Multi-wavelength fluorescence polarization immunoassays for simultaneous detection of amantadine and ribavirin in chicken and human serum

Abstract

In this study, a homologous multi-wavelength fluorescence polarization immunoassays (MWFPIA) for rapid determination of amantadine (AMD) and ribavirin (RVB) simultaneously was established. The MWFPIA could quantify AMD and RVB concentration simultaneously in 1 min. After optimisation, longwavelength tracers AEDA-AF647 with monoclonal antibody (Mab) 1A12 and RVB-CP-EDF with polyclonal antibody (Pab) G412 were chosen for the development of MWFPIA. The limit of detections (LODs) of AMD and RVB were 1.7 and 1.0 μg/kg in chickens, and 17.6 and 10.4 μg/L in human serum, respectively. Recoveries of AMD and RVB in chickens were 61.2-94.9% and 87.3-90.2%, respectively with coefficient of variations (CVs) lower than 16.7%. For human serum, recoveries were 60.2-84.3% and 63.5-74.2% for AMD and RVB, respectively, and CVs was lower than 15.3%. The results indicated that the MWFPIA could be used for the monitoring of antiviral drugs with short analysis time, great accuracy and high throughput.

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

“…According to previous reports, the length of the linker between the hapten and fluorescein would directly affect the sensitivity of FPIA [ 35 , 36 , 37 , 38 , 39 ]. Generally, the linker between the fluorescein and hapten needs to separate the antibody binding site from the fluorescein molecule, so that the antibody can recognize the hapten of tracers through its inherent affinity, with as little steric hindrance as possible.…”

“…Dong et al employed a tracer containing the longest linker to minimize the fluorescence quenching of the fluorescein molecules and to reduce the interference at the antibody binding sites by the fluorescent tags paired with antibodies to develop the best performing FPIA [ 36 ]. There are two studies on the simultaneous detection of analytes using DWFPIA that have been reported [ 25 , 38 ]. However, the effect of linker length on sensitivity was not mentioned or clearly discussed.…”

“…The detection system of DWFPIA consists of two distinct fluorescence signals; moreover, the interference between the short-wavelength fluorescein (EDF/BDF/HDF) and the long-wavelength fluorescein (DSCA/DSCH/AF647) could be ignored, according to our previous studies [ 26 , 38 ]. Furthermore, the nonspecific binding was analyzed by comparing the FP changes of each tracer (or the tracer cocktail) upon the addition of the free, specific, non-specific, and cocktail mAbs at working concentrations.…”