4.4.1. Hapten Synthesis and Conjugation

The corresponding hapten, ZON-60 -carboxymethyloxime was converted from ZON by the method of Thouvenot and Morfin [61]. The reaction mixture containing 300 mg of ZON dissolved in dry pyridine and 600 mg of carboxymethoxylamine was stirred overnight at room temperature (22 ± 0.5 ◦C), then evaporated and the residue was taken up in 50 mL of slightly alkaline (pH = 8) water. The aqueous phase was extracted with ethyl acetate (4 × 100 mL). The organic phase was dried over sodium sulfate, then separated and evaporated to afford 156 mg of the product. The process of conversion of ZON to the corresponding hapten was followed by thin layer chromatography using hexane–ethyl acetate (1:2) as an eluent. BSA and conalbumin (CONA) were applied as carrier proteins. The hapten was conjugated to these proteins through amide bonds [55], using the active

ester method for conjugation. Thus, 125 mg of the hapten ZON-60 -carboxymethyloxime was dissolved in 6.2 mL of dry tetrahydrofurane (THF), then 24 mg of *N*-hydroxy-succinimide and 73 mg of *N*,*N*0 -dicyclohexylcarbodiimide were added. The mixture was stirred for 2 h at room temperature and the precipitation formed (dicyclohexylurea) was filtered off. In the mixture of 15.5 mL of water and 0.9 mL of THF, 150 mg of the proteins were dissolved in two separate batches. To these solutions, 3.1 mL of the above THF solution of the hapten active ester was added dropwise. The mixture was stirred for 24 h at 4 ◦C, and then the products (hapten–protein conjugate) were dialyzed against water at 4 ◦C for 1 week. Conjugation was monitored by UV spectroscopy; conjugates were lyophilized and stored at −20 ◦C until the analytical measurements.
