Analytical Validation of a Direct Competitive ELISA for Multiple Mycotoxin Detection in Human Serum

Abstract

Mycotoxin exposure in humans is primarily assessed through its occurrence in external sources, such as food commodities. Herein, we have developed a direct competitive ELISA to facilitate the detection of aflatoxin B1 (AFB1), deoxynivalenol (DON), fumonisin (FUM B1/B2), ochratoxin A (OTA), and zearalenone (ZEA) in human serum. The analytical validation of the assay followed practices endorsed by the international research community and the EU directive 96/23/EC in order to examine detection capability, recovery, and cross-reactivity. The assay demonstrated a lower limit of quantitation (LLOQ) for AFB1 [0.61 ng/mL (hereon ng/mL = ppb)], DON (19.53 ppb), FUM (4.88 ppb), OTA (19.53 ppb), and ZEA (0.15 ppb). Recovery from human serum for all mycotoxins spanned from 73% to 106%. Likewise, the specificity for monoclonal antibodies against cross-reactant mycotoxins ranged from 2% to 11%. This study compares the LLOQ and recovery values with commercial and emerging immuno-based methods for detecting mycotoxins in foodstuffs. The LLOQ values from the present study were among the lowest in commercial or emerging methods. Despite the differences in the extraction protocols and matrices, the recovery range in this study, commercial tests, and other procedures were similar for all mycotoxins. Overall, the assay detected AFB1, DON, FUM, OTA, and ZEA in human serum with excellent accuracy, precision, and specificity.

Keywords: enzyme-linked immunosorbent assay; human biological monitoring; multiple mycotoxins.

Figure 1

Calibration curves with a 4PL and Gaussian fit for AFB1, DON, FUM, OTA, and ZEA. (a) The assay can accurately measure low amounts of analyte as the four-parameter logistic (4PL) curve establishes a near-perfect fit (R²) for all mycotoxins. The coefficient of determination (R²) is the ratio of the variation that the 4PL curve-fitting model explains to the total variation in the model. The R² value will equal 1 for a perfect fit and will be closer to 0 for a bad 4PL fit. Furthermore, the lower limit of quantitation (LLOQ) is the lowest amount of an analyte detectable in a sample with suitable accuracy. As a result, Figure 1a presents the LLOQ in ppb for each mycotoxin and the associated accuracy percentages at the LLOQs achieved through back-calculation. (b) Gaussian curve fit analysis and associated coefficient values used to predict the concentration (in ng/mL or ppb) for recovery and the matrix interference analysis are in Table 1. The a, b, and c Gaussian coefficients refer to the height of the curve’s peak, the position of the center of the peak, and the standard deviation, respectively. In panels a and b, %B/B0 refers to the OD value with (B) and without (B0) competition for antibody binding sites on the microplate.

Figure 2

Monoclonal antibodies against specific mycotoxins do not significantly cross-react.