## 3 Results and Discussions
### Experimental Validation of the Model  
For experimental confirmation of the established theoretical dependences, immunochemical determination of a group of structurally similar compounds from the sulfonamide class was carried out. Seven compounds were tested in two variants of the equilibrium competitive immunoassay: ELISA and FPIA. It should be noted that the competing antigen preparations, namely an SA-protein conjugate for ELISA and an SA-(fluorescein derivative) conjugate for FPIA, were synthesized using the same modification of SA coinciding with the kind of activation and orientation in the conjugates. Therefore, the demonstrated properties of the two assays are not associated with different immune recognition of SA derivatives. For both immunoassay variants, preliminary optimization of conditions was carried out, providing the highest detection sensitivity of target analytes and acceptable signal amplitude. In the final variants of the analyses, the following ratios of reagents were selected: for ELISA, the concentration of the hapten-protein immobilized on the plate was 76 nM and the concentration of specific antibodies was 0.3 nM; for FPIA, the concentration of SA-(fluorescein derivative) conjugate was 0.16 nM and the concentration of specific antibodies was 0.2 nM. It should be noted that for both assays, the time of immune interaction (5 min for homogeneous FPIA and 1 h for heterogeneous ELISA) provided the possibility to reach chemical equilibrium, and its increase for both cases did not lead to significant changes in the registered signal and analytical parameters (limit of detection, working range). The chosen concentrations of antibodies were comparable for both assays, while the concentrations of the modified haptens differed by orders of magnitude and were significantly higher for ELISA compared to FPIA. Note that the true concentrations of the active hapten bound to the surface of the microplate are unknown. The concentration of the hapten-protein conjugate used for immobilization was 475 times higher than the concentration of the SA-(fluorescein derivative) conjugate in FPIA. Even taking into account the fact that, according to existing concepts, when immobilized on a plate, more than 90% of the protein may lose activity [56; 57; 58], it can be argued that the concentration of the modified hapten (Ag* in the proposed model) is several times higher for ELISA than for FPIA. This means that the ELISA is predicted to be more cross-reactive than FPIA is. The results obtained for the determination of SAs by two methods and integrated in Table 2 confirmed the prediction of the model: the cross-reactivity of ELISA exceeded the cross-reactivity of FPIA by up to 8.5 times (for sulfadimethoxine). Thus, it was confirmed that the cross-reactivity of the immunoassay depends not only on the properties of the antibodies used, but also on the specific parameters of the analysis technique, such as the ratio of the concentrations of the antibodies and modified hapten that are used to realize the given assay format. The cross-reactivity can also be modulated by varying the concentrations and ratios of the reagents in the same assay format. We have shown this possibility considering the cross-reactivities for four sulfonamides, presented in Table 3, in the course of their determination using the FPIA technique. With an increase in the concentrations of antibodies and labeled hapten derivative by five times, the cross-reactivity increased from 2 (for sulfadiazine) to 9.5 times (for sulfadimethoxine). \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Sulfanilamide** & **IC\({}_{50}\)**, \(\mu\)g/mL** & **CR\({}_{\%}\)** & **IC\({}_{50}\)**, \(\mu\)g/mL** & **CR\({}_{\%}\)** \\  & **A** & **A** & **B** & **B** \\ \hline Sulfachlorpyridazine & 2.6 & 100 & 23 & 100 \\ Sulfadiazine & 16.5 & 16 & 75 & 31 \\ Sulfamethazine & 17 & 15 & 50 & 46 \\ Sulfadimethoxine & 121 & 2 & 120 & 19 \\ \hline \hline \end{tabular}
\end{table}