Comparison of thin-layer chromatography, spectrofluorimetry and bright greenish-yellow fluorescence test for aflatoxin detection in corn

In this study the bright greenish-yellow fluorescence test, widely used by the corn milling industry, was compared to the thin-layer chromatography (TLC) and spectrofluorimetry methods for aflatoxin detection in 40 corn samples naturally contaminated by the Aspergillus section Flavi. According to the corn processing industry criteria, all the samples were adequate for human and animal consumption by the bright greenish-yellow fluorescence test, but TLC and spectrofluorimetry analysis detected aflatoxins above the maximum tolerated limit (20 µg/kg) in 7 and 8 samples, respectively. Aflatoxins were detected in 16 (40 percent) corn samples by TLC, with levels ranging from 4.0 to 54.0 µg/kg (mean 19.97 ± 15.97 µg/kg), and in 25 (62.5 percent) samples by spectrofluorimetry, with levels ranging from 1.0 to 58.66 µg/kg (mean 17.14 ± 17.81 µg/kg). The results indicated a good correlation (ρ = 0.97) between TLC and spectrofluorimetry for aflatoxin determination in naturally contaminated corn. The bright greenish-yellow fluorescence test was simple and quick, but it showed 20 percent false-negative results, suggesting its use only as screening method for detecting the suspected lots of grains that should be tested further for aflatoxin by more sensitive methods.

Neste trabalho a contagem de fluorescência luminosa amarelo-esverdeada, amplamente utilizada pela indústria de processamento de milho, foi comparada à cromatografia em camada delgada (CCD) e espectrofluorimetria para detecção de aflatoxinas em 40 amostras de milho naturalmente contaminadas por Aspergillus section Flavi. De acordo com os critérios da indústria processadora de milho, todas as amostras estavam adequadas para o consumo humano e animal pela contagem de fluorescência luminosa amarelo-esverdeada (CFLAE), porém as análises por CCD e espectrofluorimetria detectaram aflatoxinas acima do limite máximo tolerado (20 µg/kg) em 7 e 8 amostras, respectivamente. As aflatoxinas foram detectadas em 16 (40 por cento) amostras por CCD, com níveis variando de 4,0 a 54,0 µg/kg (média 19,97 ± 15,97 µg/kg) e, em 25 (62,5 por cento) amostras por espectrofluorimetria, com níveis variando de 1,0 a 58,66 mg/kg (média 17,14 ± 17,81 µg/kg). Os resultados indicaram uma boa correlação (ρ=0,97) entre CCD e espectrofluorimetria para detecção de aflatoxinas em amostras de milho naturalmente contaminadas. A CFLAE, apesar da simplicidade e rapidez, apresentou 20 por cento de resultados falso-negativos, sugerindo seu uso apenas como método de triagem para detecção de lotes de grão suspeitos de contaminação que devem ser avaliados posteriormente por métodos mais sensíveis.

A comparison between enzyme immunoassay and HPLC for ochratoxin A detection in green, roasted and instant coffee

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for ochratoxin A (OTA) detection in green, roasted and instant coffees was developed using anti-OTA monoclonal antibody. Immunological reagents prepared were OTA-BSA (4.76 mg/mL), anti-OTA.7 MAb (2x10³-fold dilution) and HRP-anti IgG (10³-fold dilution). The detection limit was 3.73 ng OTA/g and correlation coefficients (r) between this immunoassay and high performance liquid chromatography were 0.98 for green coffee, 0.98 for roasted and 0.86 for instant. OTA levels detected by ic-ELISA were higher than by HPLC, with ELISA/HPLC ratio of 0.66 - 1.46 (green coffee), 0.96 - 1.11 (roasted) and 0.93 - 1.82 (instant). ELISA recoveries for OTA added to coffee (5 - 70 ng/g) were 81.53 percent for green coffee, 46.73 percent for roasted and 64.35 percent for instant, while recoveries by HPLC were 80.54 percent, 45.91 percent and 55.15 percent, respectively. Matrices interferences were minimized by samples dilution before carrying out the ELISA assay. The results indicate that MAb-based ic-ELISA could be a simple, sensitive and specific screening tool for OTA detection, contributing to quality and safety of coffee products.

ELISA competitivo indireto (ic-ELISA) baseado em anticorpos monoclonais foi desenvolvido para a detecção de ocratoxina A (OTA) em café verde, torrado e instantâneo. Os reagentes imunológicos necessários à reação consistiram de OTA-BSA (4,76 mg/mL), anti-OTA.7 MAb (diluído 2x10³) e anti IgG-HRP (diluído 10³), apresentando limite de detecção de 3,73 ng OTA/g. Os coeficientes de correlação (r) entre o imunoensaio e cromatografia líquida de alta eficiência (CLAE) foram de 0,98 (café verde), 0,98 (torrado) e 0,86 (instantâneo). Ic-ELISA detectou valores superestimados de OTA em relação a CLAE, com valor ELISA/CLAE variando de 0,66 - 1,46 (café verde), 0,96 - 1,11 (torrado) e 0,93 - 1,82 (instantâneo). As recuperações médias de OTA adicionada em café (5 - 70 ng/g) foram de 81,53 por cento (café verde), 46,73 por cento (torrado) e 64,35 por cento (instantâneo) por ELISA, em relação a 80,54 por cento, 45,91 por cento e 55,15 por cento por CLAE, respectivamente. A interferência de matriz no imunoensaio foi minimizada pela diluição das amostras previamente à análise por ELISA. O ic-ELISA desenvolvido pode ser considerado uma técnica alternativa simples, sensível e específica para análise de OTA, contribuindo para a qualidade e segurança de produtos de café.

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.