Development of an amplified luminescent proximity homogeneous assay for the detection of sulfonamides in animal-derived products

Abstract

In this study, we carried out an amplified luminescent proximity homogeneous assay (AlphaLISA) to detect sulfonamides (SAs) antibiotic residues in plasma, milk, pork, chicken, and fish. The SAs AlphaLISA method can detect 13 SAs with half-inhibitory concentration (IC₅₀) 2.11-29.77 ng/ml. The detection level of those SAs was 0.3-41.12 ng/ml in matrices, which satisfied the maximum residue limit (MRL) of the European Union, United States, and China. Our recoveries are in the range of 88% to 116.8% with a coefficient of variation less than 9.3% for different spiked food samples. We observed a good correlation between the AlphaLISA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) with blood samples from injected rabbits. The established AlphaLISA method provided a no-washing, rapid, high-throughput screening tool for SAs in food quality control, which is suitable for small-volume samples.

Keywords: amplified luminescent proximity homogeneous assay (AlphaLISA); food quality control; sulfamethazine; sulfonamides.

FIGURE 1

Principle of SAs detection based on AlphaLISA. (a) When there was no free SAs, the acceptor beads were in an emitting state (“bright”) owing to the singlet oxygen (¹O₂) generated by donor beads upon photo‐excitation at an effective assay distance of 200 nm; (b) in the presence of free SAs, the acceptor beads were in a nonemitting state (“dark”) because the donor beads were out of the 200 nm range

FIGURE 2

Morphology of acceptor beads as imaged by transmission electron microscopy. (a) Naked acceptor beads; (b) anti‐mouse IgG‐coated acceptor beads; and (c) monoclonal antibody‐bound anti‐mouse IgG‐coated acceptor beads. The scale bar was 50 nm

FIGURE 3

Optimization of concentration of the SAs antibody (a) and total reaction time (b). Note: B/B₀(%) equals AlphaLISA signal values of standard or sample divided by AlphaLISA signal values at zero standard concentration times 100. The vertical bars indicate standard deviation of the mean results of three replicates. The different lines represent different concentrations of the SAs antibody(a) and total reaction time (b)

FIGURE 4

Calibration curves of sulfamethazine. Each point represents the mean of three replicates

FIGURE 5

Correlation of sulfamethazine detection in plasma between the UPLC‐MS/MS and AlphaLISA methods. (Inset) Low sulfamethazine concentration range detected by UPLC‐MS/MS and AlphaLISA methods