Efficient production of human recombinant antibodies against hepatitis virus using a novel micro-well array system

虽然在各种药物发现的发展之后，由病原微生物引起的传染病似乎大大减少，但新出现的传染病，例如在世界范围内蔓延的艾滋病或在亚洲爆发的SARS，已经对人类构成威胁。抗体（Abs）作为一种理想的抗病原微生物的治疗试剂一直被期待，但由于难以有效地产生针对各种微生物的人源化单克隆Abs，它们的应用受到极大限制。本课题尝试建立一种新型的微孔阵列系统，用于高效地寻找抗原特异性的人B细胞，并将其应用于制备抗B病毒表面抗原（HBsAg）的人单克隆抗体（半平方厘米中有250，000个威尔斯孔），其中室被设计成单个B细胞的大小。为了检测Ag特异性B细胞， ...更多信息 以Fluo-4作为B细胞受体（BCR）介导的B细胞活化的标志物，用定制的DNA芯片检测细胞内Ca ~（2 ^+）i的增加，并应用此系统制备抗HBsAg的人单克隆抗体。来自用HBsAg免疫的健康志愿者的B细胞在芯片上用HBsAg刺激，通过扫描仪检测应答细胞，并通过显微操作收获。从收获的B细胞中克隆IG和L链可变区的cDNA，并在293 T细胞中产生重组Ab。通过ELISA和小鼠体内实验分别检测了重组抗体与HBsAg的结合能力和中和活性，结果表明，重组抗体对HBsAg具有较高的亲和力，同时也具有较高的中和活性。这种微孔阵列系统应该是一种高通量的系统，用于生产针对各种感染性微生物的人单克隆抗体，并有助于发现针对新出现的感染性疾病的抗体药物。少

Although infectious diseases caused by pathogenic microbes seemed extraordinarily lessened after the development of various drug discovery, newly emerging infectious diseases, such as worldwide-spreading AIDS or SARS broken out in Asia, have been a threat to humans. Antibodies (Abs) have been long expected as a desirable therapeutic reagent against the pathogenic microbe, their use has been extremely limited because of the difficulties to efficiently generate humanized monoclonal Abs to various micobes. In the present project, we have tried to develop a novel micro-well-array system to efficiently find the antigen (Ag)-specific human B cells and applied this sytem to produce human monoclonal Abs against hepatitis B virus-surface antigen (HbsAg).We have developed a micro-well-array chip consists of highly integrated micro chambers (250,000 wells in half square centimeter) on a silicon chip, of which chambers were designed to a size of a single B cell. To detect Ag-specific B cells, intr … More acellular Ca2^+ indicator, Fluo-4, was introduced to the cells as a marker for B cell activation via B cell receptor (BCR), and the increases in (Ca2^+)i were detected by the customized DNA-array.We applied this system to produce human monoclonal Abs against HbsAg. B cells from healthy volunteers who had been immunized with HBsAg were stimulated with HBsAg on a chip and the responding cells were detected by the scanner and harvested by micromanipulation. The cDNA of variable regions for Ig Hand L chains were cloned from the harvested B cells and the recombinant Abs were generated in 293T cells. The ability to bind to HbsAg or to neutralize the HB virus activity was assessed by ELISA or in vivo mouse model, respectively.As a result, we produced several human recombinant Abs with high binding affinity to HbsAg, as well as high neutralizing activity. This micro-well array system should be a high throughput system for producing human monoclonal Abs against various infectious microbes and contribute to discover antibody-medicine against newly emerging infectious diseases. Less