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，使用微孔阵列芯片分析单个淋巴细胞的细胞反应，超过230，000个微孔只能容纳单个淋巴细胞。我们还开发了新的方法，用于（1）检测具有Ca ^2+反应和Ag结合的Ag特异性B细胞，以及（2）通过荧光标记免疫斑点（FLTSPOT）试验检测产生Ab的B细胞。由于每个淋巴细胞在芯片上的地址是固定的，我们可以很容易地使用显微操作器恢复它们。用RT-PCR方法从单个淋巴细胞中扩增出抗体cDNA，并在细胞中表达。应用该系统，我们成功地从B型肝炎病毒表面抗原（HBsAg）和流感病毒HA免疫的志愿者外周血淋巴细胞中获得了抗HBsAg和抗流感病毒的抗体。目前检测Ag特异性B细胞的新方法有：（1）钙离子反应和Ag结合同时测定法，将B细胞置于芯片上， ...更多信息 d用非特异性蛋白-Cy 5染色，然后用抗原-Cy 5染色，并用细胞扫描仪监测细胞的荧光。这种方法使我们能够有效地检测Ag特异性B细胞，假阳性Ag特异性B细胞的频率低。(B)FLTSPOT检测法是将抗Ig-Ab包被在微孔阵列芯片表面，并将含有Ab分泌细胞的细胞加入到芯片中，用荧光标记的Ag检测Ag特异性Ab分泌细胞。从芯片上回收抗原特异性抗体分泌细胞，RT-PCR扩增抗体cDNA，制备重组抗体。在HBs抗原/流感-HA的情况下，在芯片上快速培养富集的CD 138 ^+人淋巴细胞，然后使用生物素化抗原检测Ab分泌细胞，然后使用链霉亲和素-Cy 3。从单克隆抗体分泌细胞中克隆的V_H和V-L cDNA转染293 T细胞，获得重组抗体。在可溶性抗原存在下，用ELISA检测抗体的Ag特异性。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