Electrochemical Immunoassay for Vitellogenin Based on Sequential Injection Using Antigen-immobilized Magnetic Microbeads
Electrochemical Immunoassay for Vitellogenin Based on Sequential Injection Using Antigen-immobilized Magnetic Microbeads
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DOI：
10.2116/analsci.22.81
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发表时间：
2006-01
影响因子：
1.6
通讯作者：
Koji Hirakawa;M. Katayama;N. Soh;K. Nakano;T. Imato
中科院分区：
文献类型：
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作者：
Koji Hirakawa;M. Katayama;N. Soh;K. Nakano;T. Imato
建立了一种快速、灵敏的卵黄蛋白原(VG)免疫分析方法。该方法包括配备安培检测器和钕磁铁的顺序注射分析(SIA)系统。将抗原(VG)固定在磁珠上，作为免疫分析的固体载体。磁珠在免疫反应池中的引入、捕获和释放是通过调节载体溶液的流量来控制的。用碱性磷酸酶(ALP)标记的抗VG单抗与固定在磁珠上的VG组分和样品溶液中的VG发生间接竞争免疫反应。VG在磁珠上的固定化包括将VG的氨基部分偶联到磁珠上，然后在涂有聚乳酸薄膜的磁珠表面激活羧酸部分。将固定在VG上的磁珠引入并捕获到装有钕磁体的免疫反应池中，然后将含有恒定浓度的碱性磷酸酶标记的抗VG抗体的VG样品溶液和对氨基苯基磷酸(PAPP)溶液依次引入免疫反应池中。将PAPP与碱性磷酸酶在抗体对氨基苯酚上的酶反应产物送入安培检测器，检测电压为+0.2Vvs。银/氯化银参比电极。用不同浓度的标准VG样品溶液(0-500 ppb)绘制VG浓度的对数与安培检测器的峰电流的对数，得到S型校准曲线。分析所需的时间不到15分钟。
A rapid and sensitive immunoassay for the determination of vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with an amperometric detector and a neodymium magnet. Magnetic beads, onto which an antigen (Vg) was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of magnetic beads in an immunoreaction cell were controlled by means of the neodymium magnet and by adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an alkaline phosphatase (ALP) labeled anti-Vg monoclonal antibody between the fraction of Vg immobilized on the magnetic beads and Vg in the sample solution. The immobilization of Vg on the beads involved coupling an amino group moiety of Vg with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactate film. The Vg-immobilized magnetic beads were introduced and trapped in the immunoreaction cell equipped with the neodymium magnet; a Vg sample solution containing an ALP labeled anti-Vg antibody at a constant concentration and a p -aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. The product of the enzyme reaction of PAPP with ALP on the antibody, p -aminophenol, was transported to an amperometric detector, the applied voltage of which was set at +0.2 V vs . an Ag/AgCl reference electrode. A sigmoid calibration curve was obtained when the logarithm of the concentration of Vg was plotted against the peak current of the amperometric detector using various concentrations of standard Vg sample solutions (0 - 500 ppb). The time required for the analysis is less than 15 min.