水中硝基苯胺类化合物酶免疫化学分析技术研究

采用戊二醛法, 将4-硝基苯乙胺与牛血清蛋白(BSA)和卵清蛋白(OVA)共价偶联, 分别合成了免疫原4-硝基苯乙胺-BSA和包被原4-硝基苯乙胺-OVA, 经紫外分光光度计及飞行时间质谱扫描鉴定. 用合成的免疫抗原免疫新西兰大白兔, 并用合成的包被原进行间接竞争酶联免疫(ELISA)试验, 获得的抗血清效价达1∶32000. 方阵实验确定了包被抗原最佳浓度(0.5 mg/L)及抗血清最佳稀释度(1∶6000), 并建立了间接竞争ELISA方法. 工作曲线表明在1～1000 μg/L浓度范围内呈良好的线性关系, 该法IC₅₀值为(52.73±2.67) μg/L, 检测限为5.12 μg/L. 其它类似结构不干扰硝基苯胺的测定. 成功地建立了硝基苯胺类化合物的间接竞争酶免疫化学分析方法．

Study on Enzyme Immuno-Chemistry of Nitroaniline in Water

4-Nitrophenethylamine was chemically linked to bovine serum albumin (BSA) or ovalbumin (OVA) by coupling with BSA through glutaraldehyde to form immunizing antigen and coating antigen. The complete antigen was determined by UV spectrometry and MALDI-TOF MS scanning method. Polyclonal antibodies against 4-nitrophenethylamine were produced from rabbits, and the antiserum was determined with an ELISA test, which showed that the titer of the antibodies was 1∶32000. The most appropriate concentration of coating anti¬gen was 0.5 mg/L, and that of antiserum was 1∶6000. Indirect competitive ELISA (icELISA) was established with this antiserum. The curve had a favorable linear relation within the concentration range of 1～1000 μg/L. The curve indicated that IC₅₀ was (52.73±2.67) μg/L, the lowest detection limit was 5.12 μg/L. No analogues of nitroaniline was found to interfere with the analysis of nitroaniline. Thus, the indirect competitive enzyme immuno-chemistry of nitroaniline was developed successfully.