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 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.
Fujii
Simone
S
Department of Food and Drug Technology, State University of Londrina, P.O.6001, 86051-990, Londrina, PR, Brazil.
Ribeiro
Ricardo Marcelo Reche
RM
Scholz
Maria Brígida Dos Santos
MB
Ono
Elisabete Yurie Sataque
EY
Prete
Cássio Egidio C
CE
Itano
Eiko Nakagawa
EN
Ueno
Yoshio
Y
Kawamura
Osamu
O
Hirooka
Elisa Yoko
EY
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
D000911:Antibodies, Monoclonal; D002273:Carcinogens; D003069:Coffee; D009183:Mycotoxins; D009793:Ochratoxins; C025589:ochratoxin A