Design, Synthesis, and Characterization of Tracers and Development of a Fluorescence Polarization Immunoassay for Rapid Screening of 4,4′-Dinitrocarbanilide in Chicken Muscle

Abstract

The compound, 4,4′-dinitrocarbanilide (DNC), is the marker residue of concern in edible tissues of broilers fed with diets containing anticoccidial nicarbazin (NIC). In this study, 25 fluorescein-labeled DNC derivatives (tracers) are synthesized and characterized to develop a rapid fluorescence polarization immunoassay (FPIA) for the detection of DNC in chickens using DNC monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of the FPIA is investigated. Our results show that after optimization, the half maximal inhibitory concentrations (IC50) and limit of detection (LOD) of the FPIA in the buffer are 28.3 and 5.7 ng mL−1, respectively. No significant cross-reactivity (CR < 0.89%) with 15 DNC analogues is observed. The developed FPIA is validated for DNC detection in spiked chicken homogenates, and recoveries ranged from 74.2 to 85.8%, with coefficients of variation <8.6%. Moreover, the total time needed for the detection procedure of the FPIA, including sample pretreatment, is <40 min, which has not been achieved in any other immunoassays for DNC from literature. Our results demonstrate that the FPIA developed here is a simple, sensitive, specific, and reproducible screening method for DNC residues in chickens.

A Europium Nanosphere-Based Time-Resolved Fluorescent Immunochromatographic Assay for the Rapid Screening of 4,4′-Dinitrocarbanilide: Aiming at Improving Strip Method Performance

“…As shown in Table 1, the CR was <0.1% and no inhibitions were observed even though the concentration of the standards was 1000 ng mL −1 . The results of the CRs were highly similar to those from the fluorescence polarization immunoassay (FPIA) using mAb 3B4 [30]. These results indicate that the developed TRFICA was specific to DNC detection.…”

A Comparable icELISA and Lateral Flow Immunoassay for the Sensitive and Rapid Detection of 4,4′-Dinitrocarbanilide in Chicken

“…In detail, the chicken was extracted with methanol, and the extraction was diluted with a buffer solution, aiming to eliminate the matrix effect. Generally, the standard curve in diluted extraction matches well with that in the buffer solution, which means the matrix effect could be eliminated [ 25 ].…”

A Robust Homogeneous Fluorescence Polarization Immunoassay for Rapid Determination of Erythromycin in Milk

“…With an increase in the concentration of analytes in the reaction solution, the antibody will be occupied by analytes, and the tracer will be bound to little or no antibody and some tracers will be free, which can lead to the decrease of the FP value. FPIAs have attracted more attention and been reported for the detection of many small molecular compounds, such as veterinary drugs [ 18 , 21 , 22 , 23 ], pesticides [ 24 ], and toxins [ 25 , 26 , 27 ] in food and environmental samples. However, to the best of our knowledge, no report of FPIA for ERY is available.…”