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Development of miniaturized fully-automated immunoassay robot using magnetic particles from recombinant magnetic bacteria

利用重组磁性细菌磁性颗粒开发小型化全自动免疫分析机器人

基本信息

批准号：
10555285
负责人：
MATSUNAGA Tadashi
金额：
$ 8.58万
依托单位：
Tokyo University of Agriculture & Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Recently, analysis of the genes concerned with magnetic particle synthesis has been undertaken in Magnetospirillum sp. strain AMB-1. The magA gene was isolated from a nonmagnetic mutant generated by transposon mutagenesis from AMB-1, and completely sequenced. MagA can be used as an anchor protein to express various foreign proteins on membrane of bacterial magnetic particle (BMP) without the need for chemical cross-linking. We have cloned a proteinA-magA hybrid gene into magnetic bacterium strain AMB-1, and produced protein A-BMP complexes. In this study, we developed a fully automated chemiluminescence enzyme immunoassay by using a magnetic separation of antibody-protein A-BMP complexes.The luminescence intensity of protein A-BMP complexes was 20 times higher than that of BMP from wild type AMB-1. Protein A was successfully expressed on the BMP membrane, and has IgG Fc region-binding activity. Human IgG concentration was measured by fully-automated chemiluminescence enzyme immunoassay system using antibody-protein A-BMP complexes and ALP-antibody. Automated immunoassay system contains a reaction station, tip rack and an automated eight pipettor that is able to attach and detach a strong magnet to a tip surface, and correspondent to 96 well microtiter plate. Microtiter plate can be mounted in the reaction station. There is one rack to hold 8 x 3 tips for reaction. Each reagents is applied to 8 lines of microtiter plate. Dose-response curve was obtained between the luminescence intensity and human IgG concentration. Detection limit was 1 pg/ml. In conclusion, chemiluminescence enzyme immunoassay using antibody-protein A-BMP complexes was developed full-automatically. The fully automated immunoassay system allows precise assay of human insulin in serum.

近年来，研究人员对磁螺旋藻AMB-1中与磁性颗粒合成有关的基因进行了分析。从AMB-1转座子诱变产生的非磁性突变体中分离到magA基因，并对其进行了完全测序。MagA可以作为锚定蛋白在细菌磁性颗粒（BMP）膜上表达多种外源蛋白，而不需要化学交联。我们将蛋白a - maga杂交基因克隆到磁性细菌AMB-1中，产生蛋白a - bmp复合物。在这项研究中，我们开发了一种全自动化学发光酶免疫分析法，使用抗体-蛋白a - bmp复合物的磁分离。蛋白A-BMP复合物的发光强度比野生型AMB-1的BMP高20倍。蛋白A在BMP膜上成功表达，并具有IgG Fc区结合活性。采用全自动化学发光酶免疫分析系统，采用抗体-蛋白A-BMP复合物和alp抗体检测人IgG浓度。自动免疫分析系统包含一个反应站，尖端架和一个自动8移液器，能够将强磁铁附着和分离到尖端表面，并对应于96孔微量滴度板。微量滴定板可安装在反应站。有一个架子容纳8 × 3的反应提示。每种试剂应用于8行微量滴度板。获得发光强度与人IgG浓度的剂量-响应曲线。检出限为1 pg/ml。综上所述，利用抗体-蛋白A-BMP复合物进行化学发光酶免疫分析是完全自动化的。全自动免疫分析系统可以精确测定血清中的人胰岛素。