tion, p: curve slope at the inflexion (IC50). 2.2.3. Effects of Light Source Intensity

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) Calibration curves were determined in the induced fluorescence method at different light source intensities. LED power can be digitally set between 0.001 µW and 4.63 mW in 256 nonequidistant steps at 532 nm wavelength, and it was tuned to provide assay signals between 20 and 30 RFU as the suggested least detectable value and 4095 RFU as the highest readable signal by the instrument. Correspondingly, the LED power values investigated ranged between 1.1 and 314 µW (1.1, 100.2, 169.4, 256 and 314 µW). As a background, 2000 ng/mL ZON solution was applied that triggered total inhibition of the antiserum. For 1.1 µW LED power there were no differences in the RFU values among different dilutions of ZON and for 314 µW LED power the RFU values reached the maximum (4095) at 0.64 ng/mL ZON concentration. Thus, effects of the LED power on ZON quantification were determined at values of 100.2, 169.4 and 256 µW. Recorded background signals were 24.7 ± 1.1, 27.9 ± 0.8 and 43.7 ± 1.0, while maximum (uninhibited) fluorescence levels were 1447, 2303 and 3131 for 100.2, 169.4, 256 µW, respectively. The analytical parameters

of the calibration curves were determined with the optimized immunoassay system (see Section 4.4.3). In the data evaluation process, RFUs were corrected with the background. Among analytical parameters, IC<sup>50</sup> values determined from the calibration curves in a concentration range of 0.0256–2000 ng/mL ZON were compared, and were found to be 2.52 ± 0.24, 3.04 ± 0.31 and 3.68 ± 0.29 ng/mL ZON for LED power values of 100.2, 169.4, 256 µW, respectively, while the LOD values did not appear to be significantly affected by the intensity of the light source.
