Danofloxacin And Pefloxacin And Lomefloxacin Enzyme-linked Immunosorbent Assay Method To Establish

Fluoroquinolones are the most important group of synthetic antimicrobials that are widely used in the veterinary industry to treat and prevent various infectious diseases. Because of concerns related to drug residues entering the food chain and contributing to bacterial resistance, more and more countries are setting MRLs (maximum residue levels) and withdrawal periods for fluoroquinolones. To avoid using labor-intensive instrumental method to detect residue of fluoroquinolones in food, a simple and convenient indirect competitive ELISA method has been developed for three fluoroquinolones including pefloxacin, danofloxain, and lomefloxacin in this study.For danofloxacin and pefloxacin, by modified cabodiimide active ester method, danofloxacin and pefloxacin were conjugated to cationized bovine serum albumin to form immunogens of cBSA-danofloxacin and cBSA-pefloxacin. The immunogens synthesized were used to prepare corresponding antibodies by immunizing New Zealand white rabbits. For the immunization of Male New Zealand white rabbits, cBSA-danofloxacin and cBSA-pefloxacin were used, which allowed the isolation of specific rabbit immunoglobulins for danofloxacin and pefloxacin. The anti-pefloxacin and anti-danofloxacin antibodies were characterized by indirect competitive ELISA method. Both antibodies show good sensitivities with IC₅₀ of 2 ppb for anti-danofloxacin and 6.7 ppb for anti-pefloxacin. And they are suitable to be used as screening assays to detect danofloacin and pefloxacin residue in foods and food products. The specificity of the antibodies has been determine by cELISA. The anti-pefloxaicn antibody prepared shows good specificity and has cross-reactivity with only a few other fluoroquinolones such as fleroxacin (116%), enrofloxacin (88%), and ofloxacin (10%). The anti-danofloxaicn antibody prepared shows even better specificity and has certain level of cross-reactivity with pefloxacin (21.7%) and fleroxacin (20.6%). The cELISA test kits were developed based on antibodies prepared. The assay measured drug residues in chicken liver spiked with pefloxacin with an inter-assay coefficients of variation of 10.4% or less and intra-assay of 8.2% or less. The average recovery rates at 0.5, 5, 10, 50, and 100 ppb were 96.2, 103.8, 94.7, 92.2, and 94.3% for inter-assay and 98.4, 102.6, 91.5, 94.6, and 95.2% for intra-assay, respectively. The assay measured drug residues in chicken liver spiked with danofloxacin with an inter-assay coefficients of variation of 17.5% or less and intra-assay of 16.4% or less. The average recovery rates at 1, 2, 3 and 5 ppb were 105.5, 84, 106.7, 98.8% for inter-assay and 7.6, 16.4, 6.9, 3.6% for intra-assay, respectively. Both of cELISA test kits have shown satisfied results in detecting residue for chicken liver.For lomefloxacin, two immunogens of cBSA-lomefloxacin were prepared by cabodiimide modified active ester method and mixed anhydride method, respectively. By using two immunogens by turns in immunizing process, a high quality of anti-lomefloxacin antibody was prepared. An indirect competitive ELISA was applied to characterize antibodt obtained. The antibody shows a good sensitivity with IC₅₀ of 0.35 ppb. The cELISA test kit developed based on antibody prepared has been used to detect milk spiked by lomefloxacin. As the results, the average percent recoveries at 0.1, 0.2, 0.3, 1, 2 ppb were 116.9, 99.7, 134.9, 128.8, 98.9% for inter-assay and 80.1, 125.0, 164.1, 145.2, 89.1% for intar-assay. The assay measured drug residues in milk spiked with lomefloxacin with an inter-assay coefficients of variation of 26.9% or less and intra-assay of 26.5% or less. The cELISA test kit developed is suitable to be used to detect residue in milk.