Chemical Enzymology Department, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, Moscow, Russia.
J Agric Food Chem. 2010 Mar 24;58(6):3284-9. doi: 10.1021/jf904338f.
An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) of sulfamethoxypyridazine (SMP) was developed. The conjugates of streptavidin with cationic horseradish peroxidase (HRP) and anionic soybean peroxidase (SbP) were used in CL-ELISA for the detection of biotinylated anti-SMP antibodies. For streptavidin-HRP conjugate-catalyzed chemiluminescence measured 20 s after the initiation of the enhanced chemiluminescence reaction (ECR), the limit of detection (IC(10)), the IC(50) value, and the working range in CL-ELISA of SMP are 0.3, 12.4, and 1.2-85.0 ng/mL, respectively. An increase in the time interval between the ECR initiation and the luminescence measurement results in the loss in the quality of analytical measurements because of the time-dependent quenching of chemiluminescence typical of the HRP-catalyzed ECR. In the case of SbP-based CL-ELISA of SMP, the limit of detection, the IC(50) value, and the working range (0.025, 0.17, and 0.045-0.63 ng/mL, respectively) are better than those for HRP-based CL-ELISA. Furthermore, the analytical parameters of SbP-based CL-ELISA remain unchanged during a long period of time (for at least 30 min). The recovery values from four spiked milk samples with different concentrations of SMP in SbP-based CL-ELISA vary from 70 to 130%.
建立了磺胺二甲嘧啶（SMP）的间接竞争化学发光酶联免疫吸附测定法（CL-ELISA）。在 CL-ELISA 中，使用链霉亲和素与阳离子辣根过氧化物酶（HRP）和阴离子大豆过氧化物酶（SbP）的缀合物来检测生物素化的抗 SMP 抗体。对于链霉亲和素-HRP 缀合物催化的化学发光，在增强化学发光反应（ECR）开始后 20 s 进行测量时，SMP 的 CL-ELISA 的检测限（IC（10））、IC（50）值和工作范围分别为 0.3、12.4 和 1.2-85.0 ng/mL。增加 ECR 启动和发光测量之间的时间间隔会导致分析测量质量下降，因为 HRP 催化的 ECR 典型的化学发光时间依赖性猝灭。在基于 SbP 的 SMP CL-ELISA 中，检测限、IC（50）值和工作范围（分别为 0.025、0.17 和 0.045-0.63 ng/mL）优于基于 HRP 的 CL-ELISA。此外，SbP 基于 CL-ELISA 的分析参数在很长一段时间内保持不变（至少 30 分钟）。在基于 SbP 的 CL-ELISA 中，从四种不同浓度 SMP 加标牛奶样品中回收的值在 70%到 130%之间变化。
