Development of a rapid, high sensitive, simple method for the determination of endocrine disrupters in environment using solid phase extraction

采用序贯注射分析（SIA）技术，将固定抗体的微珠转移到喷射环（JR）细胞中，测定鲤鱼卵黄原蛋白（Vg）。测定是基于夹心免疫分析法，其中两种类型的反应之间的抗鲤鱼Vg抗体和鲤鱼Vg使用。即，将第一步反应的抗体固定在微球（Sephadex珠）上，用辣根过氧化物酶（HRP）标记的抗体进行第二步反应。过氧化氢和邻苯二胺（OPD）的混合溶液作为反应中发色团的来源。将微珠固定抗体、Vg分析物、hrp标记抗体和显色液按程序自动导入SIA系统JR细胞，氧化后的OPD产物吸光度测定Vg的存在量。考虑到对Vg测定的敏感性，确定第一步和第二步免疫反应的最佳孵育时间分别为120和60 min。在此条件下，氧化后的OPD的吸光度与Vg浓度呈良好的线性相关。该系统测定Vg的最低检出限约为5 ng ml 1。本方法为Vg的测定提供了一种简便、准确的方法。

A sequential injection analysis (SIA) technique, in which antibody-immobilized microbeads were transferred to a jet ring (JR) cell, was used in determination of carp vitellogenin (Vg). The determination is based on a sandwich immunoassay in which two types of reactions between anti carp Vg antibodies and carp Vg are used. Namely, the antibody for the first reaction step was immobilized on microbeads (Sephadex beads), and an antibody labeled with a horseradish peroxidase (HRP) was used in the second step of the reaction. A mixed solution of hydrogen peroxide and o-phenylenediamine (OPD) was used as the source of the chromophore in the reaction. The microbeads-immobilized antibody, Vg analyte, HRP-labeled antibody and the color developing solution were introduced automatically into the JR cell of the SIA system in a programmed sequence, and the absorbance of the oxidized OPD product was used to determine the amount of Vg present. The optimal incubation times for the immunoreaction for the first and the second steps were determined at 120 and 60 min, respectively, taking into account the sensitivity to the Vg determination. Under these conditions, a good linear correlation was obtained between Vg concentration and the absorbance of the oxidized OPD. The lower detection limit for the determination of Vg was about 5 ng ml 1 in this system. The method developed here represents a simple, accurate method for the determination method of Vg.