Rapid analysis of diclofenac in freshwater and wastewater by a monoclonal antibody-based highly sensitive ELISA

Abstract

The non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF) is found worldwide in the aqueous environment. Therefore, it has raised increased public concern on potential long-term impact on human health and wildlife. The importance of DCF has been emphasized by the European Union recently by including this pharmaceutical in the first watch list of priority hazardous substances in order to gather Union-wide monitoring data. Rapid and cheap methods of analysis are therefore required for fresh and wastewater monitoring with high sample load. Here, for the first time, well-characterized monoclonal antibodies (mAbs) against DCF were generated and a highly sensitive ELISA developed. The best antibody (mAb 12G5) is highly affine (KD = 1.5 × 10(-10) M), stable to potential matrix interferences such as pH value (pH range 5.2-9.2), calcium ion concentration (up to 75 mg/L), and humic acid content (up to 20 mg/L). The limit of detection (LOD, S/N = 3) and IC50 of the ELISA calibration curve were 7.8 and 44 ng/L, respectively. The working range was defined between 11 and 180 ng/L. On average, about 10 % cross-reactivity (CR) was found for DCF metabolites 5-OH-DCF, 4'-OH-DCF, and DCF-acyl glucuronide, but other structurally related NSAIDs showed binding <1 % compared to the parent compound. While DCF concentrations at the low ppt range were measured in river and lake water, higher values of 2.9 and 2.1 μg/L were found in wastewater influents and effluents, respectively. These results could be confirmed by solid phase extraction combined with LC-MS.

Gold Nanoparticles Assembly on Silicon and Gold Surfaces: Mechanism, Stability, and Efficiency in Diclofenac Biosensing

“…Sodium dodecyl sulfate (SDS) was purchased from Fisher Scientific. The rabbit polyclonal anti-diclofenac serum was prepared in-house 32 , its analytical potential is detailed in reference 33 . Milli-Q water (18 MΩ, Millipore, France) was used for the preparation of the solutions and for all washes.…”

Development of an immunochromatographic lateral flow strip test for the rapid detection of diclofenac in medicinal wine

“…A generic mAb 12G5 against DCF was prepared. The LOD, IC 50 and the range of detection of competitive indirect ELISA were 7.8, 44 and 11-180 ng/L, respectively (Huebner et al, 2015). The CR of mAb 12G5 was 8.5-13% for DCF metabolites, but less than 1% for the structural analogues.…”

“…In contrast, immunoassays, based on the specific reaction between antigen and antibody, can overcome some of these drawbacks. Thus, the enzyme-linked immunosorbent assays (ELISAs) (Huebner, Weber, Niessner, Boujday, & Knopp, 2015) and Upconversion-Linked immunosorbent assays (ULISAs) (Hlaváček et al, 2016) were developed for the detection of DCF in wastewater, livestock carcasses, and other biological samples (Saini et al, 2012). ELISAs eliminate sophisticated sample pretreatment and can tolerate certain matrix interferences because of washes between steps.…”

Quantifying the UK Online Interest in Substances of the EU Watchlist for Water Monitoring: Diclofenac, Estradiol, and the Macrolide Antibiotics

“…Estradiol is also not fully degraded, and its presence in surface waters [39] affects sea life [41]. As wastewater treatment is suggested to not completely remove the concentrations of these substances in the aquatic environment [14,[16][17][18]39], the solution could be to manage the pollution at the source, which is the human, veterinarian, and agricultural intake and use. Steps towards this direction have been implemented, as for example in the case of Diclofenac, which is now a prescription-only drug in the UK [72].…”