Studies On Chemiluminesecence Enzyme Immunoassay Of Enrofloxacin

Enrofloxacin is a fluoroquinolone drug which is widely used in the treatment of veterinary. In recent years, more and more about enrofloxacin report of adverse reactions and resistance, the residues directly threat to human health in animal food. Most of analytical methods concerning residues of veterinary drugs were based on high-performance liquid chromatography(HPLC), gas chromatography(GC) and thin layer chromatography(TLC) et al. They take on complicated sample pre-treatment and expensive instrument, they are not easy to promote. Therefore a simple and rapid and sensitive method for residual ENR, has practical significance for veterinary drug residues in foods of animal testing. In this study, two methods based on different light-emitting systems were developed and applied to conduct the actual testing of samples.1. ALP-AMPPD system establishment and optimization of methods. Coated buffer was pH 9.6,0.05 mol/L CB. Coated antibody concentration was 2μg/mL. Hot-pack method was at 37℃for 150min. Using carbodiimide coupling method, dialysis time was 6 days. Conjugate diluent was pH7.2,0.01 mol/L PBS. Best time of chemiluminescence reaction was 8min. Brightening agent was diluted by 1:4(V:V),then was added 25μL per well. The linear regression equation and correlation coefficient were Y=-0.9961x+6.6992 and 0.9977, Y value for the logarithm of light, x was the logarithm of the concentration of ENR. The Linear range of CLEIA extended from 350pg/mL to 1000pg/mL and the limit of detection (LOD) was 239.9pg/mL. The within-run relative standard deviations (RSD) (n=5) were from 7.33% to 9.08%. Interassay RSD (n=5) were from 9.52% to 13.28%, Standard recovery were from 96.3% to 103.3%.2. HRP-Luminol-H2O2 system establishment and optimization of methods. Coated buffer was pH 9.6,0.05 mol/L CB. Coated antibody concentration was 3μg/mL. Hot-pack method was at 37℃for 150min. Using carbodiimide coupling method, dialysis time was 5 days. Conjugate Diluent was pH7.2,0.01 mol/L PBS, biocking temperature was 25℃, biocking time was 2 hours, biocking volume was 150μL each well. Optimal immune response time was 1 hour, Immune reaction temperature was 37℃. Best time of chemiluminescence reaction was 3min. Brightening agent was diluted by CB buffer 1:2(V:V), then was added 25μL per well. The linear regression equation and correlation coefficient were Y=-0.3059x+6.3904 and 0.9974, Y value for the logarithm of light, x was the logarithm of the concentration of ENR. The Linear range of CLEIA extended from 350pg/mL to 1000pg/mL and the LOD was 30.2pg/mL. Leases RSD (n=5) were from 7.75% to 9.42%, Interassay RSD (n=5) were from 9.19% to 13.05%, Standard recovery were from 99.9% to 100.0%.3. The actual sample detection CLEIA, HPLC and ELISA comparison analysis. The sensitivity of three methods were 30.2pg/mL,6ng/mL,3.6ng/mL respectively. In the determination of Milk Samples, the average recovery (n=5) of three methods were 96.7%,88.6%,94.3% respectively, RSD were 8.27%,3.72%,10.28% respectively. In the determination of egg samples, the average recovery (n=5) of three methods were 97.0%,90.8%,94.4% respectively, RSD were 7.98%,3.13%,9.40% respectively. In the determination of honey samples, the average recovery (n=5) of three methods were 97.9%,89.2%,93.7% respectively, RSD were 8.46%,3.61%, 10.07% respectively.In the determination of plasma samples, the average recovery (n=5) of three methods were 98.2%,86.6%,92.5% respectively, RSD were 8.99%, 4.09%,8.53 respectively.