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Evaluation of immunoenzymatic methods for the detection of aflatoxin M1 in ewe’s milk
Food Control 20 (2009) 1049–1052
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Evaluation of immunoenzymatic methods for the detection of aflatoxin M1 in ewe’s milk R. Rubio, M.I. Berruga *, M. Román, A. Molina Departamento de Ciencia y Tecnología Agroforestal y Genética, ETSIA-IDR, Universidad de Castilla-La Mancha, 02071 Albacete, Spain
a r t i c l e
i n f o
Article history: Received 8 October 2008 Received in revised form 15 January 2009 Accepted 20 January 2009
Keywords: Aflatoxin M1 Ewe’s milk ELISA
a b s t r a c t Ewe milk samples spiked with aflatoxin M1 (AFM1) at concentrations of 25, 50, 75 and 100 ng/kg were analysed by 5 different (Kits A to E) commercial Enzyme-Linked ImmunoSorbent Assay kits to evaluate the mycotoxin recovery average. The least accurate results of sensitivity were obtained at the 25 ng/kg concentration with underestimation in all cases. When the sample contamination-level was increased (50, 75 and 100 ng/kg), the results of four kits improved. Low coefficients of variation (3–14%) indicate good repeatability. Acceptable results of mean recoveries were obtained (47–60 ng/kg), especially at 50 ng/kg (EC Maximum Level) in all tests except Kit A. We conclude that Kits B, C, D and E are appropriate for the screening of AFM1 in ewe’s milk, but not for regulatory purposes. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Aflatoxins (AFs) are secondary metabolites produced by three species of Aspergillus, Aspergillus flavus, Aspergillus parasiticus and the rare Aspergillus nomius, which contaminate plants and plant products during growth on feeds, foods or laboratory media (Applebaum, Brackett, Wiseman, & Marth, 1982; Creppy, 2002). There is sufficient evidence in experimental animals for the carcinogenicity of naturally occurring mixtures of AFs, AFB1 and AFM1. However, the carcinogenicity of AFM1 is not so evident in the case of humans (International Agency for Research on Cancer (IARC), 1993). The acute lethal dose for adults is approximately 10– 20 mg of aflatoxins (Pitt, 2000). At specific temperatures and relative humidities, Aspergillaceae can produce AFs of groups B and G (AFB and AFG) in crops, plant products, and in all the steps of the food chain from feed to animal feeding (Virdis, Corgiolu, Scarano, Pilo, & De Santis, 2008). Stored animal feed (silage, grains and cake) are at higher risk of AFs’ contamination, most frequently AFB1 and AFB2, which is particularly true for maize, cotton seed and oil-cake (World Health Organization (WHO), 1998). AFB1 is classified as Group 1 (carcinogenic to humans) by the World Health Organisation-International Agency for Research on Cancer (WHO–IARC) (IARC, 1993). In the liver, ingested AFB1 is biotransformed by hepatic microsomal cytochrome P450 into aflatoxin M1 (AFM1), which is excreted into the milk of lactating animals (Cupid et al., 2004). The demonstrated toxic and carcinogenic effects of AFM1, led WHO–IARC to classify it as Group 2B, possible human carcinogen * Corresponding author. Tel.: +34 967599200x2830; fax: +34 967599238. E-mail address: [email protected] (M.I. Berruga). 0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2009.01.007
(International Agency for Research on Cancer (IARC), 2002). Aflatoxins are also genotoxic, teratogenic, immunosuppressive and have anti-nutritional effects (Wangikar, Dwivedi, Sinha, Sharma, & Telang, 2005; Williams et al., 2004). These characteristics of aflatoxins have led developed countries to establish limits for the maximum quantity of AFM1 allowed in milk: 50 ng/kg (Codex Alimentarius Commission, 2001; European Commission Directive, 2003). In the case of AFB1, levels are different from those for AFM1. In relation to animal feeding, the limit for AFB1 is between 5 and 50 lg/kg (European Directive, 2002/32/EC). The limit is 50 lg/kg for: feed material, complete feedingstuff for cattle, sheep and goats; complementary feedingstuffs for cattle, sheep and goats, and 5 lg/kg for feed used with dairy cattle. Within the European Union, Spain is the third largest producer of ewe’s milk with 403,000 Tons per year (FAO, 2008). The primary use of this milk (99%) is to produce cheese, a product with growing demand. Specifically, Manchego cheese from the Castilla-La Mancha region, which is internationally recognized for its high quality and protected denomination of origin (PDO). This situation has led to the development of strict routine controls of milk quality, including milk production, physical–chemical composition and somatic cells count (SCC). Recently with new Spanish legislation (RD 1728/2007), the analysis of antibiotic residues will also be compulsory. So in the future, the analysis of aflatoxins could be integrated into this routine control if we consider the fact that they represent a risk for human and animal health, and that this analysis is already regulated in the European Directive (1996/23). The official sampling and analysis methods of aflatoxins are regulated by the European Commission Regulation, 401/2006. The reference method used for the detection of aflatoxins in cow’s milk is
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high-performance liquid chromatography (HPLC) with fluorimetric detection and a previous clean-up treatment by immunoaffinity columns (International Standards Organisation (ISO), 2007). Otherwise, methods like thin-layer chromatography (Grosso, Fremy, Bevis, & Dragacci, 2004), direct fluorescence measurement after immunoaffinity clean-up (Chiavaro, Cavicchioli, Berni, & Spotti, 2005), liquid chromatography/tandem mass spectrometry (Cavaliere, Foglia, Pastorini, Samperi, & Lagana, 2006) and immunoenzymatic assays (Oveisi, Jannat, Sadeghi, Hajimahmoodi, & Nikzad, 2007; Rodríguez, Calonge, & Ordónez, 2003; Rosi et al., 2007) have been developed. Among others, Enzyme-Linked ImmunoSorbent Assay (ELISA) is a well-known assay for the detection of AFM1 in both cow’s (Rosi et al., 2007) and goat’s milk (Virdis et al., 2008), but there are no references to the use of this method with ewe’s milk. A long list of the advantages that ELISA tests offer could be mentioned as they are generally relatively accurate tests; they are considered highly sensitive and specific, and their results could be comparable with those obtained by HPLC (Abbas, 2005). One of the most important characteristics of these tests is that they are suitable for the quantitative testing of a large number of samples at the same time or for multiple screening, and the sample does not require complicated preparations for it to be submitted to the test. Given this situation, the aim of our study was to compare the efficiency of different ELISA commercial kits in the detection of AFM1 in ewe’s milk since they have not already been used to test the milk of this species. 2. Materials and methods 2.1. Ewe’s milk Bulk-tank ewe’s milk (2 L) was taken from the experimental farm of the Instituto Técnico Agronómico Provincial (Diputación de Albacete, Spain) of the Manchega breed flock in mid-lactation. Milk composition was fat 7.82%, protein 5.95% and lactose 4.31%, with a pH of 6.75. Milk was analysed to check for the absence of aflatoxin M1, according to HPLC (ISO, 2007). Ewe’s milk was prepared in aliquots of 100 mL and frozen at 25 °C for no more than 15 days before use. 2.2. AFM1 milk addition The quantity of milk needed for the experiment was thawed at room temperature. With the aflatoxin M1 standard (R-Biopharm, Glasgow, Scotland, 1000 ng/mL in acetonitrile), a stock solution and dilution series was prepared in methanol (HPLC-gradient). In the last step, AFM1 was added to milk to obtain concentrations of 25, 50, 75 and 100 ng/kg, and was left at room temperature for 30 min (Oruc, Cibik, Yilmaz, & Kalkanli, 2006). Next, 40 mL of milk were placed into opaque plastic tubes, centrifuged (4000 g for 10 min at 4 °C), and the fatty upper-layer was removed before using milk with the immunochemical kits. All the samples were isolated from light during their preparation and analysis using amber material to avoid AFM1 deterioration. Four different samples, used for all the methods, were analysed in triplicate in each ELISA kit. 2.3. Quantification of AFM1 by ELISA Five commercial ELISA kits were used for this study: (A) the Agra Quant Aflatoxin M1 test kit (Romer Labs, Singapore, Republic of Singapore), (B) the I´screen Afla M1 test kit (Tecna s.r.l., Trieste, Italy), (C) the Aflatoxin M1 test kit (Euro-Diagnostica B.V., Arnhem, The Netherlands), (D) the Transia Plate Aflatoxin M1 test kit (Raisio Diagnostics SAS, Lyon, France) and (E) the Ridascreen Aflatoxin M1
30/15 test kit (R-Biopharm AG, Darmstadt, Germany). Each kit was used following the manufacturers’ instructions. Colour development, inversely proportional to the AFM1 concentration in the sample, was determined by reading the absorbance with an Original Multiskan EX (Thermo Electron Corporation, Shanghai, China) at 450 nm. With the AFM1 standard solutions included in each kit (solutions corresponding to concentrations between 0 and 250 ng/kg), calibration curves were done for the purpose of quantifying the concentration of the samples analysed. The absorbance values obtained for the standards and samples were divided by the absorbance of the first standard (zero standard) and multiplied by 100. Therefore, the zero standard is considered to have an absorbance value of 100%, and the values of other standards and samples are expressed as percentages. The mean recoveries and the coefficients of variation (CV) were calculated for each concentration. The recovery average was expressed as the percentage relationship between the concentrations obtained and those expected. The CV was expressed as the ratio between the standard deviation and the average. 2.4. Statistical analysis All the results obtained with the ELISA kits were statistically analysed using SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA). A General Linear Model was used to determine the effect of the test and the concentration in the recovery average. A twoway analysis of variance was run on the data to simultaneously study the effect of both factors (concentration and kit), and whether the interaction between these factors was significant. When a significant difference was present among recovery averages (P < 0.05), Tukey’s test was carried out to determine disparities between the kits. 3. Results The data of the means and the CV of the four concentrations added to ewe’s milk and analysed with the five immunoenzymatic test kits are presented in Table 1. The results of the lowest concentration added (25 ng/kg,) came close to the concentrations obtained (21–23 ng/kg), except Kit E (19 ng/kg). When milk samples were contaminated with the same concentration as the Maximum Level established by the EU (50 ng/ kg), the results obtained were dependent on the test. While some kits (B and E) proved most accurate with the level of detection (47 and 49 ng/kg), others were less sensitive because some presented an underestimation of concentration (Kit A only detected 38 ng/kg), or because an overestimation was also presented (Kit C, 60 ng/kg). Similar results were obtained when the analysed samples were contaminated at the concentration of 75 ng/kg. Of the five kits checked to study the level of sensitivity in AFM1 recovery,
Table 1 Means of the concentrations of AFM1 and repeatability (CV) in samples of ewe’s milk analysed with five different ELISA kits (n = 4). Test
A B C D E
25 (ng/kg)
50 (ng/kg)
75 (ng/kg)
100 (ng/kg)
Mean
CV
Mean
CV
Mean
CV
Mean
CV
23 21 22 22 19
8 6 14 8 12
38 47 60 55 49
12 7 8 6 12
48 75 97 81 80
9 6 8 4 5
55 87 110 104 85
8 3 6 5 13
A = Agra Quant Aflatoxin M1 test kit; B = I´screen Afla M1; C = Aflatoxin M1; D = Transia Plate Aflatoxin M1; E = Ridascreen Aflatoxin M1 30/15. Mean: (ng/kg); CV: (%).
R. Rubio et al. / Food Control 20 (2009) 1049–1052
three of them showed a good recovery (B, D and E), while the other two showed less sensitivity because of an excess (C) or a deficiency (A). It should also be noted that, when samples with higher contamination levels (100 ng/kg) were analysed, a greater imprecision in the recovery capacity of the tests was observed. The underestimation was more obvious, above all, based on the results obtained with Kits A, B and E. As Table 1 clearly shows, the CV were between 3% and 14%, irrespectively of the concentration or the kit studied. This indicates good repeatability based on the studies of Botsoglou (2000), who indicated that CV values for food samples in the range of 2% to more than 20% are acceptable. Moreover, our results are closer to the aforementioned results than those obtained by Rosi et al. (2007) using Kits B and E in cow’s milk, which obtained CV from 4.9% to 18.8%. Finally, we conclude that when Kits C and D were studied, a slight overestimation in mean recoveries was detected when the concentrations of 50, 75 and 100 ng/kg were added. The comparison between different concentrations in the same kit showed that Kit A had a lower detection accuracy, in general, of the AFM1 expected (48 ng/kg detected for 75 ng/kg added or 55 ng/kg for 100 ng/kg). Kits B and E showed values very close to the real ones with concentrations of 50 and 75 ng/kg, although they were imprecise (underestimation) with the lower and the higher concentrations (25 and 100 ng/kg). These discrepancies also appear in the recovery averages (calculated using the mean concentrations in Table 1) shown in Fig. 1 for each test kit. The P value showed that a significant difference exists among the test kit (P < 0.001), the concentration (P < 0.001) and the interaction of both factors (P < 0.001). Of the five test kits used, no significant difference was found in the mean recoveries of the 25 ng/kg fortification level with values between 78% and 92%. At all the other concentrations, the test recovery levels improved, which in some cases reached up to 100% (Kit C with 50 and 75 ng/kg), with the only exception of kit A that showed a clearly significant tendency to decrease by recovering less than 56% at 100 ng/kg. As Fig. 1 shows, the concentration of 100 ng/kg presented recovery averages between 56% and 110%, which is a wide range, but one comparable to that of the mean concentrations (50 and 75 ng/kg). This situation occurs with all the kits, except kit A. So, the five test kits studied presented some variability in their development, but four of them presented similar and acceptable recovery averages, while just one gave results which cannot be considered acceptable.
Recovery average of AF M1 (%)
140 c
b
120
ab
80 60
b
b b
ab
100
b
b
b
NS
b
**
a
***
a
***
40 20 0 25
50
75
4. Discussion Based on the research done in this study, we can conclude that with the exception of Kit A, the rest of the test kits presented a good sensitivity in the recovery of AFM1 in ewe’s milk. These results are comparable to those of other studies done on cow’s milk (Rodríguez et al., 2003; Rosi et al., 2007), where a similar methodology was applied with the use of kit B or E. The data of recovery percentages were between 74.6% and 109%, when milk samples were added at a range of AFM1 concentrations of between 10 and 80 ng/L (Rodríguez et al., 2003). With added concentrations of between 30 and 100 ng/L, the range of recovery percentage obtained was 80–106% (Rosi et al., 2007). These results are similar to those obtained in the present study (excluding the results of kit A), with concentrations of between 25 and 100 ng/kg and recovery percentages of between 78% and 130%. Thus, this analysis methodology could be useful to decide whether milk must be excluded from a production chain if its AFM1 concentration exceeds 50 ng/ kg (the concentration limit set by the European Commission Directive). Despite the importance of ewe’s milk in the Mediterranean area, very few studies have been published on the analysis of AFM1 with ELISA. Kaniou-Grigoriadou, Eleftheriadou, Mouratidou, and Katikou (2005) determined the contamination of AFM1 in ewe’s milk, curd and Feta cheese by using Kit E. However, it is possible to find more studies on AFM1 occurrence analysed with Kit E in cow’s milk that had been contaminated naturally (Oveisi et al., 2007; Stoloff et al., 1975) and with goat’s milk (Virdis et al., 2008), or with the use of Kit B (Decastelli et al., 2007) for the analysis of cow’s milk and feed. Not all the kits in the market obtain the expected data results. In some cases, there is an overestimation, while an underestimation of the aflatoxin contents is shown in others. One possible explanation for this situation could be the so-called matrix effect or matrix interference, due to non-specific interaction of substances that does not relate to aflatoxin with the antibodies (Rosi et al., 2007). According to the results of our experiment, the use of commercial ELISA kits could be a reliable option to obtain acceptable results for the screening of AFM1 in ewe’s milk in a short time, but it would always be necessary to corroborate the results with official methods (HPLC) for positive or suspect samples. Acknowledgements This study was financed by the Research Project PAI06-00683875 and the predoctoral grant to R. Rubio (Ref. 07/039) from the Junta de Comunidades de Castilla-La Mancha. The authors are also grateful for the collaboration of the Instituto Técnico Agronómico Provincial of the Diputación de Albacete. References
b
a
1051
100
Concentration of AF M1 (ng/kg) Fig. 1. Recovery average (%) of AFM1 (ng/kg) in ewe’s milk for: kit A (d), kit B (s), kit C (j), kit D (h) and kit E (N) (n = 4). , , : P < 0.05, P < 0.01, P < 0.001, respectively. a, b, c: show significant differences among test kits.
Abbas, H. K. (2005). Aflatoxin and food safety. New York: Taylor & Francis Group. pp. 269–272. Applebaum, R., Brackett, R. E., Wiseman, D. W., & Marth, E. H. (1982). Aflatoxin: Toxicity to dairy cattle occurrence in milk and milk products. Journal of Food Protection, 45(8), 752–777. Botsoglou, N. A. (Ed.). (2000). Drug residues in foods: Pharmacology, food safety and analysis. New York: Marcel Dekker Incorporated. p. 775. Cavaliere, C., Foglia, P., Pastorini, E., Samperi, R., & Lagana, A. (2006). Liquid chromatography/tandem mass spectrometric confirmatory method for determining aflatoxin M1 in cow milk: Comparison between electrospray and atmospheric pressure photoionization sources. Journal of Chromatography A, 1101(1–2), 69–78. Chiavaro, E., Cavicchioli, C., Berni, E., & Spotti, E. (2005). Immunoaffinity clean-up and direct fluorescence measurement of aflatoxin B1 and M1 in pig liver: Comparison with high-performance liquid chromatography determination. Food Additives and Contaminants, 22(11), 1154–1161. Codex Alimentarius Commission (2001). Comments submitted on the draft maximum level for Aflatoxin M1 in milk. Codex Committee on food additives and contaminants. 33rd session, Hague, Netherlands.
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Creppy, E. E. (2002). Update of survey, regulation and toxic effects of mycotoxins. European Toxicology Letter, 127, 19–28. Cupid, B. C., Lightfoot, T. J., Russel, D., Gant, S. J., Turner, P. C., Dingley, K. H., et al. (2004). The formation of AFB1-macromolecular adducts in rat and human at dietary levels of exposure. Food Additives and Contaminants, 42(4), 559–569. Decastelli, L., Lai, J., Gramaglia, M., Monaco, A., Nachtmann, C., Oldano, F., et al. (2007). Aflatoxins occurence in milk and feed in Northern Italy during 2004– 2005. Food Control, 18(10), 1263–1266. European Commission (1996). Council Directive 96/23/EC of 29 April 1996 on measures to monitor certain substances and residues thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and Decisions 89/187/EEC and 91/664/EEC. Official Journal of the European Communities, L125, 10–32. European Commission (2002). Commission Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. Official Journal of the European Communities, L140, 10–21. European Commission (2003). Commission Regulation No. 2174/2003 of 12 December 2003, amending Regulation (EC) No. 466/2001 as regards aflatoxins. Official Journal of the European Communities, L326, 12–15. European Commission (2006). Commission Regulation No. 401/2006 of 23 February 2006, laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Communities, L70, 12–31. Food and Agriculture Organization (FAO) (2008). Estimation of the production of whole fresh ewe’s milk in 2006. Grosso, F., Fremy, J. M., Bevis, S., & Dragacci, S. (2004). Joint IDF-IUPAC-IAEA (FAO) interlaboratory validation for determining aflatoxin M1 in milk by using immunoaffinity clean-up before thin-layer chromatography. Food Additives and Contaminants, 21(4), 348–357. International Agency for Research on Cancer (IARC) (1993). Aflatoxins. In Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. Monographs on the evaluation of carcinogenic risk to human (pp. 245–395). Lyon: IARC. International Agency for Research on Cancer (IARC) (2002). Monograph on the evaluation of carcinogenic risk to humans (Vol. 82, p. 171). Lyon: World Health Organisation.
International Standards Organisation (ISO) (2007). Milk and milk powder. Determination of aflatoxin M1 content. Clean-up by immunoaffinity chromatography and determination by high-performance liquid chromatography. Standard 14501. Geneva, Switzerland. Kaniou-Grigoriadou, I., Eleftheriadou, A., Mouratidou, T., & Katikou, P. (2005). Determination of aflatoxin M1 in ewe’s milk samples and the produced curd and Feta cheese. Food Control, 16, 257–261. Oruc, H. H., Cibik, R., Yilmaz, E., & Kalkanli, O. (2006). Distribution and stability of Aflatoxin M1 during processing and ripening of traditional white pickled cheese. Food Additives and Contaminants, 23, 190–195. Oveisi, M.-R., Jannat, B., Sadeghi, N., Hajimahmoodi, M., & Nikzad, A. (2007). Presence of aflatoxin M1 in milk and infant milk products in Tehran, Iran. Food Control, 18, 1216–1218. Pitt, J. I. (2000). Toxigenic fungi: Which are important? Medical Mycology, 38(Suppl. 1), 17–22. Rodríguez, M. L., Calonge, M. M., & Ordónez, D. (2003). ELISA and HPLC determination of the occurrence of aflatoxin M1 in raw cow’s milk. Food Additives and Contaminants, 20, 276–280. Rosi, P., Bosari, A., Lasi, G., Lodi, S., Galanti, A., Fava, A., et al. (2007). Aflatoxin M1 in milk: Reliability of the immunoenzymatic assay. International Dairy Journal, 17, 429–435. Stoloff, L., Trucksess, M., Hardin, N., Francis, O. J., Hayes, J. R., Polan, C. E., et al. (1975). Stability of aflatoxin M in milk. Food and Drug Administration, 58, 1789–1793. Virdis, S., Corgiolu, G., Scarano, C., Pilo, A. L., & De Santis, E. P. L. (2008). Occurrence of aflatoxin M1 in tank bulk goat milk and ripened goat cheese. Food Control, 19, 44–49. Wangikar, P. B., Dwivedi, P., Sinha, N., Sharma, A. K., & Telang, A. G. (2005). Effects of aflatoxin B1 on embryo fetal development in rabbits. Food and Chemical Toxicology, 43, 607–615. World Health Organization (WHO) (1998). Safety evaluation of certain food additives and contaminants. Food additives series no. 40. Geneva: The Forty-ninth of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Williams, J., Phillips, D. T., Jolly, P. E., Stiles, J. K., Jolly, C. M., & Aggaewal, D. (2004). Human aflatoxicosis in developing countries: A review of toxicology, exposure, potential health consequences, and intervention. American Journal of Clinical Nutrition, 80, 1106–1122.
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Evaluation of immunoenzymatic methods for the detection of aflatoxin M1 in ewe’s milk R. Rubio, M.I. Berruga *, M. Román, A. Molina Departamento de Ciencia y Tecnología Agroforestal y Genética, ETSIA-IDR, Universidad de Castilla-La Mancha, 02071 Albacete, Spain
a r t i c l e
i n f o
Article history: Received 8 October 2008 Received in revised form 15 January 2009 Accepted 20 January 2009
Keywords: Aflatoxin M1 Ewe’s milk ELISA
a b s t r a c t Ewe milk samples spiked with aflatoxin M1 (AFM1) at concentrations of 25, 50, 75 and 100 ng/kg were analysed by 5 different (Kits A to E) commercial Enzyme-Linked ImmunoSorbent Assay kits to evaluate the mycotoxin recovery average. The least accurate results of sensitivity were obtained at the 25 ng/kg concentration with underestimation in all cases. When the sample contamination-level was increased (50, 75 and 100 ng/kg), the results of four kits improved. Low coefficients of variation (3–14%) indicate good repeatability. Acceptable results of mean recoveries were obtained (47–60 ng/kg), especially at 50 ng/kg (EC Maximum Level) in all tests except Kit A. We conclude that Kits B, C, D and E are appropriate for the screening of AFM1 in ewe’s milk, but not for regulatory purposes. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Aflatoxins (AFs) are secondary metabolites produced by three species of Aspergillus, Aspergillus flavus, Aspergillus parasiticus and the rare Aspergillus nomius, which contaminate plants and plant products during growth on feeds, foods or laboratory media (Applebaum, Brackett, Wiseman, & Marth, 1982; Creppy, 2002). There is sufficient evidence in experimental animals for the carcinogenicity of naturally occurring mixtures of AFs, AFB1 and AFM1. However, the carcinogenicity of AFM1 is not so evident in the case of humans (International Agency for Research on Cancer (IARC), 1993). The acute lethal dose for adults is approximately 10– 20 mg of aflatoxins (Pitt, 2000). At specific temperatures and relative humidities, Aspergillaceae can produce AFs of groups B and G (AFB and AFG) in crops, plant products, and in all the steps of the food chain from feed to animal feeding (Virdis, Corgiolu, Scarano, Pilo, & De Santis, 2008). Stored animal feed (silage, grains and cake) are at higher risk of AFs’ contamination, most frequently AFB1 and AFB2, which is particularly true for maize, cotton seed and oil-cake (World Health Organization (WHO), 1998). AFB1 is classified as Group 1 (carcinogenic to humans) by the World Health Organisation-International Agency for Research on Cancer (WHO–IARC) (IARC, 1993). In the liver, ingested AFB1 is biotransformed by hepatic microsomal cytochrome P450 into aflatoxin M1 (AFM1), which is excreted into the milk of lactating animals (Cupid et al., 2004). The demonstrated toxic and carcinogenic effects of AFM1, led WHO–IARC to classify it as Group 2B, possible human carcinogen * Corresponding author. Tel.: +34 967599200x2830; fax: +34 967599238. E-mail address: [email protected] (M.I. Berruga). 0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2009.01.007
(International Agency for Research on Cancer (IARC), 2002). Aflatoxins are also genotoxic, teratogenic, immunosuppressive and have anti-nutritional effects (Wangikar, Dwivedi, Sinha, Sharma, & Telang, 2005; Williams et al., 2004). These characteristics of aflatoxins have led developed countries to establish limits for the maximum quantity of AFM1 allowed in milk: 50 ng/kg (Codex Alimentarius Commission, 2001; European Commission Directive, 2003). In the case of AFB1, levels are different from those for AFM1. In relation to animal feeding, the limit for AFB1 is between 5 and 50 lg/kg (European Directive, 2002/32/EC). The limit is 50 lg/kg for: feed material, complete feedingstuff for cattle, sheep and goats; complementary feedingstuffs for cattle, sheep and goats, and 5 lg/kg for feed used with dairy cattle. Within the European Union, Spain is the third largest producer of ewe’s milk with 403,000 Tons per year (FAO, 2008). The primary use of this milk (99%) is to produce cheese, a product with growing demand. Specifically, Manchego cheese from the Castilla-La Mancha region, which is internationally recognized for its high quality and protected denomination of origin (PDO). This situation has led to the development of strict routine controls of milk quality, including milk production, physical–chemical composition and somatic cells count (SCC). Recently with new Spanish legislation (RD 1728/2007), the analysis of antibiotic residues will also be compulsory. So in the future, the analysis of aflatoxins could be integrated into this routine control if we consider the fact that they represent a risk for human and animal health, and that this analysis is already regulated in the European Directive (1996/23). The official sampling and analysis methods of aflatoxins are regulated by the European Commission Regulation, 401/2006. The reference method used for the detection of aflatoxins in cow’s milk is
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high-performance liquid chromatography (HPLC) with fluorimetric detection and a previous clean-up treatment by immunoaffinity columns (International Standards Organisation (ISO), 2007). Otherwise, methods like thin-layer chromatography (Grosso, Fremy, Bevis, & Dragacci, 2004), direct fluorescence measurement after immunoaffinity clean-up (Chiavaro, Cavicchioli, Berni, & Spotti, 2005), liquid chromatography/tandem mass spectrometry (Cavaliere, Foglia, Pastorini, Samperi, & Lagana, 2006) and immunoenzymatic assays (Oveisi, Jannat, Sadeghi, Hajimahmoodi, & Nikzad, 2007; Rodríguez, Calonge, & Ordónez, 2003; Rosi et al., 2007) have been developed. Among others, Enzyme-Linked ImmunoSorbent Assay (ELISA) is a well-known assay for the detection of AFM1 in both cow’s (Rosi et al., 2007) and goat’s milk (Virdis et al., 2008), but there are no references to the use of this method with ewe’s milk. A long list of the advantages that ELISA tests offer could be mentioned as they are generally relatively accurate tests; they are considered highly sensitive and specific, and their results could be comparable with those obtained by HPLC (Abbas, 2005). One of the most important characteristics of these tests is that they are suitable for the quantitative testing of a large number of samples at the same time or for multiple screening, and the sample does not require complicated preparations for it to be submitted to the test. Given this situation, the aim of our study was to compare the efficiency of different ELISA commercial kits in the detection of AFM1 in ewe’s milk since they have not already been used to test the milk of this species. 2. Materials and methods 2.1. Ewe’s milk Bulk-tank ewe’s milk (2 L) was taken from the experimental farm of the Instituto Técnico Agronómico Provincial (Diputación de Albacete, Spain) of the Manchega breed flock in mid-lactation. Milk composition was fat 7.82%, protein 5.95% and lactose 4.31%, with a pH of 6.75. Milk was analysed to check for the absence of aflatoxin M1, according to HPLC (ISO, 2007). Ewe’s milk was prepared in aliquots of 100 mL and frozen at 25 °C for no more than 15 days before use. 2.2. AFM1 milk addition The quantity of milk needed for the experiment was thawed at room temperature. With the aflatoxin M1 standard (R-Biopharm, Glasgow, Scotland, 1000 ng/mL in acetonitrile), a stock solution and dilution series was prepared in methanol (HPLC-gradient). In the last step, AFM1 was added to milk to obtain concentrations of 25, 50, 75 and 100 ng/kg, and was left at room temperature for 30 min (Oruc, Cibik, Yilmaz, & Kalkanli, 2006). Next, 40 mL of milk were placed into opaque plastic tubes, centrifuged (4000 g for 10 min at 4 °C), and the fatty upper-layer was removed before using milk with the immunochemical kits. All the samples were isolated from light during their preparation and analysis using amber material to avoid AFM1 deterioration. Four different samples, used for all the methods, were analysed in triplicate in each ELISA kit. 2.3. Quantification of AFM1 by ELISA Five commercial ELISA kits were used for this study: (A) the Agra Quant Aflatoxin M1 test kit (Romer Labs, Singapore, Republic of Singapore), (B) the I´screen Afla M1 test kit (Tecna s.r.l., Trieste, Italy), (C) the Aflatoxin M1 test kit (Euro-Diagnostica B.V., Arnhem, The Netherlands), (D) the Transia Plate Aflatoxin M1 test kit (Raisio Diagnostics SAS, Lyon, France) and (E) the Ridascreen Aflatoxin M1
30/15 test kit (R-Biopharm AG, Darmstadt, Germany). Each kit was used following the manufacturers’ instructions. Colour development, inversely proportional to the AFM1 concentration in the sample, was determined by reading the absorbance with an Original Multiskan EX (Thermo Electron Corporation, Shanghai, China) at 450 nm. With the AFM1 standard solutions included in each kit (solutions corresponding to concentrations between 0 and 250 ng/kg), calibration curves were done for the purpose of quantifying the concentration of the samples analysed. The absorbance values obtained for the standards and samples were divided by the absorbance of the first standard (zero standard) and multiplied by 100. Therefore, the zero standard is considered to have an absorbance value of 100%, and the values of other standards and samples are expressed as percentages. The mean recoveries and the coefficients of variation (CV) were calculated for each concentration. The recovery average was expressed as the percentage relationship between the concentrations obtained and those expected. The CV was expressed as the ratio between the standard deviation and the average. 2.4. Statistical analysis All the results obtained with the ELISA kits were statistically analysed using SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA). A General Linear Model was used to determine the effect of the test and the concentration in the recovery average. A twoway analysis of variance was run on the data to simultaneously study the effect of both factors (concentration and kit), and whether the interaction between these factors was significant. When a significant difference was present among recovery averages (P < 0.05), Tukey’s test was carried out to determine disparities between the kits. 3. Results The data of the means and the CV of the four concentrations added to ewe’s milk and analysed with the five immunoenzymatic test kits are presented in Table 1. The results of the lowest concentration added (25 ng/kg,) came close to the concentrations obtained (21–23 ng/kg), except Kit E (19 ng/kg). When milk samples were contaminated with the same concentration as the Maximum Level established by the EU (50 ng/ kg), the results obtained were dependent on the test. While some kits (B and E) proved most accurate with the level of detection (47 and 49 ng/kg), others were less sensitive because some presented an underestimation of concentration (Kit A only detected 38 ng/kg), or because an overestimation was also presented (Kit C, 60 ng/kg). Similar results were obtained when the analysed samples were contaminated at the concentration of 75 ng/kg. Of the five kits checked to study the level of sensitivity in AFM1 recovery,
Table 1 Means of the concentrations of AFM1 and repeatability (CV) in samples of ewe’s milk analysed with five different ELISA kits (n = 4). Test
A B C D E
25 (ng/kg)
50 (ng/kg)
75 (ng/kg)
100 (ng/kg)
Mean
CV
Mean
CV
Mean
CV
Mean
CV
23 21 22 22 19
8 6 14 8 12
38 47 60 55 49
12 7 8 6 12
48 75 97 81 80
9 6 8 4 5
55 87 110 104 85
8 3 6 5 13
A = Agra Quant Aflatoxin M1 test kit; B = I´screen Afla M1; C = Aflatoxin M1; D = Transia Plate Aflatoxin M1; E = Ridascreen Aflatoxin M1 30/15. Mean: (ng/kg); CV: (%).
R. Rubio et al. / Food Control 20 (2009) 1049–1052
three of them showed a good recovery (B, D and E), while the other two showed less sensitivity because of an excess (C) or a deficiency (A). It should also be noted that, when samples with higher contamination levels (100 ng/kg) were analysed, a greater imprecision in the recovery capacity of the tests was observed. The underestimation was more obvious, above all, based on the results obtained with Kits A, B and E. As Table 1 clearly shows, the CV were between 3% and 14%, irrespectively of the concentration or the kit studied. This indicates good repeatability based on the studies of Botsoglou (2000), who indicated that CV values for food samples in the range of 2% to more than 20% are acceptable. Moreover, our results are closer to the aforementioned results than those obtained by Rosi et al. (2007) using Kits B and E in cow’s milk, which obtained CV from 4.9% to 18.8%. Finally, we conclude that when Kits C and D were studied, a slight overestimation in mean recoveries was detected when the concentrations of 50, 75 and 100 ng/kg were added. The comparison between different concentrations in the same kit showed that Kit A had a lower detection accuracy, in general, of the AFM1 expected (48 ng/kg detected for 75 ng/kg added or 55 ng/kg for 100 ng/kg). Kits B and E showed values very close to the real ones with concentrations of 50 and 75 ng/kg, although they were imprecise (underestimation) with the lower and the higher concentrations (25 and 100 ng/kg). These discrepancies also appear in the recovery averages (calculated using the mean concentrations in Table 1) shown in Fig. 1 for each test kit. The P value showed that a significant difference exists among the test kit (P < 0.001), the concentration (P < 0.001) and the interaction of both factors (P < 0.001). Of the five test kits used, no significant difference was found in the mean recoveries of the 25 ng/kg fortification level with values between 78% and 92%. At all the other concentrations, the test recovery levels improved, which in some cases reached up to 100% (Kit C with 50 and 75 ng/kg), with the only exception of kit A that showed a clearly significant tendency to decrease by recovering less than 56% at 100 ng/kg. As Fig. 1 shows, the concentration of 100 ng/kg presented recovery averages between 56% and 110%, which is a wide range, but one comparable to that of the mean concentrations (50 and 75 ng/kg). This situation occurs with all the kits, except kit A. So, the five test kits studied presented some variability in their development, but four of them presented similar and acceptable recovery averages, while just one gave results which cannot be considered acceptable.
Recovery average of AF M1 (%)
140 c
b
120
ab
80 60
b
b b
ab
100
b
b
b
NS
b
**
a
***
a
***
40 20 0 25
50
75
4. Discussion Based on the research done in this study, we can conclude that with the exception of Kit A, the rest of the test kits presented a good sensitivity in the recovery of AFM1 in ewe’s milk. These results are comparable to those of other studies done on cow’s milk (Rodríguez et al., 2003; Rosi et al., 2007), where a similar methodology was applied with the use of kit B or E. The data of recovery percentages were between 74.6% and 109%, when milk samples were added at a range of AFM1 concentrations of between 10 and 80 ng/L (Rodríguez et al., 2003). With added concentrations of between 30 and 100 ng/L, the range of recovery percentage obtained was 80–106% (Rosi et al., 2007). These results are similar to those obtained in the present study (excluding the results of kit A), with concentrations of between 25 and 100 ng/kg and recovery percentages of between 78% and 130%. Thus, this analysis methodology could be useful to decide whether milk must be excluded from a production chain if its AFM1 concentration exceeds 50 ng/ kg (the concentration limit set by the European Commission Directive). Despite the importance of ewe’s milk in the Mediterranean area, very few studies have been published on the analysis of AFM1 with ELISA. Kaniou-Grigoriadou, Eleftheriadou, Mouratidou, and Katikou (2005) determined the contamination of AFM1 in ewe’s milk, curd and Feta cheese by using Kit E. However, it is possible to find more studies on AFM1 occurrence analysed with Kit E in cow’s milk that had been contaminated naturally (Oveisi et al., 2007; Stoloff et al., 1975) and with goat’s milk (Virdis et al., 2008), or with the use of Kit B (Decastelli et al., 2007) for the analysis of cow’s milk and feed. Not all the kits in the market obtain the expected data results. In some cases, there is an overestimation, while an underestimation of the aflatoxin contents is shown in others. One possible explanation for this situation could be the so-called matrix effect or matrix interference, due to non-specific interaction of substances that does not relate to aflatoxin with the antibodies (Rosi et al., 2007). According to the results of our experiment, the use of commercial ELISA kits could be a reliable option to obtain acceptable results for the screening of AFM1 in ewe’s milk in a short time, but it would always be necessary to corroborate the results with official methods (HPLC) for positive or suspect samples. Acknowledgements This study was financed by the Research Project PAI06-00683875 and the predoctoral grant to R. Rubio (Ref. 07/039) from the Junta de Comunidades de Castilla-La Mancha. The authors are also grateful for the collaboration of the Instituto Técnico Agronómico Provincial of the Diputación de Albacete. References
b
a
1051
100
Concentration of AF M1 (ng/kg) Fig. 1. Recovery average (%) of AFM1 (ng/kg) in ewe’s milk for: kit A (d), kit B (s), kit C (j), kit D (h) and kit E (N) (n = 4). , , : P < 0.05, P < 0.01, P < 0.001, respectively. a, b, c: show significant differences among test kits.
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