Establishment And Detection Of ?-Doping ELISA Rapid Detection Method In Animal Products

?-adrenergic agonist is a class of derivatives of phenylethanolamine.The animal body has to promote muscle tissue growth,affect the fat content,can improve the animal tissue lean meat and increase lean meat production.High residual ?-stimulants,will produce a strong side effects,and even life-threatening.Now there are more than ten kinds of synthetic ?-stimulants,clenbuterol,salbutamol,ractopamine is currently the most commonly used at home and abroad ?-stimulants.At present,Europe and the United States and other countries have limited their use in animal husba ndry,China's various departments,including the Ministry of Health,Ministry of Agriculture,the State Drug Administration issued a joint notice,are clearly defined and banned ?-stimulant drugs in the feed And the use of animal drinking water.Therefore,the development of fast detection methods for ?-stimulants in animal-derived foods is of great importance in ensuring food safety.In order to establish a rapid,sensitive,low-cost,high-throughput ?-agonist residual immunological study and analysis method,this study was based on the analysis of the immunological properties of ?-agonists and the establishment of monoclonal antibodies using ?-agonists Enzyme-linked immunoassay for ?-agonists and their application.The main results are as follows:(1)Establishment of Indirect ELISA Assay for ?-Doping.The indirect ELISA was established by optimizing the concentration of the original,the dilution of antibody,the selection of antibody dilution,the time of antibody reaction and p H value respectively.The concentration of antibody was determined to be 250 ?g/L.The concentration of antibody was 127 ?g/L and the concentration of 0.2 mol / L PB buffer was the best.The optimum p H was 7.2 and the first reaction time was 30 min.The recovery rate of porcine urine is 80.0-104.0%.The method has the advantages of high sensitivity,Good recovery rate and low false positive rate,and the determination result is accurate to meet the detection method of pig urine and other samples.(2)?-stimulant kit performance analysisAn indirect ELISA method for the detection of beta-agonists was established.The standard curve was established by chessboard method and single factor experiment.The IC50 of clenbuterol was 1.18 ?g/L and the IC50 of salbutamol was 1.42 ?g/L.The linear range of IC20-IC80 was: 0.31-4.52 ?g/L,the detection limit was 0.05 ?g/L;The linear range IC20-IC80 was 0.70-5.32 ?g/L.the detection limit was 0.10 ?g/L;The detection range was 0.05 to 4.5 ?g/L;The crossover rate was 100% with clenbuterol(CLB),83.1% with salbutamol(SAL),and 42% for mabutro(MBL),And bromoterol(BBL)was 25.82%,the rate of cross-linking with chlorpheniramine(CPL)was 9.16%,and the crossover rate was 5.41% with that of cimetidine(CML)(RAC)crossover rate of less than 5%;method specificity;kit stored at 37 ? for 6 days,stored at 4? for more than a year,the sensitivity of the kit changes smaller,better stability;The recoveries of the samples were between 80.0% and 104.0%.The method was accurate and the samples were less positive.