Soh Nobuaki, Nishiyama Hideshi, Asano Yasukazu, Imato Toshihiko, Masadome Takashi, Kurokawa Youichi
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Talanta. 2004 Dec 15;64(5):1160-8. doi: 10.1016/j.talanta.2004.06.001.
A rapid and sensitive immunoassay for the determination of carp vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a samarium-cobalt magnet. An anti-Vg monoclonal antibody, immobilized on magnetic beads, was used as a solid support for the immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by a samarium-cobalt magnet and the flow of the carrier solution. The immunoassay was based on a sandwich immunoreaction of anti-Vg monoclonal antibody (primary antibody) on the magnetic beads, Vg, and the anti-Vg antibody labeled with horseradish peroxidase (HRP) (secondary antibody), and was based on a subsequent chemiluminescence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The magnetic beads to which the primary antibody was immobilized were prepared by coupling the primary antibody with the magnetic beads after an agarose-layer on the surface of the magnetic beads was epoxidized. The primary antibody-immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the samarium-cobalt magnet, a Vg sample solution, an HRP-labeled secondary antibody solution and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photomultiplier located at the upper side of the flow cell. The optimal incubation times both for the first and second immunoreactions were determined to be 20min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2-100ngmL(-1)) were applied to the SIA system under optimal conditions. In spite of a narrow working range, the lower detection limit of the immunoassay was about 2ngmL(-1).
本文描述了一种用于测定鲤鱼卵黄蛋白原（Vg）的快速灵敏免疫分析方法。该方法采用配备化学发光检测器和钐钴磁体的顺序注射分析（SIA）系统。固定在磁珠上的抗Vg单克隆抗体用作免疫分析的固相支持物。磁珠在流动池中的引入、捕获和释放由钐钴磁体和载体溶液的流动控制。免疫分析基于磁珠上的抗Vg单克隆抗体（一抗）、Vg和辣根过氧化物酶（HRP）标记的抗Vg抗体（二抗）的夹心免疫反应，并基于随后HRP与过氧化氢和对碘苯酚在鲁米诺溶液中的化学发光反应。固定一抗的磁珠是通过在磁珠表面的琼脂糖层环氧化后将一抗与磁珠偶联而制备的。引入固定有一抗的磁珠，并将其捕获在配备钐钴磁体的流动池中，根据SIA程序序列将Vg样品溶液、HRP标记的二抗溶液和鲁米诺溶液依次引入流动池。通过位于流动池上方的光电倍增管监测化学发光发射。确定第一次和第二次免疫反应的最佳孵育时间均为20分钟。在最佳条件下，将各种浓度的标准Vg样品（2 - 100 ng mL(-1)）应用于SIA系统时，获得了Vg浓度与化学发光强度之间的凹形校准曲线。尽管工作范围较窄，但该免疫分析的检测下限约为2 ng mL(-1)。
