| It is one important way to bring about new pesticide that looking for new chemical leads and new targets from natural products. Cealangulin V, isolated from Celastrus, is a kind of active compound for insects. It may has new target in the body of insect. Important advance was made to Celastrus and ceangulin V after 20 years studies, but the target of Celangulin V was not verified and the metabolic dynamic of Celangulin V in organism was not touched because of the technique difficulty in testing and analysis of trace Celangulin V in organism. It will solve the difficulty that establishing the immunoassay of Celangulin V.The immunoassay of Celangulin V including the synthesis of artificial antigen, the preparation of antiserum, the immunoanalysis location of Celangulin V and the quantitative immunoassay of C'elangulin V were studied systematicly in this paper. The main results are as follows:1. The mount of 103 mg white powder was obtained after the reaction of Celangulin V with succinic anhydride in anhydrous pyridine, the yield rate was 89.57%. The analysis results of HPLC-MS and NMR showed that the white powder was the derivative of Celangulin V wanted , called as CA-SG.2. The two artificial antigen of Celangulin V-CA-BSA (145 mg) and CA-OVA (203 mg) were synthetized by mixed anhydride method using CA-SG and bovine serum albumin, BSA. and Ovabumin, OVA respectively. The artificial antigen CA - BSA2 ( 160 mg) was prepared by carbodiimide method using CA - SG and BSA. The characterization of hapten-protein conjugates were examined by three methods of ultraviolet (UV) spectrophotometry, sodium dodecyl sulfate -polyacrylamide gel electrophoresis ( SDS - PAGE ) and polyacrylamide gel electrophoresis respectively. The number of hapten molecules per molecule of protein estimated by UV spectrophotometry according to the optical density of compounds at 240nm for three conjugates was 11.6. 10.3 and 10.7 respectively. The molecules weight of three hapten - protein conjugates were about 74851D > 51859D and 74164D respectively.3. BSA-hapten conjugates synthetized by two methods were used as immunogens to immunize rabbits respectively and the polyclonal antisera were all emerged in rabbits. The antisera were not tested by agar double-diffusion method but the antisera were tested by indirect enzyme-linkedimmunosorbent assay (ELISA) from the fourth boosted, and the more the number of boosted times, the hihger the antisera titer.4. The characterization and location of Celangulin V was carried out using the prepared antiseia. The results showed that Celangulin V could not be examined for characterization and the location in brush border membrane vesicles fBBMV) of insect by PAGE-Western blot; that Celangulin V might be characterized but could not be located in BBMV of insects by dot blot.5. The indirect competitive enzyme-linked immunosorbent assay of Celangulin V were carried out using the polyclonal antisera. the results indicated that the probability number of inhibition rale (%) and the logarithmic number of the Ceiangulin V concentration (ug/mL) had good linear relation. A linear equation, y=4.2713+1.3908x (r=0.8169), was established within the concentration limits ranged from 0.1563 to 2.5 u g per milliliter. The tested result evaluaed by the equation of two samples known concentration of Celangulin V indicated that the ELISA of Celangulin V could be carried out using the equation.To sum up above all, The conclusions were drawn: the artificial antigen of Celangulin V were synthetized succeedly by mixed anhydride method and carbodiimide method respectively combining with succinic anhydride method, the polyclonal antisera of Celangulin V were prepared using the synthetic BSA-Celangulin V conjugates. The characterization and quantitacation analysis could be carried out using the prepared antisera.The first sutdies on immunological technique of Celangulin V, a kind of bitonic-insecticide, were carried out in China. The feasibility of immunoassay for Celangulin V was confirmed...
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