A monoclonal antibody (mAb) specific to ochratoxin A (OTA) was produced from a stable hybridoma cell line, 9C9H9, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with OTA-keyhole limpet hemocyanin. The 9C9H9 mAb belongs to the immunoglobulin G1 (kappa chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. The concentrations causing 50% inhibition of binding of OTA-horseradish peroxidase to the antibody by OTA, OTB, and OTC were found to be 0.32, 0.17, and 0.28 ng/mL, respectively, in the cdELISA. A sensitive and rapid mAb-based gold nanoparticle immunochromatographic strip was also developed using this mAb. This strip has a detection limit of 5 ng/mL for OTA and can be completed in 10 min. Analysis of OTA in coffee samples revealed that data obtained from immunochromatographic strip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunochromatographic strip assay established in this study were sensitive and accurate for rapid screening of OTA in coffee samples.

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Monascus purpureus and its fermentation products have been used in food coloring and meat preservation in Asia for centuries and have also been recently used as dietary supplements because of their cholesterol-lowering ability. However, the presence of the mycotoxin citrinin (CTN), a secondary metabolite of Monascus species, in fermentation products is a potential threat to public health. In the present study, HPLC was used to analyze CTN levels in lipid and aqueous extracts of commercialized Monascus products. CTN was detected in lipid extracts of all examined samples at concentrations varying between 0.28 and 6.29 microg/g, but was not found in aqueous extracts. When human embryonic kidney cells (HEK293) were incubated for 72 h with Monascus extracts, the concentrations causing 50% cell death by all lipid extracts were in the range of 1.8-4.7 mg/mL, whereas aqueous extracts showed a lower cytotoxicity. Incubation of HEK293 cells with 60 microM pure CTN for 72 h caused cell viability to fall to 50% of control levels. In addition, coadministration of pure CTN and lipid extracts from Monascus samples significantly enhanced CTN cytotoxicity for HEK293 cells using the MTT assay. These results provide the first information about the cytotoxic effects of various Monascus samples and CTN-Monascus mixtures on a human cell line.

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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.

Polyclonal antibodies for microcystin-leucine-arginine (MCYST-LR) were generated from rabbits after immunizing the animals with MCYST-LR conjugated with gamma-globulin. 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 the toxin in algal cultures and dietary supplements. The concentrations causing 50% inhibition (IC(50)) of binding of MCYST-horseradish peroxidase (MCYST-HRP) to the solid-phase antibodies by MCYST-LR, MCYST-arginine-arginine variant (MCYST-RR), MCYST-tyrosine-arginine variant (MCYST-YR), and nodularin (NODLN) in the cdELISA were found to be 0.10, 0.12, 0.14, and 0.20 ng/mL, respectively. In the presence of algae matrix, the detection limit is less than 10 ppb. The overall analytical recovery of MCYST-LR (25 to 500 ng/g) added to the algal dietary supplements and then extracted with 0.1 M ammonium bicarbonate in the cdELISA was found to be 83.7%. Analysis of MCYSTs in algal cultures and dietary supplements showed that six of eleven cultures produce MCYSTs, and five of the algal cultures were not MCYST producers. Eight of eleven tested commercial algal dietary supplements contained MCYSTs at a level lower than 100 ppb. The presence of MCYST-LR in the Microcystis aeruginosa culture was confirmed by high-performance liquid chromatography.

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