Gatifloxacin, Nitrofurazone Residues In Food Enzyme-linked Immunosorbent Assay Study

With the development of global economic integration and the international food trade, food safety has become an important public health problem. In recent years, Chinese food exports were blocked repeatedly because of the problem of drug residues. It caused huge economic losses, and damaged the image of Chinese food seriously. It has become an urgent task to resolve the problem of drug residue in Chinese food and food products.Veterinary drug residues lead to not only human acute and chronic toxic directly and bacterial resistance, but also potential harm to human health indirectly through the food chain. In order to protect consumers, a growing number of countries in the world have established MRLs (Maximum Residue Limits) for the drugs used in animal husbandry. Presently, a number of methods have been developed to detect drug residue, including instrumental methods such as HPLC, LC-MS, LC-MS/MS, microbial method, and immunoassays such as ELISA, RIA, EIA, FIA, MAIA.Instrumental methods are accurate, stable and reliable and can be used as standard methods, but they need expensive equipments, complex sample pre-treatment that are time-consuming and laborious. It is, thus, difficult to satisfy practical requirements. The results measured by the microbial detection method are usually higher than the MRLs. A rapid, sensitive and effective detection method must be established. Enzyme-linked immunoassay (including ELISA, MAIA etc) overcomes the shortcomings of the methods mentioned above. ELISA is a rapid, sensitive, convenient method with broad development prospects. Therefore, this research aims at establishing enzyme-linked immunosorbent assay (ELISA) for gatifloxacin (GAT) and nitrofurazone.Using the -COOH in the gatifloxacin and CPSEM, we prepared immunogens of gatifloxacin and CPSEM via the EDC method, and obtained high quality anti-gatifloxacin and anti-nitrofurazone antibodies.For gatifloxacin, three immunoassay methods including indirect ELISA, direct ELISA and MAIA were studied and compared with each other. Using the indirect ELISA, the anti-gatifloxain shows high sensitivity with an IC50 value of 2.6 ppb toward gatifloxacin and the limit of detection of 0.05 ppb with a linear range of 0.8-50 ppb. The anti-gatifloxacin shows high specificity with only a minor cross reactivity with ciprofloxacin (3.0%) among common used fluoroquinolones. Recovery rates from the gatifloxacin-fortified milk samples were in the range of 80-106 %, while the intra-assay and inter-assay coefficients of variation were < 14.3 % and < 19.6 %, respectively. Using the direct ELISA, the anti-gatifloxain shows higher sensitivity, with an IC₅₀ value of 0.45 ppb toward gatifloxacin and limit of detection of 0.01 ppb with a linear range of 0.1-10 ppb. Recovery rates from the gatifloxacin-fortified milk samples were > 90 %, while the intra-assay and inter-assay coefficients of variation were < 4.6 % and < 5.9 %, respectively. Using the MAIA, the anti-gatifloxain also shows higher sensitivity with an IC₅₀ value of 0.085 ppb toward gatifloxacin and limit of detection < 0.001 ppb with a linear range of 0.01-10 ppb. Recovery rates from the gatifloxacin-fortified milk samples were > 97 %, while coefficients of variation were < 22 %; recovery rates from the gatifloxacin-fortified swine urine samples were > 80 %, while coefficients of variation were < 16.5 %.For nitrofurazone, the anti-nitrofurazone shows relatively high sensitivity, with an IC₅₀ value of 10.5 ppb toward nitrofurazone, and high specificity with no detectable cross-reactivity with the most other similar drugs. There is a linear range of 2.5-100 ppb for nitrofurazone detection. Recovery rates from the nitrofurazone-fortified water samples were in the range of 84-106 %, while the intra-assay and inter-assay coefficients of variation were < 5.6 % and < 7.3 %, respectively.Through this study, we prepared high quality anti-gatifloxacin and anti-nitrofurazone antibodies and immunoassays were developed based on these two antibodies. The immunoassays method developed could be used to detect residues of these drugs in food s and food products for regulation purpose.