| Mylabris Cichorii Linnaeus, also known as the yellow and black cantharis, belongsto Coleoptera Meloidae insects. Modern clinical studies found that Mylabris CichoriiLinnaeus has a unique effect in the treatment of cancer and incurable diseases. Itsanti-cancer active ingredient is cantharidin which cantharis contained. So far, theresearch and reports about cantharidin transit in vivo, distribution, is very little. Eventheir biosynthesis and metabolism studies are inadequate.At early the gravimetric method and the acid-base titration were used for detectingcantharidin at home and abroad. In recent years, the more accurate methods aredeveloping such as GC-MS, HPLC and reversed-phase high performance liquidchromatography. But these methods are necessary instruments for price more expensiveand complicated to operate, require professional techniques and therefore can only beused in laboratory testing.Enzyme linked immunosorbent assay method because of its high sensitivity,specificity, good detection convenient, short cycle, and low-cost, widely used in thedetection of proteins of biological macromolecules. Early lack of immunogenicity, wecan not produce antibodies for the detection of small molecules which is mainlydependent on in the chromatographic analysis. Recently, researchers can make smallmolecules and protein macromolecules to synthetic complete antigen and obtainantibodies, making the use of immunological detection of small molecules becomepossible.The purpose of this study is to establish a rapid, sensitive the cantharidin indirectcompetitive ELISA method, and its test results conducted evaluation. The method couldprove help for cantharidin rapid and accurate detection and be basis for furtherdevelopment of cantharidin detection kit. In the meantime, using immunofluorescencewe study the cantharidin distribution of Mylabriscichorii Linnaeus in vivo, and providestrong support for the further study of cantharidin synthesis mechanisms, as well as theMeloidae insects cantharidin metabolic pathways. The study has been made in thefollowing main results:①Using already prepared the immune serum of rabbit anti-cantharidin linkedwith BSA, We obtaine high-titer antibody against cantharidin, then establishing theoptimized cantharidin indirect competitive ELISA. The cantharidin indirect competitive Elisa detection methods is: Coating microtiter plates with PBS (pH7.4) containing theCA-OVA,100μl/hole, and incubated at4℃overnight. Put in a closed1%gelatinsolution (200μl/well) in microtiter plates, and incubated at37℃for2h. While addingcantharidin standard (pH7.4, phosphate buffer dilution) and antibody againstcantharidin (1:200dilution), the50μl/hole, and incubated for1h at37℃forcompetition. Then the goat anti-rabbit IgG with HRP labeled (dilution according to1:5000with PBS) was mixed,100μl/hole, and incubated at37℃for1h. Add toconfigured OPD chromogenic fluid,100μl/hole, shading incubated at37℃for15min.Added to the reaction terminate liquid (2M concentrated sulfuric acid),50μl/hole.Place in the microplate reader, reading at492nm wavelength.The detection system of the standard curve equation was y=-13.421x+102.45, R2=0.988, detection range:0.1μg/ml to24μg/ml. The minimum detection limit is0.01μg/ml, intra-and inter-coefficients of variation were4.27%and4.56%.②About the immunofluorescence detection of the body’s main organs ofMylabriscichorii Linnaeus, we use IPP6.0and the DPS to analysis results, found that inthe experimental group of female body organs and tissues, the reproductive system ofthe average optical density values were significantly higher than that of the intestinalsystem and in reproductive system the highest average optical density value belong tothe seminal vesicle. Among the gonad, intestinal system and testes in the experimentalgroup of the male body and testis, there were significant differences in the mean opticaldensity of the intestinal system, but there is no significant difference in the averageoptical density values between the intestinal system and testes. In the whole insectbody’s tissues and organs, the average optical density of the testis is the highest. Theintestinal system of the male in the average optical density values is significantly higherthan females. In the comparison of the reproductive system, the average optical densityof males is higher than females, but there is no significant difference. It can bespeculated, the its main cantharidin storage site is located in the reproductive system,and by the high expression of the optical density values in the female seminal vesiclecan be sure that, its cantharidin from the male, is transferred by mating. Adult intestinalsystem in the average optical density values, second only to the reproductive system, thegut may also be one of the Mylabris Cichorii Linnaeus parts store cantharidin. |
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