Study On Synthesis Of Chloramphenicol Immunogen And Development Of Indirect Competitive ELISA For Detection Of CAP

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 first part, Bovine serum albumin(BSA) and egg serum albumin(OVA) were used as two protein carriers respectively to couple with the hapten CAP by improved Diazotization procedure, mixed anhydrides synthesisand carbodiimide. The complete artificial antigens acted as both immune antigen and coating antigen in ciELISA respectively. Selected a few of immunal programs to immunize the New Zealand rabbits to genenerate specific antiserum. The second part covers the topic of establishing the indirect competitive Enzyme- linked.The research method and results are as follows: artifical antigen,used as immune antigen, is made by CAP and protein carrier (BSA) through the improved Diazotization procedure, mixed anhydrides synthesis and carbodiimide. The caoting antigen is made by using the same methods.The UV scanning atlas, SDS-PAGE analysis and animal immune tests proved the success of coupling. The antiserum of Mc CAP is obtained by immuning New Zealand rabbits of 2 and 3 months by the normal immune process.The mixed anhydrides synthesis is better than the other two comparing the statistics. Thus, the antiserum is obtained by it in the later ELISA experiment.Immunosorbent assay method for detecting trace element of CAP. The most suitable working condition of ciELISA is obtained by several grid tests which are used to calculate the most optimal concentration and dilution of the coating antigen and antiserum, and the results are 1.25μg/mL and 1:12800 relatively. The standard curve of ciELISA is also established and the curve indicates that the lowest detection limit is 0.03ng/mL, which provides some valuable data for the demanded detection limit of ND (No-Detetion). The regression equation of the curve is y=1.069—0.2633x (R~2==0.9867). The curve has a favorable linearity relation within the concentration range of 10ng/mL~200ng/mL. However, the curve has an unfavorable linearity relation at the concentration exceeding200ng/mL.The accuracy of the established ELISA protocol can be determined within one batch and among batches. The error within one batch is 4.24%.The intercross ratio of CAP is 9.20%. The average rate of recovery of chicken muscle by ciELISA is 94.86%i 9.55%. No cross-reactivity was seen between antibody and five other anitbiotics including Sulfamethazine, Enrofloxacin, Streptomycin, Penicillin and Norfloxacin. The antibody shows cross-reactivity for Sulfadiazine with more than one hundred percentage.