Synthesis Of Chloramphenicol Artificial Immunogen And Identification Of Activity

CAP is the first artificial broad-spectrum antibiotic,which has been widely used in clinical or veterinary as a cheap and effective drug.The extensive researches of CAP have found that even small amount of CAP residual in the human body may also cause a direct or potential threat.Therefore,in order to control CAP abuse,a rapid.sensitive and convenient method of the determination of CAP at trace level is an urgent requirement.Immunoassay as a rapid,sensitivity,low cost and high throughput method,has tremendous advantages in the determination of CAP residues[1].The preparation of effective CAP artificial immunogen and antibody is the key of ELISA used in detection of CAP residues.In this study,artificial antigen was synthesized by three methods,the polyclonal antibody was prepared,and the CAP-ELISA test method was established.CAP was coupled with BSA respectively by MA reaction,diazotization reaction and carbodiimide reaction to get immune antigen.UV scanning and SDS-PAGE proved the coupling was achieved and the CAP immunogen has reached electrophoretic purity.Orthogonal experimental was designed to investigate mixture ratio,reaction temperature,pH of coupling and other related factors,and the optimal reaction condition was established.After optimization of experimental conditions,the highest binding ratio of CAP-BSA(CAP-HS-BSA)was 55.3,15.7,24.6,respectively.Balb/C mice were immunized by different antigen which was synthesis by MA reaction,diazotization reaction and carbodiimide reaction,recetively,to get the different serum polyclonal antibody of CAP.It was determined that the serum antibody which was get from the mouse immunized by CAP-BSA(binding ratio of 55.3,synthesized by diazotization reaction)had the highest titer,reaching 128,000.The experimental conditions of indirect ELISA were examined,the optimal condition was determined as following:The optimal dilution of serum polyclonal antibody was 16,000 times,the best CAP-OVA coating concentration was 1μg/mL,blocking buffer was 1%OVA,working temperature and times of coating antigen was 4℃ for night,working times of antibody and enzyme sign the second antibody was 1.5h,working times of reagent that develop color was 5min.The CAP antibody competitive inhibition curve was established:y=-20.212x+61.811,R2=0.9769,detection limit was 0.04ng/mL,IC50=3.84ng/mL.When concentration of CAP was between 0.5ng/mL-100ng/mL,linearity relation was better.The cross reactivity of CAP antibody and other antibiotics(thiamphenicol,florfenicol,penicillin,streptomycin,et al.)was tested by indirect competitive ELISA,the result proved that the CAP antibody specificity was good.