An ELISA for Okadaic Acid and Its Analogs among the Diarrhetic Shellfish Toxins Using Mouse Monoclonal Anti-Okadaic Acid Antibodies which Are Resistant to Organic Solvents.

Abstract

A specific and sensitive enzyme-linked immunosorbent assay using immobilized mouse monoclonal antibodies to okadaic acid (OA), OA423-3 and OA958-2, which are resistant to organic solvents, has been established to determine OA and its analogs (OAs). OA423-3 (IgG1-x), which bound only OA in 10% (v/v) aqueous methanol, is useful to determine OA selectively. The quantitative determination range of OA was 70 pg-6.0 ng/ml, and the coefficient of variation (cv) within six independent analyses was 15.4% at the OA concentration of 0.52 ng/ml. Besides, OA958-2 (IgG1-x), which equally bound OA, dinophysistoxin-1 (DTX1) and 7-O-palmitoyl-dinophysistoxin-1 (Pal-DTX1) in methanol, is successful in determing total OAs. The quantitative determination range of each toxin was 4.1 ng-2.2 μg/ml for OA, 4.9 ng-2.25 μg/ml for DTX1 and 11.0 ng-1.15 μg/ml for Pal-DTX1, respectively. The cv values were 12.9-17.5% at the toxin concentration of about 100 ng/ml. As to determine each of OAs, the mixture of OAs was first partitioned between n-hexane and 60% (v/v) aqueous methanol. Total amount of OA and DTX1 in 60% (v/v) aqueous methanol and the amount of Pal-DTX1 in n-hexane were determined using OA958-2 in methanol. And the amount of OA in 60% (v/v) aqueous methanol was determined using OA423-3 in 10% (v/v) aqueous methanol. The amount of DTX1 was calculated by subtracting the amount of OA from the total amount of OA and DTX1. The determination of the mixture of 500 ng each of OAs resulted in 1591 ng as OA for total OAs, 500 ng for OA, 474 ng for DTX1 and 519 ng for Pal-DTX1, respectively. The recovery test using nontoxic scallops and blue mussels fortified with 1.0, 2.0 and 10 μg of OA, DTX1 or Pal-DTX1/g of hepatopancreases resulted in 100.8-107.8% of recovery rates.