Development of an enzyme-linked immunosorbent assay for fentanyl and applications of fentanyl antibody-coated nanoparticles for sample preparation.

A sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of fentanyl in serum and urine. The ELISA used an indirect competitive method produced by coating the plate with thyroglobulin conjugated with fentanyl hapten. Antibodies against fentanyl-hemocyanin were detected by a goat-anti-rabbit antibody conjugated with alkaline phosphatase. Calibration standard curves ranged from 0.5ng/ml to 50mug/ml (IC(50)=10ng/ml), and the limits of detection were 0.5 and 1.0ng/ml for serum and urine, respectively. The intra- and inter-assay variations were less than 8% and 10%, respectively. The antibody produced against fentanyl completely cross-reacted with p-fluorofentanyl, thienylfentanyl and 3-methylthienylfentanyl, cross-reacted highly with carfentanil (85%), but was considered non-cross-reactive with alpha-methylfentanyl (5%), sufentanil (<1%), alfentanil (<1%) and lofentanil (<1%). Nano-sized iron oxide magnetic particles coated with the developed fentanyl antibody were capable of specific binding and releasing of fentanyl from urine samples. This enabled the drug to be effectively pre-concentrated and decreased the limit of detection by approximately one order of magnitude. The analytical background noise was significantly reduced to enable fentanyl detection at concentrations originally below chromatographic limit of detection. The change of platform for antibody binding with nanoparticles demonstrated a novel use of antibodies for sample preparation and should facilitate drug screening by traditional ELISA.