ELISA for detecting okadaic acid in model systems using purified polyclonal antibodies

Polyclonal antibodies were produced in rabbits against okadaic acid (OA) following its coupling to bovine serum albumin using standard carbodiimide condensation procedure to form the immunogen. The immunogen was dialyzed against Tris buffer pH 7.45 at 4C and used to immunize two rabbits. Each rabbit received (at four sites) 0.5 mt immunogen (i.d.) together with adjuvant followed by three repeated injections of 0.3 mt emulsified immunogen-adjuvant mixture at weekly intervals. The last injection was made 60 days after the fourth. The antiserum was collected, at intervals, the immunoglobulin fraction (IgG) isolated purified and used in an ELISA system to capture the okadaic moiety of the immunogen. Antibody titers increased following repeated immunization and the IgG recognized low levels of OA. However, ELISA was more sensitive for detecting the immunogen than for pure OA, but using the biotinylated-IgG enhanced the titration to both and the detection limit for OA war; 0.63 ng per 0.1 mL buffer-methanol and the assay linearity ranged from 0.63 to 5.0 ng OA.