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Ractopamine is double benzene alkyl amines beta-adrenergic receptor agonist (referred to as β-agonists), with a wide range of physiological effects, often used for bronchial asthma, bronchial spasms and treatment of obstetric diseases. When an amount of 5-10 times the clinical dose, but also a good nutritional redistribution agents and growth-promoting agent. In the body of the animal machine residue once enter the body through the food chain be great harm eaters security, especially for patients with heart disease, diabetes, high blood pressure, hyperthyroidism, glaucoma, prostatic hypertrophy or elderly and children even more dangerous even lead to death. China banned the β-agonist drugs such as the use of growth-promoting agents in animals. But for a long time, the illegal use of β-agonist poisoning have occurred. In order to combat illegal drug use, to protect the health and safety of consumers, there is an urgent need to improve the detection method. Ractopamine detection method compared with other β-agonists such as clenbuterol lag, China has not yet ractopamine national standard detection methods. Establish a rapid, sensitive, specific, effective ractopamine residue detection method has profound theoretical and practical significance. In this study, synthetic ractopamine artificial antigen, obtained by immunizing rabbits immune spleen using hybridoma technology to prepare a the ractopamine rabbit monoclonal antibodies Ractopamine ELISA test technology, and initial construction of of Ractopamine the ELISA detection kit. Its main findings are as follows:. Two ractopamine artificial antigen was synthesized using the mixed anhydride method, as one of ractopamine-BSA immune antigen another ractopamine-OVA as coating antigen. Artificial antigen was identified by UV scanning method, infrared scanning and Agilent 2100 biochemical analyzer lab-on-a-chip kit, confirmed the successful synthesis of artificial antigen measured immune antigen molecule mass 76.4kDa, and to estimate the hapten and carrier protein coupling ratio of 28:1. Using the Coomassie blue method on synthetic antigen protein content were measured, and the concentration of the protein in the antigen for immunization is 3.62mg/mL packet is the concentration of a protein antigen is 1.156mg/mL. Using synthetic immunogen was using subcutaneous injection, popliteal lymph node injections and ear vein injection method of combining immune WHB Black Eyed White Rabbit. Immune seven, efficient price ractopamine polyclonal antiserum, antiserum titer of 1:32 × 10 4 , 6 Hao antiserum titer of 1: 16 × 10 4 . Saturated ammonium sulfate and preliminary purification of the antiserum. Black-eyed white rabbits by immune WHB, at the same time get the rabbit immune spleen spleen cells in rabbit myeloma cells in logarithmic growth phase 240E cell fusion, and packing in 40 96-well plates (help financial The agent is 50% PEG). Fusion cells cultured with HAT selective medium. Indirect ELISA, indirect competitive ELISA and IgG quantitative methods screening clones hole, initial screening, 116 positive wells. Final screening twice Sub cloning A3-2-4, A3-2-6, B5-4-4 and B5-4-5, a total of four plant height specific hybridoma cells, after repeated freezing and recovery stability of hybridoma cells secrete antibodies. Expand production by in vitro culture of rabbit monoclonal antibodies. ELISA test conditions were optimized to get the best reaction conditions: coated and 4 ° C for 12h, an anti-action time 60min, the duration of action of the two anti-100min, the duration of action of the substrate 15min. Because the composition of the substrate have a greater impact on the sensitivity of the ELISA assay, so the test of the substrate formulation optimization, TMB and H 2 O 2 the best combination of 0.25g / L TMB 0.02% H 2 O conclusions.
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