Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads.
Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads.
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DOI：
10.1016/j.talanta.2004.06.001
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发表时间：
2004-12
期刊：
影响因子：
6.1
通讯作者：
N. Soh;Hideshi Nishiyama;Y. Asano;T. Imato;T. Masadome;Y. Kurokawa
中科院分区：
文献类型：
--
作者：
N. Soh;Hideshi Nishiyama;Y. Asano;T. Imato;T. Masadome;Y. Kurokawa
本文报道了一种快速、灵敏的鲤鱼卵黄原蛋白免疫测定方法。该方法包括顺序注射分析（SIA）系统配备了化学发光检测器和钐钴磁铁。使用固定在磁珠上的抗Vg单克隆抗体作为免疫测定的固体支持物。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。免疫测定是基于抗Vg单克隆抗体（一抗）的磁珠，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，并基于随后的化学发光反应的HRP与过氧化氢和对碘苯酚，在鲁米诺溶液。在磁珠表面上的琼脂糖层被环氧化之后，通过将一抗与磁珠偶联来制备固定有一抗的磁珠。引入固定有第一抗体的磁珠，并将其捕获在配备有钐-钴磁体的流动池中，基于SIA程序化序列将Vg样品溶液、HRP标记的第二抗体溶液和鲁米诺溶液依次引入流动池中。通过位于流动池上侧的光电倍增管监测化学发光发射。第一次和第二次免疫反应的最佳孵育时间均确定为20 min。在最佳条件下，将不同浓度（2- 100 ngmL-1）的标准样品加入SIA体系中，得到了标准样品浓度与化学发光强度之间的凹形校准曲线。尽管工作范围很窄，但免疫测定的检测下限约为2ngmL−1。
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.