title: Reliable indirect competitive ELISA used for a survey of ochratoxin A in green coffee from the North of Paraná State, Brazil.
abstract: 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 OTalpha), but cross-reacted with OTC. This ic-ELISA showed a detection limit of 3.75 ngg-1 sample, when compared to 0.80 ngg-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 3.9-7.3 ngg-1) were 1.1 to 1.6-fold higher than HPLC data (2.7-4.7 ngg-1). Five samples (7.4%) were OTA positive (range 0.84-1.30 ngg-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 0.84-7.30 ngg-1) showed OTA levels lower than 8 ngg-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.
authors.affiliation: Department of Food and Drug Technology, State University of Londrina, P.O.6001, 86051-990, Londrina, PR, Brazil.
authors.forename: Simone
authors.initials: S
authors.lastname: Fujii
authors.forename: Ricardo Marcelo Reche
authors.initials: RM
authors.lastname: Ribeiro
authors.forename: Maria Brígida Dos Santos
authors.initials: MB
authors.lastname: Scholz
authors.forename: Elisabete Yurie Sataque
authors.initials: EY
authors.lastname: Ono
authors.forename: Cássio Egidio C
authors.initials: CE
authors.lastname: Prete
authors.forename: Eiko Nakagawa
authors.initials: EN
authors.lastname: Itano
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Ueno
authors.forename: Osamu
authors.initials: O
authors.lastname: Kawamura
authors.forename: Elisa Yoko
authors.initials: EY
authors.lastname: Hirooka
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D002273:Carcinogens / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003069:Coffee / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005504:Food Analysis / Q000379:methods; D005506:Food Contamination / Q000032:analysis*; D006801:Humans; D009183:Mycotoxins / Q000032:analysis; D009793:Ochratoxins / Q000032:analysis* / Q000276:immunology; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D002273:Carcinogens; D003069:Coffee; D009183:Mycotoxins; D009793:Ochratoxins; C025589:ochratoxin A