To avoid the risk of saturatization or weak signal detection in assays, optimizations of the coating antigen concentration and serum dilution factor were performed by check-2.2.2. Immunoassay

board titration. The coating antigen, ZON-6′-carboxymethyloxime–BSA conjugate, was applied at concentrations in the range of 0.3125–2.5 µg/mL against the antiserum from rabbit 1 at dilutions in the range of 1:3375–1:1000 dilution factor. All combinations were investigated uninhibited and under inhibition by 3.2 ng/mL of ZON, as well. The coating antigen concentration and the antiserum dilution factor consistently influenced the analytical parameters (Figure 2b). The analytical signal (relative fluorescence unit, RFU) increased with increasing concentrations of the coating antigen and decreased with increasing dilution of the serum. The addition of ZON at a concentration of 3.2 ng/mL resulted in an average 40.0% ± 0.1% inhibition of the assay signal. 2.2.2. Immunoassay Indirect competitive ELFIAs were performed to established ZON calibration curves and to determine the LOD. The detection range was investigated in a concentration series of 0.004 pg/mL–2 µg/mL ZON in assay buffer. Matrix effects were determined by comparing calibration curves obtained in assay buffer and in surface water samples. No significant differences were determined among curves (*p* > 0.05), thus it has been concluded that determination of ZON in surface water can be performed without modification in sample preparation. Calibration curves and LODs were determined using both absorbance and fluorescence signals (Figure 3). For comparability of the two detection modes, assay signals are represented as relative values (signals ratios to maximal signal levels). Indirect competitive ELFIAs were performed to established ZON calibration curves and to determine the LOD. The detection range was investigated in a concentration series of 0.004 pg/mL–2 µg/mL ZON in assay buffer. Matrix effects were determined by comparing calibration curves obtained in assay buffer and in surface water samples. No significant differences were determined among curves (*p* > 0.05), thus it has been concluded that determination of ZON in surface water can be performed without modification in sample preparation. Calibration curves and LODs were determined using both absorbance and fluorescence signals (Figure 3). For comparability of the two detection modes, assay signals are represented as relative values (signals ratios to maximal signal levels). ZON at a concentration of 2000 ng/mL and above reached its full inhibition potential on the surface binding of the antibodies. This occurs because at this concentration the avidity of the primary antibody is saturated by ZON molecules in the solution, and therefore, further increases in ZON concentration cannot push the immunocomplexation equilibrium any further—ZON has reached its full capacity to block binding of the antibody to the coating antigen ZON–BSA conjugate on the surface of the immunoplate. The average relative analytical signal corresponding to the maximal assay signal produced by the uninhibited serum was set to the upper plateau level of the sigmoid standard curve. The average relative analytical signal corresponding to full inhibition of the serum was considered as the lower plateau level of the sigmoid standard curve. Analytical parameters of calibration curves are summarized in Table 1.

ZON at a concentration of 2000 ng/mL and above reached its full inhibition potential on the surface binding of the antibodies. This occurs because at this concentration the avidity of the primary antibody is saturated by ZON molecules in the solution, and therefore, further increases in ZON concentration cannot push the immunocomplexation equilibrium any further—ZON has reached its full capacity to block binding of the antibody to the coating antigen ZON–BSA conjugate on the surface of the immunoplate. The average relative analytical signal corresponding to the maximal assay signal produced by the uninhibited serum was set to the upper plateau level of the sigmoid standard curve. The LOD values were calculated for the two analytical detection modes of resorufin as a chromophore product. Thus, LOD = 0.25 and 0.09 ng/mL were determined for visual absorbance and fluorescence detections, respectively. Detection by fluorescence provided a wider and steeper dynamic range, thus ELFIA proved to be a 2.8-fold more sensitive method for ZON than the corresponding ELISA. It has to be noted that absorbance detection of resorufin by the application of QuantaRed Enhanced Chemifluorescent HRP Substrate Kit with HRP enzyme reaction (Thermo Fisher Scientific Inc., Waltham, MA, USA) provided 3.4 fold lower LOD than that of o-phenylenediamine dihydrochloride (OPD) as chromophore in a similar colorimetric ELISA (LOD for OPD = 0.85 ng/mL).

respectively.

average relative analytical signal corresponding to full inhibition of the serum was con-

**Figure 3.** Competitive indirect calibration curves for zearalenone obtained in assay buffer (hollow marker, dashed lines) and in surface water from river Danube (solid marker, solid lines) determined by absorbance (, □) and fluorescence (●, о), detected at 576 and 593 nm wavelengths, **Figure 3.** Competitive indirect calibration curves for zearalenone obtained in assay buffer (hollow marker, dashed lines) and in surface water from river Danube (solid marker, solid lines) determined by absorbance (, ) and fluorescence (•, o), detected at 576 and 593 nm wavelengths, respectively.

**Table 1.** Statistical parameters of the logistic mathematical fitting using the Rodbard equation [70] in the immunoassay format using assay signals by resorufin as a chromophore product with de-**Table 1.** Statistical parameters of the logistic mathematical fitting using the Rodbard equation [70] in the immunoassay format using assay signals by resorufin as a chromophore product with detection of absorbance and fluorescence.


 0.19 ± 0.83 1 Description of the equation parameters—ܣଵ: upper plateau, ܣଶ: lower plateau, ݔ: 50% inhibi-<sup>1</sup> Description of the equation parameters—*A*1: upper plateau, *A*2: lower plateau, *x*0: 50% inhibition, *p*: curve slope at the inflexion (IC50).
