Vol 3 (1) 2011 - UBM :: Departamentul de Chimie-Biologie

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K. Otto , T. Mihaela, F. Boltea , L. Bretan , O. Tiţa, Uuncertainties establishment in determining aflatoxin M1 analysis using enzyme immunoassay tests (Elisa) UNCERTAINTIES ESTABLISHMENT IN DETERMINING AFLATOXIN M1 ANALYSIS USING ENZYME IMMUNOASSAY TESTS (ELISA) Ketney Otto 1 2 3* , Mihaela Tiţa 1 , Florin Boltea 2,3,5 , Laura Bretan 2,3, 4 , Ovidiu Tiţa 1 1* - “Lucian Blaga” University of Sibiu , Faculty of Agricultural Sciences ,Food Industry and Environment Protection , Department of Food Biotechnology – 550024-Sibiu , Dr. Ion Ratiu Street, no. 7-9 , Romania, *otto_ketney@yahoo.com 2 - North University of Baia Mare , Faculty of Sciences, Chemistry Biology Department 430122- Baia Mare , 76, Victoriei Str, Romania 3 - Sanitary Veterinary and Food Safety Direction , Vasile Alecsandri Street , no. 66 , Baia Mare , Maramures 4 - University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur Street , No. 3-5, Cluj-Napoca 5 - University of Agricultural Sciences and Veterinary Medicine of Banat Timişoara, Food Technology Faculty, 119 A, Aradului Street, 300645, Timişoara Abstract As a result of ISO 17024 requirements regarding the accreditation of laboratories, an assessment of the uncertainty of the measurements is necessary, having to be expressed in a way that can be transmitted to the customer at request. The present paper describes the method of estimating in the case of the determining of M1 aflatoxin from milk. The uncertainty in measurement for the determining of the M1 aflatoxin through the ELISA method has been calculated starting from the Type A assessment of the standard uncertainty through the statistical analysis and through the Type B assessment of the standard uncertainty as a method of assessing of the uncertainty through other means than the statistical analysis of a series of observations and through the quantification of the two types of uncertainties the combined uncertainty and extended uncertainty have been obtained. Keywords: Aflatoxin, uncertainty, calibrators, precision, trueness, ELISA 1. Introduction The estimation of the uncertainty is a fundamental part of the ISO 17025 standard for laboratories of trials and calibration, which provides a quantitative assessment of the analytical results. According to this standard, the laboratories have to have procedures that have to be implemented when any aspect of the works tried and/or calibrated or the work results are not corresponding to their own procedures or with the requirements of the client. Calibration laboratories or the trial laboratories that make their own calibrations have to have and to apply estimating procedures of the uncertainties in measurement for all the calibrations and types of calibration [1]. This paper describes the estimating process of the uncertainty through the method of determining the aflatoxin in milk ELISA. This is describes by Daniels J. M. et. All {2]as a metabolic hydroxyl of the B1 aflatoxin, which can be encountered in milk or dairy products obtained from animals that have ingested contaminated forage, which is obtained from the B1 aflatoxin as a result of the monooxygenase microsomial that transforms the B1 aflatoxin in M1 aflatoxin at the level of the hepatocyte cells. Therefore at an international and national these issues led to a more strict imposition of new legislative limits concerning some mycotoxins that can contaminate food and raw materials in the food chain. According to the European Union regulations, the maximum level of aflatoxin M 1 in raw milk, heat-treated milk and milk-based products should not exceed [3, 4]. 7
Carpathian Journal of Food Science and Technology 2011, 3(1), 7- 11 2. Materials and methods Milk samples were analyzed using the ELISA method (Enzyme-linked immunosorbent assay) and interpreted according to the instructions of the standard or of the analysis kit (R-Biopharm AG, Ridascreen for AFM 1 , Art Nr. R1111). 39 determinations were made using this ELISA kit, samples and standards were analyzed in duplicate. The concentration of M1 Aflatoxin in milk samples was calculated using the following equation: A %Ab = 100 (1) A where: A – absorbance standard, % A 0 – Absorbance zero standard, % 0 The zero standard is thus made equal to 100 % and the absorbance values are quoted in percentages. The values calculated for the standards are entered in a system of coordinates on semi logarithmic graph paper against the aflatoxin M1 concentration [ng/l]. In order to obtain the aflatoxin M1 concentration in ng/l actually contained in a sample, the concentration read from the calibration curve must be further multiplied by the corresponding dilution factor. All the procedures for the ELISA analysis are shown in Figure 1. Figure. 1. Procedures for the ELISA analysis m- sampling quantity ; V 1 – the methanol volume used for extraction ; V 2 - the supernatant volume obtained through centrifugation; V 3 - the volume of the tampon solution for dilution ; V 4 –the volume of solution used in the analysis ; V 5 – the volume of tampon solution for blank ;V 6 –the volume of solution conjugated ; V 7 -the volume of tampon solution for standardizing ; V 8 – the volume of substrata solution ; V 9 – the volume of H 2 SO 4 solution for stopping the reaction ; -uncertain 8
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