Abstract
Aflatoxin B1 (AFB1) is a secondary metabolite produced by some Aspergillus spp. fungi affecting many crops and feed materials. Aflatoxin M1 (AFM1), the 4-hydroxylated metabolite of AFB1, is the main AFB1-related compound present in milk, and it is categorized by the International Agency for Research on Cancer (IARC) as a “group 1 human carcinogen”. The aim of this work was to evaluate and compare the analytical performances of two commercial immunoassays widely applied for the detection of AFM1 in milk, namely strip test immunoassay and enzyme linked immunosorbent assay (ELISA). Assay validation included samples at AFM1 levels of 25, 50, 75 ng/kg and blank samples (AFM1 < 0.5 ng/kg). With respect to a screening target concentration (STC) of 50 ng/kg the two assays showed cut-off values of 37.7 ng/kg and 47.5 ng/kg for strip test and ELISA, respectively, a false suspect rate for blanks <0.1% (for both assays) and a false negative rate for samples containing AFM1 at levels higher than STC, of 0.4% (for both assays). The intermediate precision (RSDip) was <32% for the strip test and <15% for the ELISA. Method verification through long-term intra-laboratory quality control (QC) measurements confirmed the results from the validation study. Furthermore, a satisfactory correlation of the results obtained with both immunoassays and the AOAC Official Method 2000.08 was obtained for the analysis of cow milk samples naturally contaminated with AFM1 at levels within “not detected” (< 0.5 ng/kg) and 50 ng/kg. Finally, the extension of the scope of the strip test method to goat and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation.
Highlights
Aflatoxins are mycotoxins found in four main chemical structures: aflatoxin B1 (AFB1 ), B2 (AFB2 ), G1 (AFG1 ) and G2 (AFG2 ); they can occur in a wide range of crops, including the major staple cereals, edible nuts and legumes and their products
The experimental design to evaluate analytical performances of strip test immunoassay and enzyme linked immunosorbent assay (ELISA) comprised the following steps, which were carried out in parallel for the two assays: i) single laboratory validation study to evaluate precision, cut-off value, false suspect and false negative rates; ii) verification of cut-off and precision values by long-term intra-laboratory quality control (QC) study; iii) evaluation of results correlation between rapid immunoassays and AOAC Official Method
The two test kits performed in a similar way, and in both cases a satisfactory correlation was observed, with results provided by the reference method (r = 0.923 and slope = 0.84 for strip test vs HPLC and r = 0.924 and slope = 1.05 for ELISA vs HPLC)
Summary
Aflatoxins are mycotoxins found in four main chemical structures: aflatoxin B1 (AFB1 ), B2 (AFB2 ), G1 (AFG1 ) and G2 (AFG2 ); they can occur in a wide range of crops, including the major staple cereals (e.g., maize), edible nuts and legumes and their products. AFB1 is converted into its hydroxylated metabolite (AFM1 ) by the liver enzymes of lactating animals [3] This toxin, like the parent compound, has been categorized by the International Agency for Research on Cancer (IARC) as a group 1 toxin, a human carcinogen [2]. Due to their carcinogenity, the aflatoxins uptake through contaminated food consumption should be as low as possible, the aflatoxin legislation is intended to implement the ALARA principle (As Low As Reasonably Achievable) and no threshold limit concerning the tolerable daily intake in humans has been established [4]
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