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

最近，已经在磁细菌属菌株AMB-1中进行了与磁性颗粒合成有关的基因的分析。从AMB-1的转座子诱变产生的突变体中分离出magA基因，并进行完全测序。MagA可作为锚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。我们将蛋白A-magA杂交基因克隆到磁性细菌AMB-1中，并制备了蛋白A-BMP复合物。在本研究中，我们建立了一种全自动化学发光酶免疫分析法，采用磁性分离抗体-蛋白A-BMP复合物，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，使用抗体-蛋白A-BMP复合物和ALP-抗体测定人IgG浓度。自动化免疫分析系统包含反应站、吸头架和自动化八个移液器，该移液器能够将强磁体附接到吸头表面和从吸头表面分离，并且对应于96孔微量滴定板。微量滴定板可安装在反应站中。有一个机架容纳8 x 3个反应尖端。将每种试剂应用于8行微量滴定板。获得了发光强度与人IgG浓度之间的剂量-响应曲线。检测限为1 pg/ml。总之，利用抗体-蛋白A-BMP复合物的化学发光酶免疫分析是全自动的。全自动免疫分析系统可精确测定血清中的人胰岛素。

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.