| Immunoassay is a new analysis technique based on the specific binding of antibody to antigen and reversible binding reaction. However, Samples are usually extracted with organic solvents or aqueous-organic mixtures in acid or basic media followed by time-consuming liquid-phase extraction (LPE), liquid-liquid extraction (LLE) and solid phase extraction (SPE)as purification steps. In some reports defatting by hexane and some other clean-up steps were also required. Such complicated and time consuming pretreatments make the EL1SA and other immunoassays not "fast and simple" enough for routine monitoring of large numbers of food samples, even in comparison to some HPLC-based methods, especially those exploiting automatic samplers. To a large extent such a problem could be attributed to the significant interference from the complex food matrix to the immunoassays, which has been confirmed by many previous studies. Thus this paper aims to establish to simplify and modify the pretreatment of food stuffs for the ELISA of FQs residues (norfloxacin and enrofloxacin), in which 5-sulfosalicylic acid dihydrate was for the first time exploited as the solvent to prepare sample extracts for direct analysis without any further purification or clean-up procedures., which made it very suitable for field analysis.In this paper, using three flatfishes (Scophthalmus maximus, Paralichthys olivaceus and Cymoglossus robustus) as samples, the elimination of protein and matrix effects on the competitive indirect enzyme-linked immunosorbent assay (ci-ELISA) of antibiotic residues by heat and organic solvents were investigated. The results were found that only the heating could not remove the effect of protein and organic solvents could eliminate the effect of protein. However, orgainc solvents have an effect on immunoassay. This could increase the complexity of sample pretreatments if measures such as heating and evaporating are taken.The effects of different kinds and concentrations acids on eliminating protein were explored and the influences on EL1SA were also investigated, which indicated that lower concentrations of 5-sulfosalicylic acid dehydrate could had little influence on ELISA performance and could eliminate the matrix interference. Based on these results, a simple, rapid sample extraction method for the determination of FQs has been developed, optimized. Influences of several parameters, such as the concentration of 5-sulfosalicylic acid dehydrate, pH values, were selected to provide a highest sensitivity on the ELISA. The efficiency of the developed technique was validated with real samples, and its potential for real application was evaluated and discussed by comparison with present methods. The FQs were determined with standards of 2% of 5-sulfosalicylic acid dihydrate in the concentration range of 0.1-25.6μg/L, and the limit of detection (LOD) was 0.1μg/L. Fishery samples were extracted with 2% of 5-sulfosalicylic acid dihydrate and the extracts were analyzed directly without any further purification or clean-up procedures. No significant matrix interference was observed as samples extracted with 2% of 5-sulfosalicylic acid dihydrate. Recoveries of FQs in fishery muscle were between 68～94% in the concentrations range of 10-50μg/kg and the variation coefficients are less than 20%.This extraction procedure was much rapider and simpler to conventional ELISA extraction procedure. Meanwhile, the clean-up of the extract was not needed in the modified procedure and the total time for the sample pretreatment was reduced from 45-60min to about only 20-30min, which indicates a significantly increased efficiency for fast screening of these antibiotics. Moreover a large quantity of organic solvents was needed to the conventional sample preparation, which could do damage to the environment and laboratory personnel.This modified method was also validated with other aquatic samples including different fishes and shrimps, and similar results that were observed. The matrix interference originated from fishery samples was eliminated by 2% of 5-sulfosalicylic acid dihydrate and did not interact with horseradish peroxidase (HRP) labeled IgG in western blotting. Recoveries of FQs in fishery muscle were between 65～95% in the concentrations range of 10-50μg/kg and the variation coefficients are less than 20%,which indicated that this modified method could be used in different fishery products. In order to validate ciELISA, samples were analyzed by high-performance liquid chromatography. The correlation between data which was obtained by using microwell assay and the one attained by HPLC was good (the R2 value was 0.97), which indicated that the modified method was capable of being applied for monitoring of FQs in fishery samples.This study establishs a new strategy to simplify and modify the pretreatment of food stuffs for the ELISA of FQs residues (norfloxacin and enrofloxacin), in which 5-sulfosalicylic acid dihydrate was for the first time exploited as the solvent to prepare sample extracts for direct analysis without any further purification or clean-up procedures. No significant matrix interference was observed as samples extracted with 2% of 5-sulfosalicylic acid dihydrate. It is supposed to be an effective method to eliminate matrix interference and make the ELISA and other immunoassays "fast and simple". |
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