Development and validation of a chemiluminescent ELISA for simultaneous determination of florfenicol and its metabolite florfenicol amine in chicken muscle

Abstract

A competitive indirect chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for florfenicol (FF) and its major metabolite florfenicol amine (FFA) residues in chicken muscle has been developed and validated according to Commission Decision 2002/657/EC criteria. The IC 50 value of the method was 0.153 mg kg À1 for FFA with a cross-reactivity of 74.3% for FF under optimum conditions, in which FFA-F-BSA (FFA-formaldehyde-BSA) and FF-G-OVA (FF-glutaric anhydride-OVA) were used as an immunogen and a coating antigen, respectively. FFA and FF were easily extracted from chicken muscle with a 40 : 1 ethyl acetate-ammonia mixture, obtaining recoveries of 70.3-100% (FFA) and 71.8-102.0% (FF). Accuracy, precision, selectivity, robustness, limit of detection (LOD), limit of quantification (LOQ) and detection capability (CCb) of the assay have been assessed during the validation process. LOD values in chicken muscle were 0.353 mg kg À1 for FFA and 0.526 mg kg À1 for FF (10-fold dilution) and 0.453 mg kg À1 for FFA and 0.657 mg kg À1 for FF (100-fold dilution). Furthermore, the CL-ELISA method gave CCb values of 1.0 mg kg À1 for FFA and FF. Finally, real chicken muscle samples were analyzed with the CL-ELISA method, traditional ELISA and a previously reported gas chromatography-negative chemical ionization mass spectrometry (GC-MS), and results confirmed the utility of this new CL-ELISA for trace determination of FF and FFA, simultaneously.

Development of a rapid chemiluminescent ciELISA for simultaneous determination of florfenicol and its metabolite florfenicol amine in animal meat products

“…The CR for FF was slightly higher (81.2%) than in the previous study (74.3%), which was ascribed to different concentrations of coating antigen and PAb in the two immunoassays. In addition, no significant CR of other structurally unrelated drugs, including clenbuterol, sulfadiazine, ciprofloxacin and penicillin, was observed, which was consistent with our previous study (Table ). Checkerboard titration experiments were used to select the appropriate concentrations of the immunoreagents and PAb to be employed in the one‐step CL‐ciELSIA.…”

“…The light intensity of enhanced chemiluminescence reaches a maximum within 3 min, thus providing rapid detection of the analytical signal. Better sensitivity was obtained in our previous study; however, the immunological reaction time was prolonged and sample pretreatment was quite time consuming. Meanwhile, only chicken muscle matrix was investigated for the chemiluminescent determination of FFA and FF in our previous study .…”

“…Better sensitivity was obtained in our previous study; however, the immunological reaction time was prolonged and sample pretreatment was quite time consuming. Meanwhile, only chicken muscle matrix was investigated for the chemiluminescent determination of FFA and FF in our previous study . Hence the chemiluminescent determination of FFA and FF in more matrices (e.g.…”

“…The one‐step CL‐ciELISA described in this study uses immunoreagents (FF‐G‐OVA) and PAb, which were previously developed and optimized for the analysis of FF and FFA in chicken muscle …”

“…Checkerboard titration experiments were used to select the appropriate concentrations of the immunoreagents and PAb to be employed in the one‐step CL‐ciELSIA. Meanwhile, the optimal physicochemical parameters of this assay were based on our previous study, with minor revision . Selected conditions were 0.20 µg mL −1 per well of coating antigen FF‐G‐OVA, 30 min of competition time, the use of 0.1 mol L −1 PB as a PAb and standard analyte diluent buffer, 1/20 000 dilution of anti‐FFA PAb in buffer d and 1/5000 dilution of peroxidase‐labeled goat anti‐rabbit immunoglobulins in PBST.…”