| Cephalosporin antibiotics and streptomycin has a broad spectrum antimicrobial,bactericidal ability, and thus been widely used in veterinary clinical treatment oflivestock bacterial infection and other diseases; these two types of drugs oftenassociated to enhance treatment effects, soincrease in livestock body of drug residuesaccumulate in the human body through the food chain, causing serious harm to humanhealth. Therefore, in order to reduce these two types of drug residues on human healthhazards, we must strengthen the detection of residues of these two types of drugs infood of animal origin, the establishment of a high sensitivity, simple method has greatsignificance.1. Synthesis of the Antigen and polyclonal antibody preparationSelect7-aminocephalosporanic acid (7-ACA), cefradine set (the CED),cephalexin (CEX) using glutaraldehyde method with bovine serum albumin (BSA),the combination of the preparation of antigens, and ovalbumin (OVA) evenThe linkswere prepared by coating antigen. The use of chemical cross-linking methodstreptomycin (SM) with BSA and OVA coupled Preparation of immunogen SM-BSAand package antigen SM-of OVA. Artificial antigen positive identification by UVscanning and SDS-PAGE protein electrophoresis, UV spectrum scanning method tocalculate the7-ACA-BSA, CED-BSA, the CEX-BSA and SM-BSA combination were14.34,15.62,12.29,15.21. These four antigens to immunize New Zealand white rabbits,the antiserum titer after by ELISA, anti-7-ACA-BSA IgG titer of anti-CED-BSA IgG,anti-CEX-BSAIgG and anti-SM-BSAIgG respectively:12800,64000,64000,64000.The results showed that: coupling, combined with more than appropriate. Income titerwith the experimental requirements.2. Antibody cross-reactivity determinationAn indirect ELISA for the detection method, optimized by determination ofcross-reactivity screening of resistance to the CEX-BSAIgG cephalosporins havegood cross-reactivity, cross-reaction rate of53.4%to101%, while resistance to theSM-IgG sulfuric acidstreptomycin and dihydrostreptomycin have goodcross-reactivity of cross-reaction rate of85%to101%. Cross-reaction rate betweenstreptomycin and cephalosporin antibiotics is less than0.1%. Therefore select againstthe CEX-BSA IgG antibodies for the development of the detection of cephalosporinstest strip3. The preparation of gold-labeled antibody and single detection test stripPurified antibody protein combined with colloidal gold of diameter18nm into gold labeled protein, the component materials and every conditions of test strip wereoptimized, every components of the test strip were assembled sequentially, and theperformance of test strip were testing, test paper of cephalexin can detect minimumcephalexin of10ng/mL and80ng/mL in PBS and milk matrix,the minimum detectionlimits of other cephalosporins in the range of500ng/mL in the milk matrix, test stripof streptomycin in PBS and milk matrix can detect minimum streptomycin of150ng/mL and400ng/mL respectively, minimum detection limits of treptomycinsulfate and dihydrostreptomycin in milk matrix is1000ng/mL. the test showed thatspecificity and sensitivity properties of test strip specificity can comply withinspection requirements.4. Assembment and detection of properties of double detection test stripThe double detection of cephalosporins and of streptomycin immunochgraphicr-omato strip is assembled,minimum detection limition of cephalexin is20ng/mL and100ng/mL in PBS and milk matrix, minimum detection limition of cephalexin is200ng/mL and500ng/mLother beta-lactam antibiotics minimum detection limits is inthe range of1000ng/mL In the milk, minimum detection line of streptomycin anddihydrostreptomycin streptomycin sulfate is in the range of1000ng/mL too,the testshowed that the development of the dual detection test strip conforms to theproduction in detection requirements of two kinds of drug residue... |
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