| Enrofloxacin(ENR) is a synthetic antibacterial agent that belongs to the fluoroquinolone group. It has been increasingly used in veterinary medicine to treat microbial infections, however, the ENR residues in animal edible tissues could cause serious public health problems. An European Commission Regulation has set a maximum residue limit(MRL) of 30ug/kg for the sum of ENR and its metabolite ciprofloxacin(CIP) in animal edible tissues. So far, several methods have been presented for the detection of ENR in animal edible tissues, including high performance liquid chromatography(HPLC) and enzyme-linked immnosorbent assays(ELISA).HPLC is time-consuming, expensive, labor-intensive and requires extensive sample clean-up. ELISA is convenient to perform, economical and reliable.The purpose of the study here is to develop an indirect competitive enzyme-linked immunosorbent assays(Ci-ELISA) to monitor ENR in chicken tissues. Bovine serum albumin(BSA) and ovalbumin(OVA) were used as protein carriers respectively to couple with hapten ENR by an active ester method, and complete antigens ENR-BSA and ENR-OVA were prepared. ENR-BSA acted as immunogen which immunized rabbits to produce antisera, while ENR-OVA acted as coating antigen in ELISA. The optimal concentration of coating antigen(ENR-OVA), antisera, horseradish peroxidase coupled to the goat IgG of anti-rabbit IgG and the optimal reaction time were determined by the phalanx titrimetric analysis. Thereafter, an Ci-ELISA was established to detect ENR residues in the experimental broiler chickens tissues and was evaluated precision, accuracy, sensitivity and selectivity of the method. The results were as followed:1.The solutions of ENR-BSA, ENR-OVA, ENR, BSA and OVA were scanned with ultraviolet ray absorbance detection at the range of 200nm~400nm, there was a maximum absorbance peak at 289, 291,333,319 and 320nm wavelength of spectrum of BSA, OVA, ENR, ENR-BSA and ENR-OVA, respectively. In addition, the spectrum of ENR-BSA and ENR-OVA both had an absorbance peak at 332nm wavelength. Their spectrum showed the fact that the spectrum of ENR-BSA and ENR-OVA are the integration of the spectrum of ENR and proteins, including ENR characteristic absorbance peak. The result suggested that it is successful to link between ENR and protein carriers.2.The antiserum litre obtained using an indirect ELISA was at the range of 1:3200 to 1:6400, its optimal working concentration was 1:800, the optimal concentration of coating antigen(ENR-OVA) and horseradish peroxidase marked to the goat IgG of anti-rabbit IgG were 1:40 and 1:1000. 3.Precision of the Ci-ELlSA employed to detect ENR indicated by coefficients of variation of intra-assay and inter-assay were 4.5~38.1% and 8.8~41.9% respectively, according to ENR different concentrations(3000, 300, 30, 3, 0.3, 0.03ng/ml) in PBS. Accuracy of the method denoted by mean recoveries of ENR at levels of 3000, 300, 30, 3, 0.3, 0.03ng/ml in spiked chicken muscle, liver and kidney samples were 35.48~63.74%, 69.08~86.97% and 51.04~72.59%, respectively. Sensitivity of the method determined by limit of detection(LOD) in muscle, liver, kidney and PBS were 7.82, 12.71, 8.75 and 1.62ng/ml, respectively. ENR concentration of 50% ENR inhibited (I50) in PBS was 16.26ng/ml, I50 of ciprofloxacin, ofloxacin, gentamicin, benzylpenicillin potassium and sulfadiazine were all over 3000ng/ml, cross-reaction between those five antimicrobial agents and ENR were all below 0.54%, that is to say, the antisera had no cross-reactivity with other fluoroquinolones and antibiotics. I50 of ENR in chicken muscle, liver and kidney samples were 63.97, 45.92 and 50.47ng/ml, respectively. The standard regression equation was Y=76.720-14.792X(r=-0.976,n=16) in muscle, Y=75.807-15.525X(r=-0.981,n=16) in liver, Y=74.631-14.461X(r=-0.992,n=16) in kidney and Y=68.006-14.865X(r=-0.997,n=16) in PBS, linear range was 7.82~3000ng/ml in muscle, 12.71~3000ng/ml in liver, 8.75~3000ng/ml in kidney and 1.62~3000ng/ml in PBS. These results mentioned above showed the method has good specifi... |
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