间接竞争酶联免疫法测定鱼体中的亚甲基蓝

Detection of methylene blue in fish by indirect competitive enzyme linked immunosorbent assay

中文关键词:  亚甲基蓝  多克隆抗体  酶联免疫法  鱼

英文关键词:methylene blue  polyclonal antibody  enzyme linked immunosorbent assay  fish

中文摘要:

      目的 建立间接竞争酶联免疫法(indirect competitive enzyme linked immunesorbent assay, IC-ELISA)快速检测鱼体中亚甲基蓝的分析方法。方法 以牛血清白蛋白(bovine serum albumin, BSA)为载体蛋白, 以天青C (Azure C)为半抗原, 采用戊二醛法合成免疫原Azure C-BSA, 免疫新西兰大白兔, 制备多克隆抗体。通过对包被浓度、抗体浓度和二抗浓度等一系列实验条件的优化, 建立检测鱼体中亚甲基蓝的IC-ELISA法。结果 获得的多克隆抗体特异性强、灵敏度高, 在5~400 ng/mL范围内与抑制率之间线性良好, 线性回归方程为Y=0.3658X–0.1867 (r2=0.9836), 半抑制浓度(50% inhibition concentration, IC50)为75.4 ng/mL, 检测限为8.3 ng/mL, 样品添加回收率为77.2%~79.3%, 相对标准偏差(relative standard deviation, RSD)为5.3%~8.1%。结论 该方法灵敏度高、特异性强, 可用于鱼体中亚甲基蓝的快速检测。

英文摘要:

      Objective To develop a method for the determination of methylene blue in fish by indirect competitive enzyme linked immunosorbent assay (IC-ELISA). Methods Using bovine serum albumin (BSA) as a carrier protein, Azure C as a hapten, the immunogen Azure C-BSA by glutaraldehyde method was synthesized, then the New Zealand white rabbits were immunized to prepare polyclonal antibody. IC-ELISA method was developed for the determination of methylene blue in fish by optimizing the coating concentration, antibody concentration and secondary antibody concentration. Results The polyclonal antibody was highly specific and sensitive. The linear regression equation was Y=0.3658X–0.1867 (r2=0.9836), with a good linearity with inhibition rate in the range of 5–400 ng/mL. The 50% inhibition concentration (IC50) value and detection limit were 75.4 and 8.3 ng/mL, respectively. The recovery of sample addition was 77.2%–79.3%, with relative standard deviation (RSD) of 5.3%–8.1%. Conclusion This method has high sensitivity and specificity, which could be used for the rapid detection of methylene blue in fish.