Study On Preparation And Applicability Of Polyclonal Antibody Of β-N-acetyl-D-glucosaminidase From Chironomus Kiiensis

Chironomids and other aquatic arthropods released N-acetyl-β-D-glucosaminidase (NAGase) which can be used as biomass indicator into the water with molting fluid. In order to study the content of NAGase that was released into the aqueous environment by Chironomus kiiensis, a type of electrophoretical pure NAGase was purified from the animals by a four-step procedure:0.05mol/LTris-HCl (pH7.2) extraction,60-100%ammonium sulfate precipitation, anion exchange column chromatography of DEAE Sephadex A-25, and molecular sieve column chromatography of Sephadex G-150. The overall purification factor and the yield, counted from crude extract, were33.03fold and10.86%, respectively. Specific activity of the purified NAGase was estimated to be8.06U/mg protein. Molecular weight of the subunits was13kD. The values of Km and Vm were determined to be0.1003mmoI/L and0.2512μmol/(L·min), respectively. The optimum pH was6.0and the optimum temperature was45℃. The purified enzyme was relatively stable at pH ranging from5.0to8.0. Activity of the enzyme remained stable even it was incubated for60min at50℃.The purified NAGase was taken to immune two New Zealand white rabbits to collect the ant(?)um. After that method of indirect non-competitive enzyme-linked immunosorbent assay (ELISA) was developed for detecting of immunoreactive content of the NAGase (NAGase-IR) from Chironomus kiiensis. Methodological studies indicated that the optimal concentration for the antigen and the rate of dilution for the antibody were7.64μg/mL and1:10000, separately. The standard curve for quantifying of the NAGase-IR could be expressed as Y=1.0886(1-e-0.2762x) with r2=0.9951and S=0.0257. The minimal detection limit of the method was estimated to be49.56ng/mL. The coefficient variation of the established ELISA was less than10%. Fortified recovery of the method was92.18-97.59%.The antiserum was employed to react with NAGase from several species of aquatic arthropod. The cross reactions were measured to be4.41%,3.12%,3.40%,4.17%,3.23%,7.50%and<0.2%, towards Daphnia carinata, Simocephalus vetulus, Moina macrocopa, Aedes albopictus, Dolerocypris sinensis, Macrobrachium nipponense, and Chlorella vulgaris. These results respected the antibody had a higher specificity which was not only between crustacean but insects. Rabbit-anti-human polyclonal antibody and Rabbit-anti-daphnia polyclonal antibody were employed to react with the NAGase from the Chironomus kiiensis. It was found that the cross reaction was1.2%and4.68%, respectively.The non-competitive ELISA was employed to detect content of NAGase that was released by Chironomus kiiensis larvae under the stress of chlorpyrifos, fenvalerate, and abamectin, respectively. It showed that release of the NAGase dropped along with the restrain in growth of Chironomus kiiensis larvae. After12day exposure, EC50of the three pesticides were1.2012,0.0043, and0.6281μg/L, respectively, as it was counted with-the polyclonal antiserum of the NAGase, and it was slightly lower than the EC50(i.e.1.4765,0.0051, and0.6756μ g/L) as it was counted with activity of the enzyme. When the mortality of Chironomus kiiensis larvae was used as the end point, LC50of three pesticides was4.8171,0.0954, and2.1340μg/L, which higher than both of the former.Results of the study suggested that the antiserum of the NAGase could be employed to detect, specifically and sensitive the effect of pesticides on bio mass of Chironomus kiiensis.