Characterization of mice antisera elicited with a ciguatoxin tetracyclic synthetic ring fragment (JKLM) conjugated to carrier proteins

As a good alternative to the lack of pure ciguatoxin (CTX), conjugates of J KLM ring fragment, a carboxylic derivative of the right-hand tetracyclic te rminus portion of CTX-IB (the most potent CTX) with two carrier proteins ha ve been synthesized. Two procedures using different amount of hapten were e valuated: (i) a bulk technique (3-5 mg) via the N- hydroxysuccinimide ester of the carboxylic fragment in the presence of a water-soluble carbodiimide according to the standard method in aqueous buffer, or (ii) a micro-scale technique (300 pg) via the mixed anhydride method performed in a reversed m icellar medium. In both cases, bovine serum albumin and ovalbumin were resp ectively used for immunization of BALB/c mice and antibody screening by a s olid phase enzyme-linked immunosorbent assay (ELISA). Using the conjugates obtained through the micro-scale procedure, a long-term immunization schedu le appeared to be more efficient to specifically trigger the mice immune sy stem. These antisera liters determined in an end-point titration standard E LISA format were found around 1/128,000 as compared to 1/16,000 obtained in the short-term protocol (immunogen prepared via the bulk procedure). In co mpetitive inhibition ELISA experiments, both types of antisera did not sign ificantly cross-reset with a brevetoxin congener (PbTx-3). okadaic acid (OA ), monensin or other polyether compounds, but only sera from the short-term protocol did show high cross-reactivity to CTX-IB (133%). With sera from t he long-term protocol, a lower detection limit for JKLM (1.23 x 10(-9) hi) was achieved by implementation of a biotin-avidin amplification system rath er than by miniaturization of the assay in Terasaki plates. This study conf irms the feasibility of the immunological approach for CTXs assay in fish t issues, but also emphasizes the importance of (i) the choice of the hapten to construct a relevant well-defined immunogen. (ii) the immunization sched ule to obtain hapten-specific Abs still exhibiting high cross-reactivity to CTXs. (C) 1999 Elsevier Science Ltd. All rights reserved.