Preparation Of Polyclonal Mice Antiserum Against Goldfish Vitellogenin And The Application In The Detection Of Pesticide With Environmental Estrogens Activities

Pesticide production and consumption in China are tremendous. Annual production is more than 500,000 tons with the annual consumption of 400,000 tons or more since mid-1990s and they are ascending year by year. 80% use of pesticide could affect the environment directly resulting in the severe environment pollution. Most pesticides not only have toxicity, but also disturb endocrine system. Large proportion of the chemical with environmental estrogen activity is pesticide. 86 of 125 or 68.8% of environmental estrogen reported by World Wide Fund for Nature is pesticide. Thus, it is important to establish the environment estrogen screening and evaluation system in order to reduce the environment pollution. Nowadays, fish vitellogenin induction, recombinant yeast bioassay, and yeast-two hybrid technique are the relatively mature screening and evaluation system around the world. Male fish Vtg is widely used as an important bioindicator to detect environmental estrogens. However, the Vtg qualitative and quantitative detection depends on the preparation of antibody. Because fish Vtg in different species has species difference, it is necessary to prepare diverse antibody to detect Vtg. We have acquired the polyclonal rabbit antiserum against goldfish, in order to improve the specificity of the antibody. We selected goldfish as model animal preparing the polyclonal mice antiserum against goldfish Vtg, and screened the environmental estrogen activity of eight species of pesticides by applying the western-blot technique. The result shows as follows:Goldfish was received 0.05 mg/(g·BW) 17β-estradiol intraperitoneal injection. After 14 day induction, its blood drawn from caudal vessel was isolated and purified by gel filtration and ion-exchange chromatography. After native-PAGE, we recognized this protein as Vtg by the CBB G250-, phosphor-, lipo-, and glycol-protein staining methods. The vitellogenin showed a molecular weight of approximately 460kDa by Native-PAGE, and it was broken into four subunits in SDS-PAGE, with molecular weight of approximately 130,106,81 and 54 kDa, respectively.Purified Vtg was used to immunize male mice by intraperitoneal injection. After the first immunity, the second was implemented 2 weeks later. In the next 3 weeks, it was implemented once a week. Each male mice was injected 50μg purified Vtg(Vantigen: VFreund's complete adjuvant=1:1) each time. After the fifth injection, the antigen was injected twice a week to enhance the immune effect with the immune cycle was 6 weeks. The antiserum was collected and isolated. By agar double immunodifuson, the effect of the polyclonal mice antiserum against goldfish Vtg was measured to be at least 1:16. With the western-blot for Vtg detection, the detection lower limit is 1μg/L(polyclonal antiserum 1:500 dilute).Using the polyclonal mice antiserum against goldfish Vtg as the first antibody and goat anti-mose IgG/HRP as the second antibody,with the western-blot, we screened the environmental estrogen activity of eight pesticides (which are Beta cypermethrin, triadimefon, xiujwelin, omethoate, dichlorvos, monocrotophos, methomyl, and glyphosate). The positive reaction was detected between the polyclonal mice antiserum against goldfish Vtg and male goldfish plasma which was exposured by Beta cypermethrin, triadimefon, xiujwelin, omethoate, dichlorvos, and monocrotophos, with three doses (0.01 mg/L, 0.1 mg/L and 1.0 mg/L), respectively. The result showed that they could induce male goldfish synthesizes Vtg, so they are potential environmental estrogen. No reaction was detected when male goldfish plasma was exposured by Methomyl and glyphosate. Thus, they may not have environmental estrogen activity.In conclusion, goldfish Vtg induced by E2 was isolated and purified. Male mice was immunised by purified Vtg to acquire the polyclonal mice antiserum against goldfish Vtg, which was used to screen the environmental estrogen activity of eight pesticides. The isolated and purified goldfish Vtg showed high purity. The polyclonal mice antiserum against goldfish Vtg had a better specificity against goldfish Vtg. Beta cypermethrin, triadimefon, xiujwelin, omethoate, dichlorvos, monocrotophos may have environmental estrogen activity. Methomyl and glyphosate may not have environmental estrogen activity.