| Coccidiosis is one of the main contact parasitosis harmful for poultry industry, caused by Eimeria Globidium, as an effect anti Coccidiosis medicine, Nicarbazin be application to clinic for near half a century. Result of residual in chicken and eggs, are harmful for human heathy. On account of this, in order to development a method to detect residual in chicken or eggs fastly, experiments makes some studies as following.Firstly, according to construction features of 4,4-dinitrocarbanilide (DNC), take certain proportion quality of p-nitroaniline and Succinic Anhydride, and then dissolve into Acetic Acid, radiated by microwaves, rinse reaction product repeatedly with double steams water, suction filtration and drying, synthesizes the mimics structure 3-[N-(4-nitropheny)amido]-propenoic acid of DNC, the returns-ratio is 94%, yield 91%, analysis confirmation to its structure by mass spectrum analysis, nuclear magnetic resonance, far infrared and ultraviolet analysis. Results show melting point is about 192~194℃. Secondly, the hapten conjugated with the macro-molecule carrier protein, such as bovine serum albumin(BSA), ovalbumin(OVA), by EDC methods, the whole reaction system was stayed for 12 hours, at 4℃. The conjucion was purified by ultrafiltration separation. All of the far infrared, ultraviolet and SDS-PAGE analysis have confirmed the result of the hapten was conjugated with the macro-molecule carrier protein, which satisfied with immune requestion. The complete antigen was prepared successfully.Mixed aequales cubic capacity conjugated antigen into Complete Freund’s adjuvant (CFA) or Incomplete Freund’s adjuvant (IFA), by emulsification, immunized Balb/C mouse aged 9 weeks, immune dosage 0.1mL/only and protein content 120~150μg.mL-1 each time, the interval among immuno-time was average 30d/times and immunity 6 times in total. To establishment indirect ELISA examination method during immuno-time. The optimization results indicate stay coating plate in 37℃for 4 hour, and over night for 4℃, block for 1 hour with 1% gelatin, Antigen-antibody reactions of 2h, then react to horse radish peroxidase(HRP) for 1 hour.The optimization coating concentration and blood serum dilution are separation 2μg.mL-1 and 0.6μg.mL-1. The spleen cell got from Balb/C mouse of booster 7 days , titer of blood serum was above 1:10000, after immune programme finished, was fused with myeloma cell NS0 by 50% polyethylene glycol 2000(PEG2000), screening positive holes from clone cells by 5 limited dilutions, obtained 3 strain hybridoma cell line secreting the anti-DNC antibody, named 03E-01, 02F-02 and 10H-03. Supernatant titer was between 1:600 and 1:800, ascites titer was above 1:6400, ascites titer average between 1:12800~1:15000 after purification, the light and gamma chain is about 24KD and 50KD. The block testing obviously confirms the antibody has the function of blocking DNC.Study prepared antibody against DNC residual of Nicarbazin based on preparing for conjugate antigen and immunity, support technical to establish the ELISA methods of detecting residual Nicarbazin in foods,and development a simp quickly kit combining with colloidal gold, application to production and monitoring Nicarbazin residual work of our country. |
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