Quantification of Thiram in Honeybees: Development of a Chemiluminescent ELISA

Abstract

International audienceA Chemiluminescence Enzyme-Linked Immuno-Sorbent Assay (CL-ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti-thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC50 and the limit of detection (LOD) values were 60 ng mL-1 and 9 ng mL -1, respectively, similar to those of colorimetric ELISA with a calibration range of 9 – 15,000 ng mL-1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid-liquid extraction or the graphitized carbon-based solid phase extraction. The calibration curves in honeybee extracts from liquid-liquid procedure gave an IC50 of 141 ng mL-1 and a LOD of 17 ng mL-1. In case of extracts obtained by SPE these values were 139 ng mL-1 and 15 ng mL-1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL-1 of thiram was 72% for SPE, higher than for liquid-liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid-liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia

Determination of thiram in natural waters using flow‐injection with cerium(IV)–quinine chemiluminescence system

“…On the other hand, FI techniques allow the rapid and reproducible mixing of sample and reagent near the detector, which makes this methodology particularly well suited to monitoring transient light emission from CL reactions. [16,17] Girotti et al [18] recently reported a chemiluminescent enzymelinked immunosorbent assay (CL-ELISA) using luminol, horseradish peroxidase as labeling enzyme and polyclonal antibodies for the detection and quantification of thiram in honeybee extracts. Recently, we have reported [19] an FI-CL method for the determination of dithiocarbamate fungicides (maneb, nabam and thiram) based on CL detection.…”

High performance Au/Ag core/shell bipyramids for determination of thiram based on surface‐enhanced Raman scattering

“…In the body, carbon disulfide is formed from the breakdown of thiram, which contributes to the toxicity of thiram to the liver . Many methods have been reported for the determination of thiram, including gas chromatography/mass spectrometry, UV–visible spectrophotometry, high‐performance liquid chromatography, liquid chromatography–atmospheric pressure chemical ionization mass spectrometry, chemiluminescence analysis, and surface‐enhanced Raman scattering (SERS) spectroscopy . Table gives a comparison among some different methods for the determination of thiram.…”

Photodegradation and Flow-Injection Determination of Dithiocarbamate Fungicides in Natural Water with Chemiluminescence Detection

“…Girotti et al 22 recently reported a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) using luminol and horseradish peroxidase as labeling enzyme and polyclonal antibodies for the detection and quantification of thiram in honeybee extracts. Comprehensive reviews on thiram pesticide in environmental and pharmaceutical samples have also been reported.…”