Antibody-based methods for surfactant screening
Antibody-based methods for surfactant screening
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DOI：
10.1007/s002160101079
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发表时间：
2001-10-01
期刊：
影响因子：
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通讯作者：
Emnéus, J
中科院分区：
文献类型：
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作者：
Fránek, M;Zeravík, J;Emnéus, J
本简要概述总结了欧洲 INCO-Copernicus 项目 BIOTOOLS 两年来获得的基于免疫测定的结果。该项目旨在简化表面活性化合物（SAC）的检测程序，使用基于抗体的方法，即基于微量滴定板的酶联免疫吸附测定（ELISA）、偏振荧光免疫测定（PRA）和酶流注射免疫测定（FIIA）。用与蛋白质免疫原缀合的五种不同的磺基苯基部分和三种对羟基苯基部分对三十三只兔子进行免疫，以产生针对直链烷基苯磺酸盐（LAS）和壬基酚（NP）的分析抗体。尽管大多数抗体在间接 ELISA 中表现出结合反应，但只有少数抗体表现出所需的检测灵敏度。在间接 ELISA 中，LAS 的最佳抗体在 IC50 19.8 mug L-1 时表现出 50% 的结合抑制。使用过氧化物酶示踪剂的直接 ELISA 观察到类似的抑制作用。针对 NP 的抗体允许建立在 mg L-1 范围内操作的间接测定。描述了使用同质 PFIA 筛选 NP 和 LAS 的快速且简单的方案。 10 个样品的测定时间为 7 分钟，因此可以在 mg L-1 水平上快速检测所选 SAC。开发了一种通用竞争性 FIIA 系统，使用蛋白 G 柱分离游离和抗体结合的 β-半乳糖苷酶 (β-Gal) 示踪剂，用于筛选 LAS、NP 和壬基酚十乙氧基化物 (NPEO10)。 FIIA 的样品吞吐量 (STP) 为每小时 5-10 个样品，LAS、NP 和 NPEO10 的检测限 (LOD) 分别为 19.5、52 和 2.4 mug L-1。开发的 FIIA 应用于暴雨和地表水。
This brief overview summarises the immunoassay-based results obtained in the course of two years of the European INCO-Copernicus project BIOTOOLS. The project is aimed at simplifying the procedures for detection of surface active compounds (SAC) using, among others, antibody-based methods, i.e., microtiter plate-based enzyme-linked immunosorbent assays (ELISA), polarisation fluoro immunoassays (PRA), and enzyme flow injection immunoassays (FIIA). Thirty-three rabbits were immunised with five different sulphophenyl moieties and three p-hydroxyphenyl moieties conjugated to protein immunogens to produce analytical antibodies against linear alkylbenzene sulphonates (LAS) and nonylphenol (NP). Although most of the antibodies exhibited binding reaction in indirect ELISA, only a few showed the required assay sensitivity. The best antibodies for LAS exhibited a 50% binding inhibition at IC50 19.8 mug L-1 in indirect ELISA. Similar inhibition was observed for direct ELISA using peroxidase tracers. Antibodies against NP allowed the establishment of an indirect assay operating in the mg L-1 range. A rapid and simple protocol for the screening of NP and LAS using homogeneous PFIA is described. The assay time for 10 samples was 7 minutes, thus allowing fast detection of the selected SAC at the mg L-1 level. A generic competitive FIIA system, using a protein G column for separation of free and antibody-bound beta -galactosidase (beta -Gal) tracer, was developed for the screening of LAS, NP, and nonylphenol decaethoxylate (NPEO10). The FIIA had a sample throughput (STP) of 5-10 samples per hour, with limits of detection (LOD) for LAS, NP, and NPEO10 of 19.5, 52, and 2.4 mug L-1, respectively. The developed FIIAs were applied to spiked rain and surface water.