间接竞争酶联免疫法测定鱼体中的亚甲基蓝

目的 本实验旨在建立快速检测鱼体中亚甲基蓝的间接竞争酶联免疫分析方法。方法 以BSA载体蛋白,以天青C(Azure C)为半抗原,利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间接竞争酶联免疫吸附方法(ic-ELISA)。结果 获得的多克隆抗体特异性强、灵敏度高,在5 -500 ng/mL范围内,线性良好,线性回归方程为y=0.3658x-0.1867 (R²=0.98),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%,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。

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

Objective To develop an indirect competitive enzyme linked immunosorbent assay (ic-Elisa) for determination of Methylene blue in fish. Method Using BSA as a carrier protein, Azure C as a hapten, to synthesize immunogen Azure C-BSA by glutaraldehyde method, then the New Zealand white rabbits were immunized to prepare polyclonal antibody. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the determination of Methylene blue in fish by optimizing the coating concentration, antibody concentration and antibody concentration. Results The polyclonal antibody was highly specific and sensitive. The linear regression equation was y = 0.3658x -0.1867 (R_² = 0.98), with the range of 5 to 400 ng/mL. The IC ₅₀ value and detection limit were 75.4 and 8.3 ng/mL respectively. The recovery of sample addition was 77.2%-79.3% , with RSD of 5.3%-8.1% . Conclusion The method has high sensitivity and specificity, which could be used for the rapid detection of Methylene blue in fish.