Research On Rapid Detection Of Fluoroquinolones In Meat Based On Immunochromatographic Assay

Fluoroquinolones(FQs),a kind of synthetic antimicrobials with 4-quinolone as the basic structure,are commonly used in the prevention and treatment of bacterial infectious diseases in livestock.FQs mainly inhibit the DNA synthesis of bacteria by disrupting the DNA gyrase or topoisomerase IV,thereby producing antibacterial effects on gram-negative bacteria and gram-positive bacteria.However,in recent years,the problem of unreasonable use of FQs has get more and more serious.The accompanying environmental pollution,bacterial resistance,drug residues and other problems have seriously threatened human health.Therefore,in China,the rectification of excess FQs residues in animal-derived foods has been strengthened,and the requirements for detection methods of FQs have also been continuously improved.Moreover,different FQs may be added for different purposes during the process of breeding,resulting in multiple FQs residues in livestock,so the detection method only for a single FQs is also likely to cause false negatives.In order to avoid false negative,there are higher requirements for the sensitivity and broad-spectrum of immunoassays.The broad-spectrum monoclonal antibody(m Ab)of high-quality is a key factor.Mabofloxacin(MBF)is a kind of FQ_S.The molecular conformation of its hapten is the same as that of most FQ_S.Using MBF to synthesize immunogen for immunity is conducive to obtaining broad spectrum antibody.In the second chapter,the immunogen and coating antigens of MBF were synthesized through the carbodiimide method.Mice were immunized with the immunogen of MBF for a total of 5 times.After the fifth immunization,spleen cells of positive mice with good inhibition and high titer were selected for fusion with myeloma cells SP2/0 according to the results of serum detection by indirect competitive enzyme linked immunosorbent assay(ic-ELISA).After three times of subcloning and screening,four cell lines were obtained from the fused hybridioma cells.Then the four cell lines were used to prepared ascites through the method of inducing ascites tumor in vivo.After the ascites was purified via caprylic acid-saturated ammonium sulfate method,a total of 4 high-quality m Abs against various FQs were obtained,which were named M4E3,M7A6,M3C7,and M5C6,respectively.The sensitivity and specificity of these m Abs were evaluated by ic-ELISA.The results showed that the half maximal inhibition concentration of M4E3,M7A6,M3C7,and M5C6 was 0.07 ng m L^-1,1.03 ng m L^-1,0.77 ng m L^-1,and 0.88 ng m L^-1,respectively.The linear ranges based on M4E3,M7A6,M3C7,and M5C6 were0.01-0.47 ng m L^-1,0.02-47.18 ng m L^-1,0.30-2.00 ng m L^-1,and 0.08-10.23 ng m L^-1,respectively.In particular,the cross reaction rate between M4E3 antibody and lomefloxacin,ofloxacin,enrofloxacin,fleroxacin,danofloxacin,pefloxacin,and difloxacin was higher than 21.21%,indicating that M4E3 antibody could detect MBF and these seven kinds of FQs.In the third chapter,the M4E3 antibody screened in the second chapter was coupled with colloidal gold and then sprayed on the conjugate pad.The sample pad,conjugate pad,nitrocellulose membrane and absorbent paper were assembled into a complete colloidal gold immunochromatographic test strip.A series of experimental conditions had been optimized.The results showed that the optimal p H,labeling amount of m Ab,concentration of complete antigen,and the detection time were 6.0,10?g m L^-1,0.6 mg m L^-1,and 10 min,respectively.Besides,five kinds of sample pretreatment methods were compared.Finally,the Melvaine buffer containing 5%Tween-20 was selected to extract FQs in pork and duck.Then the protein and other impurities in meat were removed by boiling for 5 min.By combining the pretreatment method with colloidal gold immunochromatographic test strips effectively,eight kinds of FQs,including MBF,ofloxacin,lomefloxacin,pefloxacin,daffloxacin,enrofloxacin,difloxacin and fleroxacin,could be detected rapidly and qualitatively in pork and duck;The detection thresholds of them were 2?g kg^-1,2?g kg^-1,0.5?g kg^-1,2?g kg^-1,2?g kg^-1,5?g kg^-1,5?g kg^-1 and 5?g kg^-1 in pork,and 1?g kg^-1,1?g kg^-1,0.5?g kg^-1,5?g kg^-1,2?g kg^-1,5?g kg^-1,5?g kg^-1 and 2?g kg^-1 in duck,respectively.In summary,the anti-multiple FQs of M4E3 antibody prepared in this study have high cross reaction rate with MBF,ofloxacin,lomefloxacin,pefloxacin,daffloxacin,enrofloxacin,difloxacin and fleroxacin,while the colloidal immunochromatographic assay established based on this antibody can quickly and sensitively detect these eight kinds of FQs in pork and duck on-site.