*4.2. Immunogen and Antibody Production*

Since ZON is low-molecular-weight hapten, it is non-immunogenic and should be conjugated to a protein carrier for immunisation. ZON was converted to the corresponding hapten, ZON-60 -carboxymethyloxime, and was conjugated to carrier proteins BSA and CONA by the method from the literature [26]. Polyclonal antibodies directed against ZON were produced in female, 3-month-old New Zealand white rabbits immunised periodically and intradermally with a standard mixture of 25 µg of the ZON-CONA conjugate immunogen per kg body weight and 50 µL of Freund's complete or incomplete adjuvant. Rabbit immunisation was carried out under the supervision of the Ethics Committee of Research on Animals (Food Science Research Institute, National Agricultural Research and Innovation Centre, Budapest, Hungary) and under the authorisation and inspection by the Government Office for Pest County in Hungary (Official permit for animal testing # PE/EA/45-6/2020, last date of approval: 21 February 2020). Serum from the whole blood obtained was centrifuged at 2400 g for 15 min, and its IgG fraction was purified by sodium-sulphate precipitation [80].
