Study On CLEIA And LFM-ICA Immunoassay Technology Of Sarafloxacin And Difloxacin In Animal-Derived Foods

Fluoroquinolones are widely used in the fields of human medicine and veterinary medicine due to their wide antibacterial spectrum and good efficacy.Among them,Sarafloxacin（SAR）is the first fluoroquinolone drug approved for use in food animals,and it is frequently used in the treatment and prevention of animal disease infections by the livestock and aquaculture industries.Overdose or irregular use leads to accumulation of SAR drug residues in livestock and poultry,which in turn endangers human health through the food chain.In order to ensure the food safety of peopel,it is imperative and general trend to establish fast,sensitive and simple detection methods of SAR and difloxacin（DIF）.To further improve the sensitivity of detection of SAR and DIF,shorten the detection time,and simplify the detection operation,this study prepared SAR monoclonal antibodies through monoclonal antibody preparation technology,and then established an indirect competitive chemiluminescence enzyme-linked immunoassay method,direct competition chemiluminescence enzyme-linked immunosorbent assay method and preparation of lanthanide fluorescent micro-immunochromatographic test strips by using SAR monoclonal antibody.TestⅠPreparation of sarafloxacin monoclonal antibodyTo obtain sarafloxacin（SAR）monoclonal antibody,the study synthesized the complete immunogen SAR-1-BSA and coating antigen SAR-1-OVA after molecular modification of sarafloxacin hydrochloride.The successful coupling was identified by UV scanning.After immunizing mice with complete immunogen for four times,their spleen cells and SP2/0 cells preserved in the laboratory were tested for cell fusion.After indirect ELISA and indirect competitive ELISA,After indirect ELISA and indirect competitive ELISA,the B1 cell line that can stably secrete antibodies and has the best drug inhibitory effect was screened,and inoculated into the abdominal cavity of BALB/c mice to induce ascites to obtain SAR monoclonal antibodies.The ascites titer determined by indirect ELISA reached 4.0×10⁶;the inhibition standard curve is y=-0.3637x+0.746（linear fit R²=0.9916）,the linear range is 0.3125-20 ng/m L,and its sensitivity IC₅₀is 4.75 ng/m L;specificity test results show that SAR monoclonal antibody has no cross-reactivity with the other four antibiotics,the cross-reaction rate with difloxacin is as high as 99.16%,and the cross-reaction rate with other fluoroquinolones is less than 10%.The above experimental results show that the SAR monoclonal antibody was successfully prepared,not only with high titer but also good specificity.TestⅡEstablishment of chemiluminescence enzyme-linked immunoassay（CLEIA）detection method for sarafloxacin and difloxacinTo establish a rapid detection method for sarafloxacin（SAR）and difloxacin（DIF）drug residues in animal-derived foods.An indirect competitive chemiluminescence enzyme-linked immunoassay（ic-CLEIA）detection method and a direct competitive chemiluminescence enzyme-linked immunoassay（dc-CLEIA）detection method were established by the prepared sarafloxacin（SAR）monoclonal antibody.The above method was evaluated by sensitivity,precision,cross-reaction rate,and additive recovery test.The results show that the ic-CLEIA method is in the linear range:0.0625～10 ng/m L,the regression equation is:y=-0.2803+0.5456（R²=0.9951）.The sensitivity IC₅₀is:1.45ng/m L;the lowest detection limit（LOD）of SAR and DIF in chicken samples are 0.46μg/kg and 0.43μg/kg,respectively;the recovery rates of SAR and DIF in chicken samples are respectively 88.3%～106.7%and 84.8%～106%,the coefficient of variation is≤12.2%.The linear range of the dc-CLEIA method is 0.3125～20 ng/m L,the regression equation is y=-0.3734x+0.6883（R²=0.985）,and its sensitivity IC₅₀is calculated to be 3.19ng/m L;The LOD of SAR and DIF in chicken samples were 1.25μg/kg and 1.36μg/kg,respectively;The recoveries of SAR and DIF in chicken samples were 88.2%-108.3%,93.36%-102.7%,and the coefficient of variation was≤13.4%.The average intra-assay and inter-assay differences between the two methods are less than 7%;Except for the high cross-reaction rate with DIF,the cross-reaction rate with other fluoroquinolone drugs is less than 8%,and there is no cross-reaction with other non-fluoroquinolone drugs.The above results show that the two established detection methods are not only fast,but also sensitive.They can be used for the quantitative detection of SAR and DIF drug residues in animal-derived foods,and provide a new detection method for SAR and DIF drug residues.TestⅢEstablishment of a rapid detection method for lanthanide fluorescent microsphere immunochromatography（LFM-ICA）for sarafloxacin and difloxacin drug residuesThe purpose of this experiment is to establish a lanthanide fluorescent microsphere immunochromatographic detection method（LFM-ICA）for the rapid detection of sarafloxacin（SAR）and difloxacin（DIF）.Firstly,the SAR monoclonal antibody was coupled to the nanospheres,and the goat anti-mouse Ig G and the original SAR-1-OVA were sprayed on the NC membrane,and then assembled with the sample pad,absorbent pad and bottom plate to form test strips.the antibody microsphere conjugate is preliminarily reacted with the sample to be tested and then added to the loading pad,and finally detected on the high-sensitivity fluorescence analyzer to establish a rapid detection method for lanthanide fluorescence micro-immunochromatography.According to the principle of single factor variables,the reaction time of the microspheres and the sample,the detection time,the amount of microspheres and the T-line coating concentration were optimized respectively.Under each optimized condition,the SAR and DIF standard dilution solution of gradient dilution was used as a competitive inhibition test to establish a standard inhibition curve within the linear range of 32～0.5 ng/m L.The standard inhibition curve of SAR is y=-0.2112x+0.5286,R²=0.9765,the sensitivity IC₅₀is 1.37 ng/m L,and the lowest detection limit（LOD）IC₂₀is 0.05 ng/m L.While the standard inhibition curve of DIF is y=-0.3447x+0.5316,R²=0.9605,the IC₅₀is 1.24 ng/m L and IC₂₀is 0.17 ng/m L.The precision test results showed that the average intra-assay coefficient of variation and inter-assay coefficient of variation were both lower than 7%.The specificity results showed that except for the high cross-reaction rate with DIF,which was 106%,the cross-reaction rate with other fluoroquinolones was less than 10%,and there was no cross-reaction with the other four antibiotics.The results of the addition recovery test showed that the recovery rates of SAR and DIF in chicken samples were 87.71%～114.39%,85.91%1～02.84%,and the coefficient of variation was≤7%.The above results indicate that the method established in this study has good stability and reproducibility,and can be applied to the detection of SAR and DIF residues.In addition,its sensitivity is much higher than that of ELISA,and its speed is its b Ig Gest advantage.