Polyclonal and monoclonal antibodies to PbTx-2-type brevetoxins using minute amount of hapten-protein conjugates obtained in a reversed micellar medium

Minute amount of Brevetoxin PbTx-3 (400 mug; 0.446 mu mol) was converted in to an hemisuccinate derivative (PbTx-3 HS) then covalently linked to bovine serum albumin (BSA) and ovalbumin (OVA) in a reversed micellar medium. Acc ording to the efficient cyclic synthetic procedure described, the epitope d ensity of the conjugates was around 10 and 20 for OVA and BSA carriers, res pectively. The kinetics of antibody production in sequential sera harvested from a single BALB/c mouse immunised by multiple intraperitoneal (i.p.) in jections of PbTx-3-BSA conjugate was performed by enzyme-linked immunosorbe nt assay (ELISA). Two monoclonal antibodies (MAbs) against PbTx-3 were sele cted from fusion of the mouse immune splenocytes with the P3-X63-Ag 8.653 m yeloma cells. In competitive inhibition ELISA experiments, both polyclonal antibodies and MAbs exhibited strong cross-reactivity (greater than or equa l to 100%) to other PbTx-2-type toxins (PbTx-2 and -9) but low or moderate cross-reactivity (6-15%) to a PbTx-1-type toxin (PbTx-1). Moreover, using t hese two MAbs, a low cross-reactivity with okadaic acid (3%) was noticed bu t no significant cross-reactivity was observed with two ciguatoxins (CTX-1B and CTX-3C) over the concentration range studied. The apparent dissociatio n constant (KD) for the interaction of these MAbs with free PbTx-2-type tox ins was in the 10(-6)-10(-7) M range. The performance of this MAb-based ass ay (limit of detection approximate to 5 ng/well; working range = 8-150 ng/w ell) coupled with adequate extraction methods would provide an alternative assay to the mouse i.p. bioassay for routine shellfish monitoring. This pro duction and characterisation of MAbs using small amount of polyether toxins in a reversed micellar medium appear most valuable for the development of immunoassays to other highly potent but poorly available marine polyether t oxins like ciguatoxins (CTXs). (C) 2001 Elsevier Science Ltd. All rights re served.