Study On Enzyme Link Immunoassay For Group-Specific Determination Of Chloramphenicol And Clenbuterol Hydrochloride

Chloramphenicol (chloramphenicol, CAP) is a broad-spectrum antibiotic to Gram-positiveand negative bacterial. As a result of abusing drug, the residue problem appeared seriously.The chloramphenicol residue can inhibit human hematopoietic function, leading to renewableobstacles anemia.Clenbuterol (clenbuterol hydrochloride, CL) is aβ2-adrenergic receptor agonist. In the1964, Clenbuterol, which belongs toβtype cordial, was synthesized in the United States, withclinical purposes of prevention and treatment of Bronchial asthma and spasm of human andanimal. Consumption over Clenbuterol residues limit will induce the acute poison symptoms.Clenbuterol long-term consumption of contaminated pork and offal will cause humancardiovascular system and nervous system diseases.Therefore, the study on chloramphenicol and Clenbuterol ELISA kit plays an significantsocial and economic role for protecting consumers' interests, for food safety testing as well asfor international trade. This study focused on synthesizing the artificial antigen andestablishing ELISA method. Here are results as following:In this study, the hapten chloramphenicol and Clenbuterol were linked to the carrierprotein Albumin Bovine Serum (BSA) by Diazo binding methods, and two immunogens wereobtained. The successful coupling of the haptens and the BSA were proved by ultraviolet scan,infrared spectrogram scan and SDS-PAGE electrophoresis. Then two antigens weresynthesized with carrier protein Albumin Chicken egg (OVA) likewise.Based on the immunogens we obtained, the chloramphenicol mouse monoclonalantibodies and Clenbuterol rabbit polyclonal antibodies were got from the cooperationcompany. Antibodies, envelope antigens, enzyme-labelled secondary antibodies were used inorder to establish the indirect competitive ELISA detection. Considering the factors includingsensitivity, linear range, detection process and detection time, indirect competitive ELISAwhich was reasonable sensitivity and convenience was decided as the detection method forthe kits products.Depending on mono-factor remarkably and optimization tests about certain ELISA detection method the chloramphenicol ELISA parameters were decided as followings: 10mLBuffer, 450μL TMB, 5μL H202 and 5% glycin was used as confining liquid; the dilution ratio ofmouse monoclonal antibodies and antigens were 1:1600 and 1:100 respectively withphosphatic buffer(PBS, 0.01M pH7.4); the antigens were placed in the refrigerator at 4℃about 12 hours to be enveloped; the chloramphenicol mouse monoclonal antibodies wereincubated at 37℃for 60min to be confined; the enzyem-antibodies were incubated at 37℃for60min; the mixture reacted at 37℃for 15 min to color developed and it could achieve the bestcoloration effect. For the lowest detection limits of chloramphenicol detected by the optimizeddirect competitive ELISA were at 0.3837ng/mL, which is close to the request of EU(EC-decisions 2003/181/EC and 2004/25/EC).Relying on mono-factor remarkably and optimization tests about certain ELISA detectionmethod the Clenbuterol ELISA parameters were decided as followings: 10mL Buffer, 400μLTMB, 7μL H₂O₂ and 5% glycin was used as confining liquid; the dilution ratio of Clenbuterolrabbit polyclonal antibodies and antigens were 1:8000 and 1:800 respectively with phosphaticbuffer (PBS, 0.01M pH7.4); the antigens were placed in the refrigerator at 4℃about 12 hoursto be enveloped; the Clenbuterol rabbit polyclonal antibodies were incubated at 37℃for 60minto be confined; the enzyem-antibodies were incubated at 37℃for 40min; the mixture reactedat 37℃for 15 min to color developed and it could achieve the best coloration effect. For thelowest detection limits of Clenbuterol detected by the optimized direct competitive ELISA wereat 1.52ng/mL. All these results achieved the detection standards of Clenbuterol residues infood.