The Preparetion Of Group-specificity Antibodis And Immunoaffinity Column For Penicillins

Penicillins,the high efficient antibiotics,are widely used in veterinary medicine for animal bacterial infections which caused by intestinal and respiratory diseases,for producting resistant strains and allergic reactions,many countries monitor penicillins strictly which used in animals and residued in food.As a class of antibacterial drugs widely and the biggest used in veterinary medicine,the residue of penicillins was a very outstanding problems to the food safty. It is very necessary for the developments of adequate analytical procedures to determine the residues in products of animal Origin to penicillin drugs.Group-specificity synthetic antigens against penicillins were prepared by choosing the 6-Aminopenicillanic Acid,are immunoantigens and coating antigens.and polyclonal antiserums were prepared by immunization of rabbits.Then,Biological activities of the antiserums were determined and IgG was isolated and purified from the antiserum.Then,which was coupled to Sepharose 4B activated by CNBr to develope immonoaffinity chromatography column,and the preliminary study on its Evaluation has been carried out.The results are as follows.1.The Preparetion and identifition of artificial Group-specificity antigenics against penicillins6-Aminopenicillanic Acid was coupled to BSA and OVA by a glutaraldehyde method, 6-APA-BSA was immunoantigen and 6-APA-OVA was artificial coating antigen.The synthetic antigens were scanned and identied by UV spectrum and Fourier transform infrared spectrometry(FTIR).which showed that the couplings were successed and the rates of coupled antigens were 13.53,8.25,calculated by UV method.2.The Preparation of Group-specificity polyclonal antibody against penicillins and Detecting for immunological activitiesGroup-specificity polyclonal antibodies against penicillins were prepared by immunization of rabbits with artificial 6-APA-BSA.Indirect ELISA was established to screen antiserums,by optimization,optimal reactive condition of I-ELISA for detecting coated with artificial 6-APA-OVA was determined as follows,the quantity of antigen coated was 5μg/ml,the positive serum dilution and the negative control were both 1:4000,and the dilution of IgG—HRP was 1:10000.and the Titre of three antiserum were 5.12×10⁵,6.4×10⁴,3.2×10⁴ in the method of I-ELISA.In constrast,Cross-reactivity rates of Penicillin G,Ampicillin,Cloxacillin,Oxacillin, Amoxicillin were in the range from 13.5%to 101%.The rates of Other antibiotics (Enrofloxacin,Sulfadiazine,Chloramphenicol,Terramycin and Lincomycin) were all very low.The coefficients of variation for the Ci-ELISA were all low 5%in intra-assay and 10%in inter-assay.3.The preparation for Immunoaffmity Column against penicillins and The dynamic capacity of IAC was preliminarily evaluatedIgG was isolated and purified from the antiserum by a series procedure of precipitation with saturated ammonium sulfate and chromatography with DEAE-Sephadex A-50.After SDS-PAGE electrophoreisis,purified IgG was reach the electrophoretic purity.The purified IgG was coupled to sepharose 4B activated by CNBr to prepare multi- immuno -affinity chromatography columns.the rate of coupling was 90.58%.The regression equation of Penicillin G in HPCE was y = 468.66x-8.4299,The liner range was 1～100μg·ml^-1,and the coefficient was 0.9971.The recovery is within 99.3%to 101.5%. The relative standard deviation(RSD) of within day and between day were within 4%,The dynamic capacity of the resulting immunosorbents was 1433.8 ng ml^-1 gel,and the absolute capacity of IAC was 24.42 ng/mg IgG,respectively.The recovery of penicil -lin G was 94.67%～76.38%,with CV of 5.67%～15.91%.and the eluent was Methanol- Acetonitrile-Acetate /acetate buffer(60:25.15).The Accurate and Precision were all meet the requireements of the detection to the residue of penicillins.