Study Of The Polyclonal Antibody Against Ceftiofur And Chloramphenicol

Ceftiofur(CEF) is cephalosporin used on veterinarian clinic at first,because it is used extensively day by day in veterinarian,its residue in animal food has already aroused great concern from people gradually.How to detect the CEF residues quickly has became the problem resolved urgently.Chloramphenicol(CAP) is a highly-effective broad-spectrum antibiotics which is widely used in stockbreeding. But it has been found that CAP residues in animal tissues and/or animal products has toxic effects to human body.Therefore,it is necessary to work out an analytical method to monitoring CAP residues.In the paper,the technique of molecular immunology was used to the preparation of the anti-CEF polyclonal antibody and the anti-CAP polyclonal antibody,The two above polyclonal antibody establish the foundation for the preparation of their monoclonal antibody and the exploitation of their ELISA Kit.The main content and results of the study are descripted as follow:1 The anti-ceftiofur polyclonal antibody was prepared successfully.1.1 The chemical crosslinking was used to conjugate ceftiofur(CEF) to BSA and obtained artificial immunogen CEF-BSA,the coating antigen CEF-OVA was obtained in the same way.UV and SDS-PAGE were used to identify CEF-BSA.The process of synthetic antigen was optimized by orthogonal decomposition.1.2 Balb/c mice was immunized with CEF-BSA,the titre of polyclonal antibody(pAb) was 135000 by indirect ELISA,The linear range of calibration curves to detect CEF was 100 ng/mL-1000 ng/mL,The curve equation is y=-2.6x+206.0,coefficient correlation r=0.98,its IC₅₀ is 206.0 ng/mL and the limit of detection was 49.1 ng/mL, The recoveries ranged from 79.2%to 94.5%for detecting CEF residues in milk and the coefficients of variation was 5.1%to 13.4%.2 The anti- chloramphenicol polyclonal antibody was prepared successfully.2.1 The active group carboxyl was introduced to Chloramphenicol(CAP) and formed CAP half succinaate(CAP-HS).The carbodiimide(EDC) was used to conjugate CAP-HS to BSA and obtained artificial immunogen CAP-HS-BSA,the coating antigen CAP-HS-OVA was obtained in the same way.UV and SDS-PAGE were used to identify CAP-HS-BSA.The process of synthetic antigen was optimized by orthogonal decomposition.2.2 Rabbits and Balb/c mice were immunized with CAP-HS-BSA,the titre of polyclonal antibody(pAb) was 162000 by indirect ELISA,The linear range of calibration curves to detect CAP was 10 ng/mL-100 ng/mL,The curve equation is y=-4.3x+32.4,coefficient correlation r=0.98,its IC₅₀ is 32.4 ng/mL and the limit of detection was 0.94 ng/mL,The recoveries ranged from79.4%to 98.5%for detecting CAP residues in eggs and the coefficients of variation was 0.5%to 4.3%.