Colour-encoded paramagnetic microbead-based direct inhibition triplex flow cytometric immunoassay for ochratoxin A, fumonisins and zearalenone in cereals and cereal-based feed

Abstract

A combined (triplex) immunoassay for the simultaneous detection of three mycotoxins in grains was developed with superparamagnetic colour-encoded microbeads, in combination with two bead-dedicated flow cytometers. Monoclonal antibodies were coupled to the beads, and the amounts of bound mycotoxins were inversely related to the amounts of bound fluorescent labelled mycotoxins (inhibition immunoassay format). The selected monoclonal antibodies were tested for their target mycotoxins and for cross-reactivity with relevant metabolites and masked mycotoxins. In the triplex format, low levels of cross-interactions between the assays occurred at irrelevant high levels only. All three assays were influenced by the sample matrix of cereal extracts to some extent, and matrix-matched calibrations are recommended for quantitative screening purposes. In a preliminary in-house validation, the triplex assay was found to be reproducible, sensitive and sufficiently accurate for the quantitative screening at ML level. The triplex assay was critically compared to liquid chromatography-tandem mass spectrometry using reference materials and fortified blank material. Results for the quantification of ochratoxin A and zearalenone were in good agreement. However, the fumonisin assay was, due to overestimation, only suitable for qualitative judgements. Both flow cytometer platforms (Luminex 100 and FLEXMAP 3D) performed similar with respect to sensitivity with the advantages of a higher sample throughput and response range of the FLEXMAP 3D and lower cost of the Luminex 100.

The priciple of the direct inhibition microbead immunoassay using fluorescent mycotoxin-reporter conjugates

Fig. 1

Comparison of the direct assay (this work) and indirect microsphere immunoassay [33]. In an indirect assay (A), sample, antibodies and mycotoxin-BSA conjugated beads (a) are incubated so that there is competition between the conjugated mycotoxins on the bead and the free mycotoxins in the sample (b). After incubation, the beads are trapped by a magnet and the non-bound reagents washed away (c). The beads are released and an anti-mouse-RPE is added (d). After incubation, the beads are trapped again and non-bound anti-mouse-RPE is washed away (e). After release, the beads are measured (f). In the much simpler direct assay presented in this work (B), sample, mycotoxin-RPE conjugate labels and antibody-coupled beads are incubated (g). Labelled and free mycotoxins compete for antibodies on the beads (h). Then beads are trapped by a magnet and the non-bound reagents washed away (i). Beads are released and measured (j). This is done all-in one for three different mycotoxins in one well (C)

Fig. 2

Dose–relative response curves for FB₁ (a), OTA (b) and ZEN (c) in the triplex microsphere immunoassay in buffer and maize and wheat extracts (n = 3)

Fig. 3

Relative responses (n = 9) obtained in the triplex microsphere immunoassay by multi-fortification of six different blank wheat samples with FB1 (2,000 μg/kg, blue bars), OTA (5 μg/kg, red bars) and ZEN (100 μg/kg, green bars)