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首页> 外文期刊>Journal of Immunological Methods >Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles.
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Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles.

机译：从用荧光病毒样颗粒选择的单个抗原特异性B细胞中产生轮状病毒重组人单克隆抗体。

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Technical difficulties have severely limited the yield of methods for the generation of human antiviral monoclonal antibodies (Mabs) in the past. We describe here a novel method for the efficient development of human Mabs against viruses. Rotavirus (RV) is a major cause of gastroenteritis in infants and adults worldwide. We generated fluorescent virus-like particles (VLPs) to identify and physically sort single RV-specific B cells from healthy adult blood donors, or RV-infected infants or adults. We expanded the sorted single B cells in culture, tested for RV-specific antibody secretion, and cloned and sequenced the antibody heavy and light chain variable region (VH and VL) genes. The percentage of wells that produced antibodies after sorting and expanding RV-specific adult B cell clones was high at 23%. The overall efficiency of RV-specific antibody gene recovery after the isolation, confirmation, and cloning of RV-specific VH segments was 1.3% of sorted cells in adults. RV-specific variable gene segments also were obtained from acutely infected infants, although infant B cells did not proliferate and differentiate in culture as well as adult B cells. We expressed recombinant Fabs incorporating the VH and VL genes from RV-specific B cell clones using a new modified bacterial Fab expression vector that we describe. Finally, we demonstrated binding of purified Fabs to RV proteins by immunofluorescence and ELISA. This method for the generation of recombinant human Mabs to RV from single antigen-specific B cell clones selected with fluorescent VLPs could be used to generate human Mabs to many other viruses whose proteins can self-assemble into VLPs.
机译：过去，技术困难严重限制了产生人抗病毒单克隆抗体（Mabs）的方法的产量。我们在这里描述了一种新的方法，可以有效开发针对病毒的人类单克隆抗体。轮状病毒（RV）是全世界婴儿和成人胃肠炎的主要原因。我们生成了荧光病毒样颗粒（VLP），以识别健康的成人献血者或RV感染的婴儿或成人的单个RV特异性B细胞并对其进行物理分选。我们在培养中扩增了分类的单个B细胞，测试了RV特异性抗体的分泌，并克隆并测序了抗体重链和轻链可变区（VH和VL）基因。在分选并扩增RV特异性成年B细胞克隆后，产生抗体的孔百分比很高，为23％。 RV特异性VH片段的分离，确认和克隆后，RV特异性抗体基因恢复的总体效率为成人分选细胞的1.3％。 RV特异性可变基因片段也从急性感染的婴儿中获得，尽管婴儿B细胞和成年B细胞均未在培养物中增殖和分化。我们表达了重组Fabs，其中包含了我们描述的新的修饰的细菌Fab表达载体，从RV特异性B细胞克隆中整合了VH和VL基因。最后，我们通过免疫荧光和ELISA证明了纯化的Fab与RV蛋白的结合。这种从用荧光VLP选择的单个抗原特异性B细胞克隆向RV产生重组人单克隆抗体的方法可用于为许多其他病毒（其蛋白质可自组装成VLP）产生人单克隆抗体。

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