Reliable indirect competitive ELISA used for a survey of ochratoxin A in green coffee from the North of Paraná State, Brazil
The performance of an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a monoclonal antibody (mAb) for ochratoxin A (OTA) detection was evaluated in a comparative study with high-performance liquid chromatography (HPLC) analysis using 68 freshly harvested coffee samples from the North of Paraná State, Brazil. The anti-OTA mAb showed high specificity and low cross-reactivity with OTA analogues (OTB and OTα), but cross-reacted with OTC. This ic-ELISA showed a detection limit of 3.75ngg −1 sample, when compared to 0.80ngg −1 by HPLC, with an ic-ELISA/HPLC correlation coefficient of 0.90. As regards OTA analysis of these coffee samples, natural contamination was detected in 10 samples (14.7%) by both methods, where the ic-ELISA values (range 3.9–7.3ngg −1 ) were 1.1 to 1.6-fold higher than HPLC data (2.7–4.7ngg −1 ). Five samples (7.4%) were OTA positive (range 0.84–1.30ngg −1 ) only by HPLC assay, probably due to the higher detection limit reached by ic-ELISA. OTA was undetectable in 53 samples (77.9%) by both methods, while all positive samples (range 0.84–7.30ngg −1 ) showed OTA levels lower than 8ngg −1 (maximum limit recommended by the European Union). The matrix interference of green coffee was minimized by dilution of sample extracts before carrying out the ELISA assay. This mAb-based ic-ELISA can be effectively applied for OTA screening in coffee, because it is simple, sensitive and sample preparation is easy.
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Document Type: Research Article
Department of Food and Drug Technology, State University of Londrina, P.O.6001, 86051-990, Londrina, PR, Brazil
Agronomic Institute of Paraná, P.O.481, 86001-970, Londrina, PR, Brazil
Department of Biochemistry and Biotechnology, State University of Londrina, Brazil
Department of Agronomy, State University of Londrina, Brazil
Department of Pathological Sciences, Science University of Tokyo, Shinjukuku, Tokyo 162-0812, Japan
Faculty of Pharmaceutical Science, Science University of Tokyo, Shinjukuku, Tokyo 162-0812, Japan
Department of Biochemistry and Food Science, Faculty of Agriculture, Kagawa University, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0765, Japan
September 1, 2006
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