兔血清乙酰胆碱酯酶用于农药残留速测的最佳条件研究

Study on Optimal Conditions of Assaying Pesticides Residue through Inhibition of Rabbit Serum Acetylcholinesterase

研究了磷酸缓冲液pH值、反应时间、反应温度、底物、酶量对兔血清乙酰胆碱酯酶(AChE)活力测定的影响,结果表明,供试AChE活力最佳条件是:磷酸缓冲液(pH8.0)3.0ml,显色剂0.6%二硫双对硝基苯甲酸(DTNB)50μl,0.75%酶液(pH8.0)100μl,反应温度35℃,混匀稳定后加入底物1%碘化硫代乙酰胆碱(BTCI)50μl,反应180s后在412nm处测定光密度。在上述条件下,测定表明有机磷杀虫剂丙硫磷(Prothiofos)浓度在0.0125～0.4μg/ml,保育时间5～30min之间与AChE抑制率之间存在良好的相关性,可根据检测需要调整保育抑制时间。本试验结果将为应用兔血清AChE研制、改进有机磷和氨基甲酸酯类农药残留速测卡,实现农产品、食品农药残留快速检测提供重要参考。

The conditions were studied for assaying of acetylcholinesterase （ACHE） activity from rabbit serum, as the target enzyme of organophosphate and carbamate pesticide. The results showed that the optimal reaction conditions were： 3.0ml of pH8.0 buffer solution of phosphoric acid, 50μl of 0.6% DTNB and 100μl of 0.75% AChE were mixed with 50μl of 1% BTCI at 35℃, and then value detected of OD412 after 180s. The AChE activity showed good inhibition effect on the prothiofos, an organophosphate pesticide, with 0.0125 ～0μg/ml as the concentration range and / or 5-30 min as incubation time under the above condition. It indicated that the assay time could be adjusted. These results would be important to the R＆D of rapid detection card for assaying pesticide residue by rabbit serum ACHE and improveing test accuracy.