## 4 Conclusions  
This work proposes a method for determining the content of nonylphenol using the developed chemuluminescent competitive enzyme immunoassay. By optimizing the analysis conditions, it was possible to decrease the detection limit of the target analyte to 9 ng/mL, compared to the colorimetric EIA (55 ng/mL), in a buffer solution. In addition, the chemiluminescence method allows one to evaluate the kinetics of substrate oxidation in microplate wells with different label contents.  
The advantages of chemiluminescenceence are high signal values, wide operating ranges, absence of background luminescence, and the possibility to evaluate the signal in kinetic mode. The main disadvantage is a high risk of nonspecific substrate oxidation. The proposed option for detecting and analyzing an analytical signal clearly demonstrates its performance in the case of choosing the optimal time of interaction with the substrate and establishing equilibrium in the system. This work shows that the maximum convergence of the curves in various water samples is observed 20 min after adding the substrate mixture to the wells. The proposed method and approach have good prospects for further application for the detection of analytes in water samples by CL-EIA.  
Conceptualization, A.N.B.; validation, N.S.K.; formal analysis, N.S.K. and K.V.S.; investigation, A.N.B.; resources, A.N.B.; writing--original draft preparation, A.N.B. and N.S.K.; writing--review and editing, A.N.B., N.S.K., K.V.S., A.V.Z. and B.B.D.; visualization, A.N.B.; supervision, A.V.Z. and B.B.D.; project administration, A.V.Z. and B.B.D.; funding acquisition, A.N.B. and B.B.D. All authors have read and agreed to the published version of the manuscript.  
This work was financially supported by the Russian Science Foundation (grant 22-13-00293).  
The animal study protocol was approved by the Institutional Ethics Committee of the Research Center of Biotechnology.  
Not applicable.  
The raw data supporting the conclusions of this article will be made available by the authors on request.  
The measurements were carried out on the equipment of the Shared-Access Equipment Centre \"Industrial Biotechnology\" of Research Center of Biotechnology of the Russian Academy of Sciences.  
The authors declare no conflicts of interest.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**No.** & **Technique** & **Detection** & **Sample** & **Antibody** & **IC\({}_{\mathbf{10}}\) or IC\({}_{\mathbf{50}}\)** & **Ref.** \\ \hline
1 & ELISA & Colorimetry & \begin{tabular}{c} Prawn and clam \\ samples (seafood) \\ \end{tabular} & \
\begin{tabular}{c} polyclonal \\ \end{tabular} &
\begin{tabular}{c} 10 \(\mu\)g/L \\ 20 \(\mu\)g/L \\ \end{tabular} & [48] \\
2 & ELISA & Colorimetry & Lake water & Monoclonal, polyclonal &
\begin{tabular}{c} 76 ng/mL--OP \\ 24 ng/mL—NP \\ \end{tabular} & [49] \\
3 & LFIA & Colorimetry & - & \
\begin{tabular}{c} polyclonal \\ \end{tabular} &
\begin{tabular}{c} 1 \(\mu\)g/mL \\ 1.1 and 0.4 \(\mu\)g/mL \\ \end{tabular} & [50] \\
5 & EIA & Chemuluminescent & Spring water & \
\begin{tabular}{c} polyclonal \\ \end{tabular} &
\begin{tabular}{c} 7 ng/mL \\ \end{tabular} & This work \\ \hline \hline \end{tabular}
\end{table}
Table 5: Works on the immunochemical determination of alkylphenols in the most recent 10 years.