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

最近，对磁螺菌中与磁性颗粒合成相关的基因进行了分析。AMB-1菌株。MAGA基因是从AMB-1转座子诱变产生的一个非磁性突变体中分离出来的，并进行了全序列测定。MAGA可以作为锚定蛋白在细菌磁性颗粒(BMP)膜上表达各种外源蛋白，而不需要化学交联。我们已将蛋白A-MAGA杂合基因克隆到磁性细菌AMB-1中，并产生蛋白A-骨形态发生蛋白复合体。在这项研究中，我们建立了一种全自动的化学发光酶免疫分析方法，通过磁分离抗体-蛋白A-BMP复合物，其发光强度比野生型AMB-1的BMP高20倍。蛋白A在BMP膜上成功表达，并具有Ig G Fc区结合活性。利用抗体-蛋白A-BMP复合体和碱性磷酸酶抗体，采用全自动化学发光酶免疫分析系统测定人免疫球蛋白浓度。自动免疫分析系统包括一个反应站、一个滴定架和一个自动化的八个移液器，该移液器能够将强磁铁附着和分离到尖端表面，并对应于96孔微量滴定板。微量滴定板可以安装在反应站内。有一个机架可容纳8x3个反应提示。每种试剂加于8条微量试板上。获得了发光强度与人免疫球蛋白浓度之间的剂量-反应曲线。检测下限为1pg/ml。综上所述，建立了基于抗体-蛋白A-骨形态发生蛋白复合体的化学发光酶免疫分析方法。全自动免疫分析系统可以精确地检测血清中的人胰岛素。

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.