Development Of One-step Enzyme-linked Immunoassay For Rapid Detection Of Enrofloxacin Residues In Seafood

Enrofloxacin is a synthetic broad-spectrum antimicrobial agent and beloged to the family of fluoroquinolones (FQs). Because of its ease of use, low-cost and high efficiency, now enrofloxacin is widely used in aquaculture for the treatment of bacterial infections. Many studies have confirmed that FQs could induce potential hazards to human health such as microbial resistance and residual toxicity. Therefore many countries or areas have set strict maximal residue limits (MRLs) for enrofloxacin and other FQs in animal foods. Now chromatography basd methods are usually used for confirmatory analysis of enrofloxacin residues. Immunoassays have been recently reported as rapid screening tools for their high specificity, sensitivity and simplicity.This study is designed to set up one-step ELISA techniques for rapid detection of enrofloxacin residues in sea foods. Based on optimization of competition mode, reaction time and washing time of the ELISA procedures, an one-step indirect competitive ELISA was established and tested with aquatic products; horseradish peroxidase(HRP) labeled anti-enrofloxacin antibodies were prepared using different conjugation methods, and using this HRP-labeled antibodies as receptors, an one-step direct ELISA was established and tested with aquatic products. Main results were listed as following:1. For establishment of one-step indirect ELISA, a saturated competition reaction mode was exploited and the best sequence to add reagents was found to be: Goat anti-mouse IgG-HRP ----samples to be tested ---- anti-enrofloxacin antibody. The established method was tested with incurred aquatic products. In the spiking range of 10μg/kg～200μg/kg, the average recoveries were above 70% and the relative standard deviationes were below 15%, and the detection time could be reduced from 4 hours to2 hours or less. PEG demonstrated little effect to promote the determination efficiency.2. HRP labeled anti-enrofloxacin antibodies were prepared via sodium periodate (NaIO4) oxidation method. The molefraction of the HRP-antibody, the HRP concentration of conjugates and the HRP binding ratio were estimated to be 1.6, 1.2mg/mL and 40%, respectively. The titer of the HRP labeled antibodies was determined higher than 10000×with a working titer about 1000×. The labeled antibodies demonstrated no significant cross reactivities (less than 1%) to other FQs, and the conjugation proved to have no effect on the binding activities and specificity of antibodies. All these results indicated the success of conjugation performance.3. Two bifunctional crosslinkers SPDP and Sulfo-SMCC were also used to prepare HRP-antibody conjugate. The results indicated that the crosslinking efficiency was pH dependent, and with increased length of space arms, is the HRP-antibodies demonstrated higher binding activity to enrofloxacin. But the overall conjugation efficiency was much less that performed via sodium periodate oxidation.4. Based on HRP-labeled anti-enrofloxacin antibodies prepared by sodium periodate oxidation, an one-step direct ELISA (Dc-ELISA) was established and tested with incurred fish and shrimp samples. The lowest detection limit was estimated to be in the range of 5μg/kg～10μg/kg, depending on the resource and purification method of antibodies. Within spiking concentrations from 10μg/kg to 40μg/kg, the average recovery was above 70% and the relative standard deviation was below 10%.5. The efficiency of established Dc-ELISA was further validated with high performance liquid chromatogaraphy (HPLC). A significant positive correlation (R2 higher than 0.95) was observed between the determination resuluts of these two methods for three incurred aquatic products. The detection time of established Dc-ELISA was reduced to about 2hours, which was only half of usually exploited two-step indirect ELISA.At present, the immunoassays for FQs residues in food are mainly based on the indirect competitive ELISA; few studies about direct competitive ELISA were indicated. In this paper a direct competitive ELISA based on HRP-labeled antibody is for the first time developed for fast detection of ennuoshaxing residues. The established methods demonstrated satisfactory sensitivity, accuracy and precision, and proved much simpler and more rapid than present techniques, which allowed us to suggest it potential application as a more efficient fast screening tool for routine monitoring of enroflocacin residues in sea foods.