As a good alternative to the lack of pure ciguatoxin (CTX), conjugates of JKLM ring fragment, a carboxylic derivative of the right-hand tetracyclic terminus portion of CTX-1B (the most potent CTX) with two carrier proteins have been synthesized. Two procedures using different amount of hapten were evaluated: (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 μg) via the mixed anhydride method performed in a reversed micellar medium. In both cases, bovine serum albumin and ovalbumin were respectively used for immunization of BALB/c mice and antibody screening by a solid phase enzyme-linked immunosorbent assay (ELISA). Using the conjugates obtained through the micro-scale procedure, a long-term immunization schedule appeared to be more efficient to specifically trigger the mice immune system. These antisera titers determined in an end-point titration standard ELISA 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 competitive inhibition ELISA experiments, both types of antisera did not significantly cross-react 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-1B (133%). With sera from the long-term protocol, a lower detection limit for JKLM (1.23 x 10-9 M) was achieved by implementation of a biotin-avidin amplification system rather than by miniaturization of the assay in Terasaki plates. This study confirms the feasibility of the immunological approach for CTXs assay in fish tissues, but also emphasizes the importance of (i) the choice of the hapten to construct a relevant well-defined immunogen, (ii) the immunization schedule to obtain hapten-specific Abs still exhibiting high cross-reactivity to CTXs.
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