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

我们开发了一个单细胞微阵列系统，使用微孔阵列芯片来分析单个淋巴细胞的细胞反应，该芯片有超过23万个微孔，只能容纳单个淋巴细胞。我们还开发了新的方法(1)检测具有钙离子反应和Ag结合的Ag特异性B细胞，以及(2)用荧光连结免疫斑点(FLTSPOT)法检测产生抗体的B细胞。由于芯片上每个淋巴细胞的地址是固定的，我们可以使用微操作器轻松地恢复它们。RT-PCR可从单个淋巴细胞扩增出抗体基因，并在细胞中表达。应用该系统，我们成功地从HBs-Ag和流感-HA免疫志愿者的外周血淋巴细胞中获得了抗HBs-Ag和流感病毒的抗体。新的检测方法如下：(1)在芯片和…上应用B细胞同时进行Ca~(2+)反应和Ag结合试验用非特异性蛋白Cy5染色，然后用抗原Cy5染色，用细胞扫描仪监测细胞的荧光。(2)将抗Ig-Ab包被于微孔阵列芯片表面，在芯片上加入含有抗体分泌细胞的细胞，用荧光标记的银检测抗原特异性抗体分泌细胞。从芯片中回收抗原特异性抗体分泌细胞，RT-PCR扩增抗体基因，制备重组抗体。在HBs抗原/流感-HA的情况下，在芯片上培养浓缩的CD138^+的人淋巴细胞，用生物素化的抗原检测抗体分泌细胞，然后用链霉亲和素-Cy3检测。将从单个抗体分泌细胞中克隆的V_H和V-L基因分别导入293T细胞，获得重组抗体。在可溶性抗原存在的情况下，用酶联免疫吸附试验检测抗体的抗原特异性。较少

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 an … More d 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. Less