Abstract:

Chloramphenicolsuccinate and keyhole limpet hemocyanin (KLH) were mixed at 3 initial molar ratios, namely 30:1, 20:1 and 10:1, to synthesize chloramphenicol (CAP) immunogens (hapten-KLH conjugates) with coupling ratios of 25:1, 16:1 and 8:1, respectively. Additionally, chloramphenicolsuccinate was mixed with ovalbumin (OVA) at 7 initial molar ratios, namely 40:1, 30:1, 20:1, 10:1, 7:1, 4:1 and 1:1, to synthesize CAP coating antigens (hapten-OVA conjugates) with coupling ratios determined by the UV spectrometric method of 23:1, 20:1, 14:1, 6:1, 3:1, 1:1 and 1:3, respectively. Indirect competitive enzyme linked immunosorbent assay (ic-ELISA) experiments were carried out to choose a CAP coating antigen with an optimal coupling ratio. CAP was determined with the CAP coating antigens to have IC50 values ranging from 15 to 36 ng/mL. As the coupling ratio between chloramphenicolsuccinate and OVA decreased from 26:1 to 3:1, there was a decrease in the IC50 value of CAP. When the coupling ratio was less than 3:1, the IC50 value of CAP exhibited a slight increase. These findings demonstrate that a CAP coating antigen with an optimal coupling ratio should be synthesized for the ic-ELISA assay of IC50 significantly influenced by coupling ratio.

Key words: chloramphenicol, immunogen, coating antigen, coupling ratio