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 OTα), 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.

Keywords:

• Ochratoxin
• immunoassay
• HPLC
• monoclonal antibody
• coffee

Acknowledgements

The authors thank the CNPq (Brazilian Government Organization for grant aid and fellowship to Brazilian researchers), CAPES Foundation – Brazil (Foundation for the Coordination of Higher Education and Graduate Training), JICA (Japan International Cooperation Agency) and the UGF-SETI (the Paraná State–Brazil Government Secretary for grant aid) for financial support.