| Sulfonamides and fluoroquinolones are widely used antibiotics in animal husbandry, prophylactics and growth promoters in animal feeds and also as treatment for infectious diseases. As a result, residues of sulfonamides and fluoroquinolones were usually found in animal-derived foods. However, unreasonable use of homologous drugs for medicine as animal-derived food could result in resistance of pathogenic bacteria which could transfer. The treatment effects and residuals were left in animal products which was harmful for human health.Thus, development of rapid and sensitive detection methods is in need with the ultimate goal of monitoring the risks of sulfonamides and fluoroquinolones residues in the scope of food safety management.In this study, new monoclonal antibodies against sulfonamides and fluoroquinolones were first produced by screening of rabbit hybridoma with proposed high affinity, respectively. And then the related enzyme linked immunosorbent assays were further optimized based on antibodies SAs-80-8, FQs-111-2and FQs-31-3, respectively. Finally, the developed approach was applied to real sample analysis, with the objective of confirmation its performance in practical uses.1. Immunogens of SD-BSA and SMX-BSA were synthesized by diazonium coupling which SD and SMX were coupled to BSA. Mixed immunization was used for injection four New Zealand rabbits. The structure TS was synthesized and coupled to OVA as coating antigen. Rabbit No.11421was selected for cell fusion through antiserum screening.2. Splenocytes from rabbit were fused with myelomas240E,122of positive clones were obtained for primary screening, and9of which were selected for subcloning. Finally, the clone80-8was chosen for production as rabbit monoclonal antibody against sulfonamides.3. RabMAb SAs-80-8had good competition with four sulfonamides and the IC50values for sulfathiazole, sulfapyridine, sulfadiazine, sulfamethoxazole and the mixture of the four sulfonamides were0.68ng mL-1,1.15ng mL-1,1.11ng mL-1,5.27ng mL-1,3.31ng mL-1. Spiked experiment of mixed sulfonamides for swine urine and milk, the recoveries (CV) were92.6%-104.3%for swine urine and61.1%-81.6%for milk, and the coefficient of variation were less than20%.4. Based on the available RabMAb SAs-80-8, a rapid and sensitive lateral flow immunoassay (LFA) platform has been developed for quantitative detection of four sulfonamide residues (sulfadiazine, sulfathiazole, sulfapyridine and sulfamethoxazole). Within the designed LFA format, the hapten conjugate of TS-OVA and goat anti-rabbit antibody were sprayed as capture and control reagents, respectively, and then the antibody was conjugated to colloidal gold particles as the detector reagent. With quantitative assessment aided by a colorimetric strip reader, the IC50values were5.19ng mL-1for SD,1.25ng mL-1for STZ,0.66ng mL-1for SP and24.14ng mL-1for SMX, respectively. The recoveries at three spiked levels (5,20,50ng mL-1for SD, STZ, SP and20,50,100ng mL-1for SMX) were in the range of78.02-135.10%for milk and76.40-137.16%for swine urine. More importantly, the detection performance of the established platform was consistent with those of in-parallel LC-MS/MS analysis. In conclusion, the proposed LFA platform has showed the great potential of fast, sensitive and relatively accurate quantitation on four involved sulfonamide residues in practical use.5. Ofloxacin, ciprofloxacin, danofloxacin, norfloxacin, enrofloxacin, flumequine were chosen as hapten for coupling with BSA, and mixed immunization was used for injection four New Zealand rabbits. Rabbit No.11413was selected for cell fusion through antiserum screening.6. Splenocytes from rabbit were fused with myelomas240E,194of positive clones were obtained for primary screening, and5of which were selected for subcloning. Finally, the clone111-2and31-3were chosen for production as rabbit monoclonal antibody against FQs.7. RabMAb FQs-111-2had good competition with six fluoroquinolones, and the IC50values of enrofloxacin, ofloxacin, difloxacin, pefloxacin, danofloxacin and fleroxacin were below10ng mL-1. The IC50values were below100ng mL-1for norfloxacin, sarafloxacin and enoxacin, and below200ng mL-1for ciprofloxacin and lomefloxacin. The IC50for the mixture of FQs was0.32ng mL-1with LOD0.10ng mL-1. In spiked experiment of mixed FQs for swine urine and milk, the recoveries were53.44%-97.31%for swine urine with the CV value3.32%-14.67%for milk and93.07%-139.69%for swine urine with the CV value2.32%-5.51%for milk.RabMAb FQs-31-3had good competition with ofloxacin with the IC50value of0.24ng mL-1. The IC50values of pefloxacin and fleroxacin were6.51ng mL-1and0.99ng mL-1. In spiked experiment of ofloxacin for swine urine and milk, the recoveries were61.12%-135.68%for swine urine with the coefficient of variation of3.31%-8.87%and76.10%-99.36%for milk with the coefficient of variation of1.75%-9.73%.In addition, through recombinant technology, anti-sulfa monoclonal antibodies were produced. Nevertheless, its sensitivity was lower than the antibody produced by hybridoma production. |
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