Study Of Maduramicin Immunological Rapid Detection Method

Maduramicin(MD) is a kind of polyether ionophore antibiotics, which isseparated from Actinomadura yumaensis’ fermentation product by Liu and Labeda in1983. MD is widely added in animal feed as result of wide anticoccidial spectrum,high activity, small dosage and coccidium resisting. MD is not easy to produce drugresistance. It can cause people who eat these animal tissues which contain MDvasodilation, particularly evoked that heart coronary artery dilatation and increasedblood flow. It can also cause people who are suffering from coronary artery diseaseprevalence of coronary artery ectasia, deterioration, which bring great harm to people’shealth. With the development of science and technology and national attention, moreefficient detecting technology is developing and in application. There aremicrobiological method, ultraviolet spectrophotometer, atomic absorptionspectrophotometry, thin layer chromatography, high performance liquidchromatography, liquid chromatography-mass spectrometry technology andimmunological detection methods in current detection method. It is significant for foodsafety to establish accurate, rapid, and effective determination of MD residueanalytical method.Based on the analysis of MD immunological characteristics, MD monoclonalantibody is prepared. Indirect competitive ELISA kit is assembled. At the mean time,its performance is evaluated. Common rapid sample treatment method is established,which lay a foundation for the application of MD rapid detection kit. The main resultsare as follows:1. Preparation and identification of MD artificial antigenUsing mixed anhydride method to couple BSA and OVA, and artificialImmunogen MD-BSA. Coating antigen MD-OVA are synthesized. It showed that theMD-BSA is coupled successfully by identification of spectrophotometry(UV), gelelectrophoresis(SDS-PAGE) and ELISA after immunization of mice with antiserum. 2. Preparation of monoclonal antibodies and immunological characterization ofMDUsing ELISA method to select rats that for hybridoma technology to make a cellfusion of immune spleen cells of mice and NS0tumor cells. Five specific, sensitiveand hybridoma cell lines1D11,2F9,3A5,4H5,4D9were screened after detection,screening and clone. The McAb of4H5showed good sensitivity with an IC50of1.455μg/L.25%cross-reactivity to monensin and little or no cress-reactivity to othercompounds. High sensitivity and specificity of MD monoclonal antibodies wasobtained by the test, which can be used in ELISA detection test of MD.3. Development of rapid determination of MD residue methodBased on the anti-SAL McAb and the enzyme-linked immunosorbent assayprinciple, MD residue fast detecting indirect competitive ELISA Kit was developed byMD mAb. The calibration curve of MD-Kit with standard MD inhibitor was typicalsigmoid curve fitted to the four parameters logistic equation with the lowestdetermination of0.186μg/L. The half of the inhibition of IC50is0.175μg/L. Thecoefficient of variation is of less than15%. The chicken liver and chicken meat wereadded5,10,100,200ng/ml MD, respectively. The average recoveries were71.225%and74.550%. The performance of MD is stable, and can be preserved for3monthsunder4℃.