Development of a multiplex flow cytometric microsphere immunoassay for mycotoxins and evaluation of its application in feed

Abstract

A multi-mycotoxin immunoassay-using the MultiAnalyte Profiling (xMAP) technology-is developed and evaluated. This technology combines a unique color-coded microsphere suspension array, with a dedicated flow cytometer. We aimed for the combined detection of aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, zearalenone and T-2-toxin in an inhibition immunoassay format. Sets of six mycotoxin-protein conjugates and six specific monoclonal antibodies were selected, and we observed good sensitivities and no cross-interactions between the assays in buffer. However, detrimental effects of the feed extract on the sensitivities and in some cases on the slopes of the curves were observed and different sample materials showed different effects. Therefore, for quantitative analysis, this assay depends on calibration curves in blank matrix extracts or on the use of a suitable multi-mycotoxin cleanup. To test if the method was suitable for the qualitative detection at EU guidance levels, we fortified rapeseed meal, a feed ingredient, with the six mycotoxins, and all extracts showed inhibited responses in comparison with the non-fortified sample extract. Contaminated FAPAS reference feed samples assigned for a single mycotoxin showed strong inhibitions in the corresponding assays but also often in other assays of the multiplex. In most cases, the presence of these other mycotoxins was confirmed by instrumental analysis. The multiplex immunoassay can be easily extended with other mycotoxins of interest, but finding a suitable multi-mycotoxin cleanup will improve its applicability.

Fig. 1

Typical flow cytometric output of the StarStation software showing all microspheres in the Doublet Discriminator (DD) plot (upper left part), classification and counting of the microspheres based on the log CL1 (red) and log CL2 (infra-red) ratio in the Classifier plot (upper right part) and 2 of the 6 response plots (lower two parts) showing the reporter signals (RPTs) for the DON and ZEA assay in the multiplex flow cytometric immunoassay

Fig. 2

Average dose-response curves (n = 9) of the six mycotoxin assays in the multiplex microsphere inhibition assay format in buffer (a) and in two times diluted sample extract (b)

Fig. 3

Average (n = 3) maximum responses (MFI) for the OTA (a) and AFB₁ (b) assays in different sample extracts and buffer