Study On The Preparation Of McAb Against Chloramphenicol And Its Electrochemical Methods For Detecting Chloramphenicol Residues

Chloramphenicol(CAP), one of the broad-spectrum antibiotics, was once used widely in husbandry. However, due to its strong toxicity and side-effects, CAP was restricted or even banned in use for edible animal and its maximal residual was strictly regulated in animal foodstuff in most of the developed western countries. The Ministry of Agriculture of the People's Republic of China declared formally that CAP and other kinds of CAP derivates should not be detected out in the edible tissues of animal food. Nevertheless, because of its low price and steady effectiveness against bacteria, CAP is still illegally used in livestock and aquaculture. Up to now, the main approaches for the detection of CAP residue include immunoassay, chromatography, multiple techniques of chromatography linked with mass spectrum. However, these methods have their own disadvantages, such as complex analyzing processes, long time preparation of samples and expensive equipments.In order to obtain the anti-CAP Monoclonal antibody(McAb) for Biosensor research, Chloramphenicol Succinate-Bovine Serum Albumin(CAPS-BSA) and Chloramphenicol Succinate-Ovalbumin(CAPS-OVA) were synthetized by the method of mixed anhydride. The binding ratios of these two conjugations were identified through ultraviolet spectrophotometry according to the method reported by Chunyan Chai. The results showed that binding ratios of CAPS-BSA and CAPS-OVA is 33:1 and 6:1 seperately. Balb/c mice were immunized using CAPS-BSA as an immunogen. Two cell strains(named 20060723-1D11 and 20060723-4D2) secreting specific antibodies against CAP were obtained by cell fusion technology. Because the titration and specificity of the antibody secreted by 20060723-1D11 is higher than those by 20060723-4D2, the antibody from the former cell strain was chose for the whole experiment. The antibody titrations produced by this strain in the supernatant fluid of the culture and in the peritoneal fluid were 1:200 and 1:12800 respectively, based on indirect ELISA detection.To explore the immuno-voltammetric techniques with high sensitivity and specificity for determination of CAP residues in milk, the conjugation of CAPS-OVA was anded onto the micro-reaction plate and the monoclonal anti-chloramphenicol antibody was used to set up competitive Enzyme-linked immunoabsorbent assays(ELISA). After the procedure of competitive combination of CAPS-OVA and CAP in sample with anti-chloramphenicol monoclonal antibody during the incubation period, the plate was rinsed and alkaline phosphatase(ALP)-labeled goat anti-mouse IgG added into the microcuvettes. p-Nitrophenylphate(pNPP) was applied as substrate after the second incubation and rinse. The oxidative peak currents(OPC) were recorded by Differentiated Pulse Voltammetry(DPV) after the reaction on the plate which was terminated by 2mol/L NaOH. The result show that hydrolytic production of pNPP(ALP catalyzed), p-Niteophenyl(PNP) could be oxidized on glass-carbon electrode(GCE) at 1.20V(vs.Ag/AgCl). This method allows the detection limit as low as to 0.064μg/L with the linear range of 0.15-600μg/L CAP and the average recovery rate reaches 89.8% based on milk sample. Furthermore, the immuno-voltammetric apparatus are portable and can be used for the determination of CAP residues in milk on the spot due to its small size and easy operation.A stabilized molecularly imprinted film was prepared through the electropolymerization of o-phenylenediamine on a glass-carbon electrode in the presence of template(Chloramphenicol succinate), based on molecularly imprinting technology and electropolymer, under the condition of weak acidity. And the characteristics of this molecularly imprinted film were determined by differential pulse voltammentry. The results showed that the molecularly imprinted film exhibited quick response, good sensitivity, selectivity and recurrence. This film has shown approved prospect in the research of the electro-polymerizations for the detection of chloramphenicol.On the base of the preparation of chloramphenicol monoclonal antibody, we combined electrochemistry sensor and the insoluble enzyme-linked immuno assay technique together to establish the Fast, efficient and accurate ALP-pNPP system for the examination of chloromphenicol residue that is concidered to be the point of innovation of this experiment.