Synthesis and characterization of tracers and development of a fluorescence polarization immunoassay for amantadine with high sensitivity in chicken

Abstract

Fluorescence polarization immunoassay (FPIA) is a homogeneous and rapid analytical method that is suitable for high-throughput screening of large numbers of samples. However, FPIA typically suffers from lower sensitivity than the well-established enzyme-linked immunosorbent assay (ELISA), limiting its wide application as an analytical tool that can be run with trace levels of an analyte. Herein, a highly sensitive FPIA for detecting amantadine (AMD) in chicken is described. To achieve high sensitivity, nine chemical tracers of AMD that employ different fluoresceins, fluorescein derivatives, and haptens were synthesized and paired with four previously produced monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of FPIA was investigated and discussed. We found that the tracers with a linear and shorter bridge between adamantane and fluorescein generally provided higher sensitivity. After optimization, N′-(1-adamantyl) ethylenediamine (AEDA), an AMD structural analogue labeled with fluorescein isothiocyanate (FITC), achieved the lowest IC 50 value (1.0 ng/ml) in the FPIA, which was comparable to that of the heterologous ELISA format that used the same mAb7G2. We also investigated the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. The developed FPIA was applied to chicken to detect AMD residue, demonstrating a limit of detection (LOD) of 0.9 µg/kg with recoveries of 76.5-89.3% and coefficients of variation (CVs) below 14.5%. These results show that the proposed FPIA is an efficient, accurate, and convenient method for the rapid screening of AMD residues in chicken. Practical Application: The fluorescence polarization immunoassay (FPIA) was developed to determine and quantify amantadine (AMD) in chicken samples with high sensitivity. This homogeneous method avoids coating and washing steps and may provide high-throughput AMD screening in chicken in 10 min with high accuracy and precision. FPIA can be used as a monitoring tool and contribute significantly to the rapid detection of AMD in chicken.

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

“…In this study, ERY-CMO was obtained using ERY modified by CMO at the C9 position and introducing a reactive carbonyl group as previously described [ 28 ]. Tracer synthesis is important for the establishment of a highly sensitive and specific FPIA, once the antibody is prepared [ 29 , 31 ]. Therefore, to achieve highly sensitive detection of ERY, five new tracers including ERY-CMO-4′-AMF, ERM-FITC, ERM-DTAF, ERM-SRSC, and ERM-AF647 were designed using two haptens and five fluoresceins ( Figure 2 ).…”

“…In FPIA, limited tracer and antibody concentrations are considered to achieve the desired performance [ 29 ]. In addition, the dyes applied in this study were pH-sensitive reagents, and the combination between the mAb and tracers could be significantly affected by buffer pH [ 31 , 32 ].…”

“…ERM-FITC was synthesized according to our previously published method with minor modifications ( Figure 2 B) [ 29 ]. Briefly, ERM (1 mg) was solubilized in 1 mL DMF, and then FITC (1 mg) and triethylamine (50 µL) were added to 200 μL of the above solution and react at RT for seven days in dark conditions.…”

Dual-Wavelength Fluorescence Polarization Immunoassay for Simultaneous Detection of Sulfonamides and Antibacterial Synergists in Milk

“…Different antibody–tracer pairs may have a crucial impact on the sensitivity and accuracy in the development of FPIA [ 29 , 35 ]. To obtain the best antibody–tracer pair, we calculated the antibody titers of each mAb–tracer pair according to the antibody dilution curves ( Table 1 and Figure S2 ).…”

“…For more than a decade, our group has reported many FPIA methods to detect hazardous compounds, such as aflatoxins, zearalenone, sulfamethazine, orbifloxacin, and amantadine. [ 17 , 25 , 27 , 28 , 29 ]. However, to the best of our knowledge, no DWFPIA method has been reported for the simultaneous detection of SAs and ASGs in foodstuffs.…”