell microarray-based analysis offers one way for analyzing cytomics; it has been conventionally performed with a single parameter (11)(12)(13)(14)(15)(16)(17)(18). Here, for the first time, we present a method of two-parameter cell microarray-based time-lapse analysis.…”

“…After that, the MAC-CCD system detected the position of each microwell on the chip again. Fifteen seconds after the stimulation, the fluorescence images of the fluo-4 signals were acquired on the whole microwell area every 3 s for an additional 45 s (scanning points [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The cells inside the wells did not move out of the wells after four gentle washes.…”

“…The Cy-5 and fluo-4 signal doublepositive cells that were retrieved with a micromanipulator, TransferMan NK2 (Eppendorf, Hamburg, Germany), from a glass capillary micropipette (Primetech, Tokyo, Japan) under a fluorescence microscope, BX51WI (Olympus, Tokyo, Japan). HyHEL10 antibody cDNA was amplified by single-cell RT-PCR as described previously (17,18).…”

“…Recently, we reported two methods for identifying rare antigen-specific B cells: one is detecting the binding of a fluorescence-labeled antigen to cell-surface antibodies on B cells with a confocal laser scanner (17) and the other is monitoring intracellular Ca 21 mobilizations with a CCD camera-based imager (MAC-CCD system) (18). Both methods enabled us to efficiently detect antigen-specific B cells, although they could not detect the cells whose frequency was less than 0.1% due to the nonspecific binding of fluorescence-labeled antigen to B cells (17) and spontaneous increase in intracellular Ca 21 concentration (18)(19)(20)(21). Thus, the detection efficiencies were not sufficient for detecting antigenspecific B cells whose frequency was below 0.1%.…”