Study On Applicability Of Polyclonal Antibodies Of NAGase From Daphnia Magna To Detect Abundance Of Aquatic Arthropods

For purpose of developing antibodies to quantify β-N-Acetyl-D-glucosaminidase (NAGase) released by Daphnia magna in its development and reproduction, a type of electrophoretical pure NAGase was purified from the animals by a four-steps procedure:i.e. O.1mol/L PBS (pH7.4, with0.2mol/L NaCl) extraction,35-75%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 yield, counted from crude extract, were20.09fold and25.2%, respectively. Specific activity of the purified NAGase (approximately6.5mg) was estimated to be2243380U/mg protein. Molecular weight of the subunits was128,99, and71kD, respectively. The values of Km and Vm were determined to be0.300mmol/L and0.072μ mol/mg/min, respectively. The optimum pH was7.0and the optimum temperature was45℃. The enzyme was relatively stable at pH ranging from4.2to7.8. Activity of the enzyme remained stable even it was incubated for30min at35℃.As for the free enzyme separated from the media, the optimum pH was7.4and optimum temperature was40℃. The enzyme was relatively stable at pH ranging from4.6to7.8. Activity of the enzyme remained stable even it was incubated for30min at temperature of40℃. This suggests that the purified NAGase and the free NAGase separated from the media were similar in terms of their responses towards temperature and pH, and it is reasonable to infer that the two types of enzyme belong to the same molecular form.The purified enzyme was taken to immune mice of BALB/c to gain the antiserum. After that method of indirect non-competitive enzyme-linked immunosorbent assay (ELISA) was developed for detecting of immunoreactive content of NAGase (NAGase-IR) in D. magna.Methodological studies indicated that the optimal concentration for antigen and antibody were9.1ug/mL and1:8000, separately. The standard curve for quantifying of NAGase-IR could be expressed as Y=1.650·(1-e-0.172x) with r=0.9984and S=0.0255. The minimal detection limit of the method was estimated to be2.12ng/mL. The coefficient variation of the established ELIS A was less than10%. Fortified recovery of the method was95.4-97.6%.The antiserum was employed to react with free NAGase released by several species of aquatic Arthropod. The cross reactions were measured to be87.5%,7.75%,6.25%,72.5%,80%and0.25, respectively, for D. magna, Mosquito, Chironomus sp., Cyclops sp.,Simocephalus exspinosus and Chlorophyta. As antigens, the NAGase released by D. magna, Chironomus sp., Cyclops sp., Simocephalus exspinosus and Chlorophyta had low reaction with rabbit-anti-human antibody developed from gene recombination polypeptide of β-N-Acetyl-D-glucosaminidase (0.25%-19.09%).The non-competitive ELISA was employed to detect impact of sub-lethal concentrations (0.02μg/L,0.02μg/L,0.81μg/L) of thiourea insecticide diafenthiuron on population development of D. magna. Result of the21d experiment showed that the free NAGase released by the populations declined significantly compared with that of the control. The rate of the declinations was in accord with that of the activity of the enzyme for all of the three concentrations. This suggests that the NAGase-IR can serve as indicator of population biomass.