ABSTRACT
Polyclonal antibodies for ochratoxin A (OTA) were generated from rabbits after the animals had been immunized with either OTA-gamma-globulin or OTA- keyhole limpet hemocyanin (KLH). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of OTA in various agricultural commodities. The antibody titers in the serum of rabbits immunized with OTA-gamma-globulin were considerably higher than those in rabbits immunized with OTA-KLH. The antibodies from the rabbits immunized with OTA-gamma-globulin were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of OTA-horseradish peroxidase to the antibodies by OTA, ochratoxin B (OTB), and ochratoxin C (OTC) were found to be 0.90, 110, and 0.54 ng/mL, respectively. When 10 to 250 ng/g of standard OTA was spiked to soybean samples and then extracted with 50% aqueous methanol, the recovery rate of OTA was found to be 85.9% in the cdELISA. Analysis of OTA in various agricultural commodities showed that 12 of the 20 examined samples were contaminated with OTA at levels from 16 to 160 ng/g. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Ochratoxins/analysis , Animals , Antibodies/blood , Antibody Specificity , Hemocyanins/immunology , Mycotoxins/analysis , Ochratoxins/immunology , Rabbits , gamma-Globulins/immunologyABSTRACT
Polyclonal antibodies for domoic acid were generated from rabbits after the animals had been immunized with either domoic acid-keyhole limpet hemocyanin (KLH) or domoic acid-bovine serum albumin (BSA). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of domoic acid in blue mussels and clams. The antibody titers in the serum of rabbits immunized with domoic acid-KLH were considerably higher than those in rabbits immunized with domoic acid-BSA. The antibodies from the rabbits immunized with domoic acid-KLH were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of domoic acid-horseradish peroxidase to the antibodies by domoic acid and a domoic acid analogue, kainic acid, were found to be 0.75 and 200 ng/mL, respectively. In the presence of blue mussel matrix, the detection limit of domoic acid was <25 ng/g. The overall analytical recovery of domoic acid (25-500 ng/g) added to the blue mussels and then extracted with 50% aqueous methanol in the cdELISA was found to be 81.1%. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method. Analysis of domoic acid in shellfish samples showed that 10 of the 15 shellfish examined were contaminated with domoic acid at levels of <50 ng/g.
