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

开发一种快速、高灵敏度、简单的固相萃取测定环境中内分泌干扰物的方法

基本信息

批准号：
12555238
负责人：
MASADOME Takashi
金额：
$ 7.04万
依托单位：
Ariake National College of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

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.

将抗体固定化微球转移到喷射环（JR）池中，采用顺序注射分析（SIA）技术测定鲤鱼卵黄原蛋白（Vg）。该测定是基于夹心免疫测定法，其中使用抗鲤鱼Vg抗体和鲤鱼Vg之间的两种类型的反应。即，将用于第一反应步骤的抗体固定在微珠（Sephadex珠）上，并且在反应的第二步骤中使用用辣根过氧化物酶（HRP）标记的抗体。过氧化氢和邻苯二胺（OPD）的混合溶液被用作反应中的发色团源。将微珠固定化抗体、Vg分析物、HRP标记抗体和显色溶液以程序化顺序自动引入SIA系统的JR池中，并使用氧化OPD产物的吸光度来确定存在的Vg的量。考虑到对Vg测定的灵敏度，第一步和第二步免疫反应的最佳孵育时间分别确定为120和60 min。在此条件下，氧化OPD的吸光度与Vg浓度之间具有良好的线性关系。在此系统中，用于测定Vg的检测下限为约5 ng ml-1。该方法简便、准确，适用于Vg的测定。