Schmitt A, Hingst V, Erdinger L, Helmbold W, Sonntag H G
Abt. Hygiene und medizinische Mikrobiologie, Universität Heidelberg.
Zentralbl Hyg Umweltmed. 1992 Oct;193(3):272-86.
Since December 1990, strict limits for pesticide content in drinking water have been in effect in the FRG. These limits have to be enforced and controlled regularly. Because the classical analytical methods, like GC/MS and HPLC are relatively expensive and complicated, concentration and clean up of the samples are necessary. Therefore, the significance of Enzyme Immunosorbent Assays has increased in environmental analysis. The topics of our research are synthesis of suitable Metolachlor conjugates for immunization and as coating antigen, as well as development of an Enzyme Immunosorbent Assay. The chloroacetanilide Metolachlor was coupled with an AMSA-Spacer on Sheep IgG and with AHT-Spacer on BSA. The epitope density was measured photometrically with TNB-derivate. Polyclonal antibodies were produced in white New Zealand rabbits. Metolachlor-AMSA-Sheep IgG conjugate was served as immunogen. The test is indirect and competitive. Microtiter plates are coated with the coating antigen (Metolachlor-AHT-BSA conjugate). In the next step the Metolachlor sample competes with the coating antigen for the antibodies. Detection of bounded antibodies is indirect with a peroxidase-labelled secondary antibody. The chromogenic substrates ABTS, TMB and OPD were tested. TMB showed the best qualities, fast turnover, as well as good and steady colour. Titer of serum and crossreactivity of other chloroacetanilide pesticides and their metabolites were tested. With the exception of Alachlor and Dimethachlor, no pesticide showed a high interaction with serum. Quantitative interpretation was done with the logit/log transformation, which allows comparison of different sera and test series. The antibodies are specific but the affinity is not good enough to detect the limits assigned by the "deutsche Trinkwasserverordnung". Raising the concentration, as is done in classical analytical methods, would solve the problems so that the test can be used as a screening test.
自1990年12月以来，德国对饮用水中的农药含量实施了严格限制。必须定期执行和控制这些限制。由于气相色谱/质谱联用仪（GC/MS）和高效液相色谱（HPLC）等传统分析方法相对昂贵且复杂，因此需要对样品进行浓缩和净化。因此，酶联免疫吸附测定法在环境分析中的重要性日益增加。我们的研究主题是合成适合用于免疫和作为包被抗原的异丙甲草胺缀合物，以及开发一种酶联免疫吸附测定法。氯代乙酰苯胺类异丙甲草胺与羊免疫球蛋白（IgG）上的AMSA间隔臂以及牛血清白蛋白（BSA）上的AHT间隔臂偶联。用TNB衍生物通过光度法测量表位密度。在新西兰白兔体内制备多克隆抗体。异丙甲草胺-AMSA-羊IgG缀合物用作免疫原。该检测为间接竞争法。微量滴定板用包被抗原（异丙甲草胺-AHT-BSA缀合物）包被。下一步，异丙甲草胺样品与包被抗原竞争抗体。用辣根过氧化物酶标记的二抗间接检测结合的抗体。测试了显色底物ABTS、TMB和OPD。TMB表现出最佳性能，周转速度快，颜色良好且稳定。检测了血清效价以及其他氯代乙酰苯胺类农药及其代谢物的交叉反应性。除了甲草胺和二甲草胺外，没有其他农药与血清表现出高相互作用。采用logit/log变换进行定量解释，这使得不同血清和测试系列之间能够进行比较。抗体具有特异性，但亲和力不足以检测到《德国饮用水条例》规定的限值。像传统分析方法那样提高浓度将解决这些问题，从而使该检测可作为一种筛查检测方法。
