*4.5. Enzyme-Linked Immunosorbent Assay*

Enzyme-linked immunosorbent assays (ELISAs) were carried out in 96-well microplates (see Section 4.1) in a competitive immunoassay format. Microplate wells were coated with 1 µg/mL ZON–BSA conjugate in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH = 9.6) for overnight (≈8 h) at 4 ◦C. The unbound conjugate was washed out 4 times with phosphate buffer saline (PBS) (137 mM NaCl, 2.7 KCl, 10 mM Na2HPO<sup>4</sup> × 2H2O, pH = 7.4) with 2% Tween20. Blocking was carried out with 150 µL/well 1% gelatine in PBS for 1.5 h of incubation at 37 ◦C. After washing, competition was performed by adding 50 µL/well of both ZON analytical standard and purified rabbit anti-ZON serum (in PBS buffer with 5% Tween20, dilution 1:1000). After 1 h of incubation at 37 ◦C and four times washing, 100 µL/well goat anti-rabbit IgG-HRP (horseradish peroxidase, dilution 1:7500) conjugate were added as secondary antibody and incubated for 1 h at 37 ◦C. Unbound secondary antibodies were washed out 4 times with PBS, and enzymatic activity was measured using 100 µL/well substrate solution containing 1.3 mM hydrogen peroxide as a substrate and 0.42 mM TMB as a chromophore in 100 mM citrate buffer (pH = 6.0). After 10 min of incubation, the enzymatic activity was stopped by 50 µl of 4 M sulphuric acid (H2SO4), and absorbance was measured at a wavelength of 450 nm.

**Author Contributions:** Conceptualisation, A.N. and A.S.; methodology, A.N.; validation, A.M.A.-J., B.G. and E.T.; formal analysis, A.M.A.-J. and B.G.; investigation, A.N.; writing—original draft preparation, A.M.A.-J., B.G. and E.T.; writing—review and editing, A.N. and A.S.; visualisation, A.M.A.-J. and B.G.; supervision, A.N. and A.S.; project administration, A.S.; funding acquisition, A.N. and A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Hungarian National Research, Development and Innovation Office, project NVKP\_16-1-2016-0049 "*In situ*, complex water quality monitoring by using direct or immunofluorimetry and plasma spectroscopy" and TKP2020-NKA 24 "Tématerületi kiválóság program" in the Thematic Excellence Programme 2020.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy reasons.

**Acknowledgments:** A.M.A.-J. would like to thank Babylon University, College of Sciences, Hilla, Iraq for sponsoring his PhD project at Sheffield Hallam University, UK.

**Conflicts of Interest:** The authors declare no conflict of interest.
