Development Of Ripid Immunoassay Of Melamine,Berberine Hydrochloride,Hygromycin B

Feed safety is the basis of ensuring the safety of livestock products and people's health.At present,the abuse and illegal use of some drugs in feed has caused widespread concern in society.The Ministry of Agriculture of China and relevant departments of other countries have also promulgated corresponding rules and regulations for feed management to control the illegal addition and abuse of drugs.Immunoassay technology has favourable selectivity and specificity,which is easy to use and low cost.It can simultaneously detect large quantities of samples.It is of great significance in the food safety rapid detection market.In this paper,three kinds of contaminants and additives in feed were studied:melamine,berberberine hydrochloride and hygromycin B.The corresponding monoclonal antibodies with high affinity and sensitivity were prepared.The ELISA and colloidal gold chromatographic strips were established by using the corresponding antibodies,which can be used for rapid detection of real samples in the field.In this study,complete antigens of melamine,berberine hydrochloride and hygromycin B were designed and synthesized.The corresponding monoclonal antibodies were obtained by immunizing mice,cell fusion,ascites preparation and antibody purification.The antibody MEL-2H4 for melamine has the affinity constant of 2.288×10¹11 L/mol,the antibody subtype of IgG2b,the IC₅₀0 value of 1.804 ng/mL under the optimum indirect competitive ELISA conditions,and the recovery rate of between 80%and 120%.The antibody BER-3H10forberberine hydrochloride has the affinity constant of 8.390×10¹00 L/mol,the antibody subtype is IgG2b,the IC₅₀0 value of 3.469 ng/mL under the optimum indirect competitive ELISA conditions,and the recovery rate of between 80%and 120%.The antibody HB-1G12for hygromycin B has the affinity constant of 2.390×10¹00 L/mol,and the antibody subtype of IgG1,the IC₅₀0 value of 9.100 ng/mL under the optimal indirect competitive ELISA conditions,and the recovery rate of between 80%and 120%.Colloidal gold immunochromatographic strips based on melamine monoclonal antibodies were prepared for the detection of feed and milk samples.The results of affix recovery test showed that the cut-off value of the melamine test strip in feed was 100 ng/g,and the cut-off value of the melamine test strip in milk was 10 ng/mL,the sensitivity of which is better than that reported.Colloidal gold immunochromatographic strip was prepared based on monoclonal antibodies against berberine hydrochloride and hygromycin B for the first time.The cut-off value of berberine hydrochloride strip was 2.500?g/g,which was 100 ng/g for colorimetric use,and that cut-off value of hygromycin B strip was 250 ng/g.