The Ci-ELISA Determination Methodes Of Three Fluoroquinolone Drugs Residue And Their Elimination In Chicken Tissues

Levofloxacin,ciprofloxacin and enrofloxacin belong to the fluoroquinolones drug of the third generation,this kind of medicines generally have such characteristic as having wider antibacterial spectrum and stronger antibacterial activity,in addition that can be better absorbed by digest and have no cross reation with the other categeries of antibacterial drugs which now have been widely used as the clinical medication in the cultivation and production of the domestic animal and poulty. Because of this category of drugs,the hazard brought by the residue of the fluoroquinolones drug have been paid more and more attention to and to make sure of the product of chicken safety,the establishment of high performance and sensitive detective technology for the residual drugs is in need.This study use the method of immune analytical,and take levofloxacin,ciprofloxacin and enrofloxacin respectively as the half-antigen to prepare antiserum,then use the three drugs to establish the enzyme-linked immunosorbent assay of the indirect competitive of fluoroquinolones, as to find a method of sentitive and high performance to detective the residual drugs and to study their elimination.The result of experience as follows:1.The synthesis indentity on the artificial antigen and preparation of antibody For levofloxacin,enrofloxaein and ciprofloxacin.Take the levofloxacin,enrofloxacin and ciprofloxacin respectively to couple with the apoprotein to prepare the immunizing antigen by the N-hydroxide succinimide method;take the levofloxacin,enrofloxacin and ciprofloxacin respectively to couple with the OVA by the isobutyl chlonoformate method to prepare the peridiun detection antigen,make identifification on the synthetical antigen by the violet scanning method. The results indicate that immunizing antigen and coating antigen are all successful prepared.The conjugating ratios for LVL-BSA,ENR-BSA,CIF-BSA LVL-OVA,ENR-OVA,CIF-OVAis 10.6,11.6,13.6,5.1,5.2,6.8,respectively.Use three kinds of artifical immunity antigen like LVL-BSA,ENR-BSA,CIF-BSA to immune the rabbit respectively to prepare the antiserum,make the antibody titer detection with ELISA method indirectly,if the antibody titer of the antiserum all are above 1:2¹² and the preparation of antiserum is successful.2.The establishment of enzyme-linked immunosorbent assay by indirect competition for three fluoroquinolonesFirstly,the experiment determinate the cross-reactive of LVL,CIF,ENR.The consequence indicate that the three kinds of drug all have unvarying cross-reaction with drugs of fluoroquinolones,and among the total the cross-raection between ENR and CIF have the best reflection.Through the Ci-ELISA method optimized experimental conditions the best response to the following parameters.Coating antigen was optimal concentration of antigen were 1:200,1:200,1:400;Anti-LVL,ENR,CIF serum concentration of the best work 1:3200,1:6400,1:3200;and the best incubation time is 2h.The dilution of the enzyme symbolize to adope goat toresist rabbit IgG-HRP is 1:5000.On the basis of the optimization of Ci-ELISA method reaction parameters.Use the serum of anti-LVL,anti-ENR,anti-CIF to establish the Ci-ELISA standard curve of LVL,ENR,CIF respectively,and we know that the equation of linear regression of the LVL standard curve is Y=-0.2894X+0.0495(R²=0.9878),the midvalue(IC₅₀) is 64ng/ml,and the lowest detectable limit(LOD) is 10 ng/ml,the linear range of standard curve is 10～1000 ng/ml;the equation of linear regression of the ENR standard curve is Y=-0.2186X+0.216(R²=0.9843),the midvalue(IC₅₀) is 36ng/ml, and the lowest detectable limit(LOD) is 10 ng/ml,the linear range of standard curve is 10～1000 ng/ml;the equation of linear regression of the CIF standard curve is Y= -0.219X+0.1661(R²=0.9885),the midvalue(IC₅₀) is 24ng/ml,and the lowest detectable limit(LOD) is 10 ng/ml,the linear range of standard curve is 10～1000 ng/ml.By adding recovery rate test,make corresponding detect on the tissue of chicken (muscle,liver,kidney included),at the hand of recovery rate and variation coefficient, on average,the recovery rate of the kind of LVL,ENR,CIF drugs all are above 80%, the interassay and intrassay coefficient of variation are lowen then 15%,it seem that the recovery rate and variation coefficient of this experience both get to the requinement of the residual anylysis of the drugs.3.Use Ci-ELISA detection method to study the residual drugs of three fluoroquinolones in the tissue of chicken and their eliminationThe research take the LVL,ENR,CIF as the object,the edible chicken are drencked by 30mg/kg,the result shows that:96h after discontinuation,the drug concentration has been already lower then the limit of quantication in muscle but can be still found in liver and kidney;only more then 120h after discontinuation,the concentration of drug is lower then the limit of quantication in each tissue.The result of the experiment indicate that the article use three kinds of drugs as the antiserum to establish the Ci-ELISA method to detect the residual drugs,which have the characteristic of high sensitivity and better degree of precision,wider detective range and lowest detectable limit,has achived the requirement of the residual drugs like fluoroquinolones also have determinate practical significance.