| An indirect non-competitive ELISA was established to quantify the brain acetylcholinesterase (AChE, EC3. 1,1.7) from a freshwater fish, topmouth gudgeon (Pseudorasbora parva).Effect of two factors (i.e., weight of fish and raising temperature) on immunoreactive AChE protein (AChE-IR) was studied. It also studied the change of AChE specific activity (A), AChE-IR, and A/AChE-IR after the fish was treated singly by an organophosphorus insecticide triazophos and a pyrethroid insecticide fenvalerate at different levels, and the effect of an insecticide fipronil on recovery of AChE-IR inhibited by triazophos. The main results are as follows:Aftera PEG2000/phosphate-salt two aqueous phase extraction, DEAE-Sephadex A-50 and Sephadex G-200 chromatography, the purified AChE from the brain of topmouth gudgeon indicated a single peak by HPLC, and the AChE activity have increased by 533 folds.The coefficient variation of the created indirect non-competitive ELISA was less than 10%, and it held a sensitivity of 0.004 g/mL. Fortified recovery of the method was 90~105%. The method was, therefore, suitabled to assay AChE-IR.At 10.5 ~30 , the AChE specific activity in brain of the topmouth gudgeon was increasing with the raising temperature. The AChE-IR showed the same change as the AChE specific activity; It showed the raising temperature had no influence on the ratio of AChE specific activity to AChE-IR (A /AChE-IR).In brain of topmouth gudgeon which weighed 1.0~1.5g, the AChE specific activity and AChE-IR both significantly less (P<0.05) than that of fish which weighed 3.0~3.5g; 5.0~5.5g, and 7.0~7.5g, but there were no significant difference (P>0.05) among latter three group of fish. There were no significant difference in A/AChE-IR among all the fish (P>0.05) .After treated with 0.1 g /L and 0.4 g /L triazopos, the in vivo AChE specific activity of topmouth gudgeon was significantly higher than that of the control,showing the AChE activity to be induced by triazopos. Inhibition of AChE activity was up to 90% in vivo when up to 3.0~48.0ug /L triazopos was added.; Result showed that the AChE-IR was induced by triazopos at all level. Reversely, the A/AChE-IR of topmouth gudgeon was inhibited by triazopos, even at low exposure (0.1 ug /L or 0.4ug/L) .It was indicated that 0.2-2.0ug /L fenvalerate induced AChE activity and AChE-IR, but A/AChE-IR did not affected.When the topmouth gudgeon was treated with 2.0-8.0jag /L fenvalerate, the inhibition of AChE activity and A/AChE-IR was evident and the toxic symptoms, such as, paralysis, death, and so on, could be observed.It was detected that fipronil delayedrecovery of AChE inhibited by triazophos as the fish was treated with a mixture of fipronil and triazophos. This delay could be explained by the fact that less enzyme was synthesized in presence of fipronil. |
