# Determination of AflatoxinM1 in Milk by ELISA Technique in Mashad (Northeast of Iran)

Authors: Ali Mohamadi Sani; Mohamad Khezri; Halime Moradnia
Journal: ISRN Toxicology
 (2012)

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## Abstract

The aim of this study was to detect the amount of aflatoxin M1 (AFM1) in pasteurized milk samples in Mashad in northeast of Iran. For this purpose, 42 milk samples were collected from retail stores during fall 2011 and analyzed for AFM1 by enzyme-linked immunosorbent assay (ELISA) technique. All the analyses were done twice. Results showed presence of AFM1 in 97.6% of the examined milk samples by average concentration of 23 ± 16 ppt and contamination level ranging between 6 and 71 ppt. The concentration of AFM1 in all the samples was lower than the Iranian national standard and Food and Drug Administration limits (500 ppt), and, only in 3 (1.6%) samples, AFM1 concentration was more than the maximum tolerance limit (50 ppt) accepted by European Union and Codex Alimentarius Commission. According to our findings and previous studies, AFM1 contamination of milk is not a concern in this region, and the regional standard of AFM1 contamination in milk might be changed to lower than 100 ppt.


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## Body

## 1. Introduction

Mycotoxins are secondary metabolites of molds which are associated with certain disorders in animals and humans. In addition to being acutely toxic, some mycotoxins are now linked with the incidence of certain types of cancer, and it is this aspect which has evoked global concern over feed and food safety, especially for milk and milk products [1]. Aflatoxin M1 (AFM1) is a hepatocarcinogen found in milk of animals that have consumed feeds contaminated with aflatoxin B1 (AFB1), the main metabolite produced by fungi of the genus Aspergillus, particularly A. flavus, A. parasiticus, and A. nomius [2]. About 0.3–6.2% of AFB1 in animal feed is transformed to AFM1 in milk [3]. Due to serious health concerns, many countries have set maximum limits for aflatoxins, which vary from country to country [4]. The European Community prescribes that the maximum level of AFM1 in liquid milk should not exceed 50 ppt. However, according to the US standard, the level of AFM1 in liquid milk should not be higher than 500 ppt [5]. There have been several studies on AFM1 concentration in milk samples in different regions of the world and also in Iran, but this study was done to evaluate the occurrence of AFM1 in milk distributed in Mashad in northeast of Iran in order to evaluate the potential of changing the regional standard on AFM1 contamination of milk.


## 2. Materials and Methods

### 2.1. Materials

#### 2.1.1. Samples

In this study the AFM1 content of pasteurized milk samples in retail stores in Mashad (northeast of Iran) was determined in fall 2011. Forty-two pasteurized milk samples (1000 mL milk packets, heat treated at 72–74.4Ć for 15–20′′) from different brands were collected by simple random sampling method. The samples were transported to the laboratory in an insulated container at about 4°C and analyzed upon arrival.


#### 2.1.2. Reagents

Most of the reagents used to detect AFM1 were contained in the RIDASCREEN test kit, which included microtiter plate coated with capture antibodies, AFM1 standard solutions used for the construction of the calibration curve (1.3 mL each 0, 5, 10, 20, 40, and 80 ppt), peroxidase-conjugated AFM1, substrate (urea peroxidase), chromogen (tetramethylbenzidine), and stop reagent contains 1N sulphuric acid. Methanol used was of analytical grade and provided by Merck.


### 2.2. Methods

#### 2.2.1. AFM1 Detection

The quantitative analysis of AFM1 in pasteurized milk samples was performed by competitive ELISA (RIDASCREEN AFM1, R-Biopharm) procedure as described by R-biopharm GmbH [6]. Prior to analysis of the samples, the ELISA method was validated to ensure data quality. Validation of ELISA was carried out by determination of recoveries and the mean variation coefficient for fresh milk spiked with different concentrations of AFM1 (5, 10, 20, 40 and 80 ppt). The results are expressed in Table 1.Table 1
Validation data of the competitive ELISA for AFM1.
AFM1 spiked (ppt)(n=5)
AFM1 found (ppt)
Recovery(%)
Variation coefficient (%)
5
5
100
0.0
10
9.98
99.8
0.2
20
20.11
100.55
0.5
40
39.84
99.60
0.4
80
79.90
99.87
0.1Milk samples were centrifuged at 3500 g for 10 min at 10°C. The upper creamy layer was completely removed by aspirating through a Pasteur pipette and from the lower phase (defatted phase) 100μL was directly used per well in the test. One hundred μL of the AFM1 standard solutions (100 μL/well) and test samples (100 μL/well) in duplicate were added to the wells of microtiter plate and incubated for 60 min at room temperature in the dark. After the washing steps, 100 μL of the enzyme conjugate was added and incubated for 60 min at room temperature in the dark. The washing step was repeated three times. Fifty μL of substrate and 50 μL of chromogen were added to each well and mixed thoroughly and incubated for 30 min in the dark. Following the addition of 100 μL of the stop reagent to each well, the absorbance was measured at 450nm in ELISA reader (ELX-800, Bio-Tek Instruments, USA). According to the RIDASCREEN kit guidelines, the lower detection limit is 5 ppt for milk.


#### 2.2.2. Evaluation of AFM1

The absorbance values obtained for the standards and the samples were divided by the absorbance value of the first standard (zero standards) and multiplied by 100 (percentage maximum absorbance). Therefore, the zero standard is thus made equal to 100%, and the absorbance values are quoted in percentages. The values calculated for the standards were entered in a system of coordinates on semilogarithmic graph paper against the AFM1 concentration in ppt (Figure 1). The equation of the trendline in Figure 1 is as follows:
(1)y=0.016x2-1.940x+91.34.Figure 1
Calibration curve of standard solutions of AFM1 with concentrations of 5, 10, 20, 40, and 80 ppt by ELISA analysis.


## 2.1. Materials

### 2.1.1. Samples

In this study the AFM1 content of pasteurized milk samples in retail stores in Mashad (northeast of Iran) was determined in fall 2011. Forty-two pasteurized milk samples (1000 mL milk packets, heat treated at 72–74.4Ć for 15–20′′) from different brands were collected by simple random sampling method. The samples were transported to the laboratory in an insulated container at about 4°C and analyzed upon arrival.


### 2.1.2. Reagents

Most of the reagents used to detect AFM1 were contained in the RIDASCREEN test kit, which included microtiter plate coated with capture antibodies, AFM1 standard solutions used for the construction of the calibration curve (1.3 mL each 0, 5, 10, 20, 40, and 80 ppt), peroxidase-conjugated AFM1, substrate (urea peroxidase), chromogen (tetramethylbenzidine), and stop reagent contains 1N sulphuric acid. Methanol used was of analytical grade and provided by Merck.


## 2.1.1. Samples

In this study the AFM1 content of pasteurized milk samples in retail stores in Mashad (northeast of Iran) was determined in fall 2011. Forty-two pasteurized milk samples (1000 mL milk packets, heat treated at 72–74.4Ć for 15–20′′) from different brands were collected by simple random sampling method. The samples were transported to the laboratory in an insulated container at about 4°C and analyzed upon arrival.


## 2.1.2. Reagents

Most of the reagents used to detect AFM1 were contained in the RIDASCREEN test kit, which included microtiter plate coated with capture antibodies, AFM1 standard solutions used for the construction of the calibration curve (1.3 mL each 0, 5, 10, 20, 40, and 80 ppt), peroxidase-conjugated AFM1, substrate (urea peroxidase), chromogen (tetramethylbenzidine), and stop reagent contains 1N sulphuric acid. Methanol used was of analytical grade and provided by Merck.


## 2.2. Methods

### 2.2.1. AFM1 Detection

The quantitative analysis of AFM1 in pasteurized milk samples was performed by competitive ELISA (RIDASCREEN AFM1, R-Biopharm) procedure as described by R-biopharm GmbH [6]. Prior to analysis of the samples, the ELISA method was validated to ensure data quality. Validation of ELISA was carried out by determination of recoveries and the mean variation coefficient for fresh milk spiked with different concentrations of AFM1 (5, 10, 20, 40 and 80 ppt). The results are expressed in Table 1.Table 1
Validation data of the competitive ELISA for AFM1.
AFM1 spiked (ppt)(n=5)
AFM1 found (ppt)
Recovery(%)
Variation coefficient (%)
5
5
100
0.0
10
9.98
99.8
0.2
20
20.11
100.55
0.5
40
39.84
99.60
0.4
80
79.90
99.87
0.1Milk samples were centrifuged at 3500 g for 10 min at 10°C. The upper creamy layer was completely removed by aspirating through a Pasteur pipette and from the lower phase (defatted phase) 100μL was directly used per well in the test. One hundred μL of the AFM1 standard solutions (100 μL/well) and test samples (100 μL/well) in duplicate were added to the wells of microtiter plate and incubated for 60 min at room temperature in the dark. After the washing steps, 100 μL of the enzyme conjugate was added and incubated for 60 min at room temperature in the dark. The washing step was repeated three times. Fifty μL of substrate and 50 μL of chromogen were added to each well and mixed thoroughly and incubated for 30 min in the dark. Following the addition of 100 μL of the stop reagent to each well, the absorbance was measured at 450nm in ELISA reader (ELX-800, Bio-Tek Instruments, USA). According to the RIDASCREEN kit guidelines, the lower detection limit is 5 ppt for milk.


### 2.2.2. Evaluation of AFM1

The absorbance values obtained for the standards and the samples were divided by the absorbance value of the first standard (zero standards) and multiplied by 100 (percentage maximum absorbance). Therefore, the zero standard is thus made equal to 100%, and the absorbance values are quoted in percentages. The values calculated for the standards were entered in a system of coordinates on semilogarithmic graph paper against the AFM1 concentration in ppt (Figure 1). The equation of the trendline in Figure 1 is as follows:
(1)y=0.016x2-1.940x+91.34.Figure 1
Calibration curve of standard solutions of AFM1 with concentrations of 5, 10, 20, 40, and 80 ppt by ELISA analysis.


## 2.2.1. AFM1 Detection

The quantitative analysis of AFM1 in pasteurized milk samples was performed by competitive ELISA (RIDASCREEN AFM1, R-Biopharm) procedure as described by R-biopharm GmbH [6]. Prior to analysis of the samples, the ELISA method was validated to ensure data quality. Validation of ELISA was carried out by determination of recoveries and the mean variation coefficient for fresh milk spiked with different concentrations of AFM1 (5, 10, 20, 40 and 80 ppt). The results are expressed in Table 1.Table 1
Validation data of the competitive ELISA for AFM1.
AFM1 spiked (ppt)(n=5)
AFM1 found (ppt)
Recovery(%)
Variation coefficient (%)
5
5
100
0.0
10
9.98
99.8
0.2
20
20.11
100.55
0.5
40
39.84
99.60
0.4
80
79.90
99.87
0.1Milk samples were centrifuged at 3500 g for 10 min at 10°C. The upper creamy layer was completely removed by aspirating through a Pasteur pipette and from the lower phase (defatted phase) 100μL was directly used per well in the test. One hundred μL of the AFM1 standard solutions (100 μL/well) and test samples (100 μL/well) in duplicate were added to the wells of microtiter plate and incubated for 60 min at room temperature in the dark. After the washing steps, 100 μL of the enzyme conjugate was added and incubated for 60 min at room temperature in the dark. The washing step was repeated three times. Fifty μL of substrate and 50 μL of chromogen were added to each well and mixed thoroughly and incubated for 30 min in the dark. Following the addition of 100 μL of the stop reagent to each well, the absorbance was measured at 450nm in ELISA reader (ELX-800, Bio-Tek Instruments, USA). According to the RIDASCREEN kit guidelines, the lower detection limit is 5 ppt for milk.


## 2.2.2. Evaluation of AFM1

The absorbance values obtained for the standards and the samples were divided by the absorbance value of the first standard (zero standards) and multiplied by 100 (percentage maximum absorbance). Therefore, the zero standard is thus made equal to 100%, and the absorbance values are quoted in percentages. The values calculated for the standards were entered in a system of coordinates on semilogarithmic graph paper against the AFM1 concentration in ppt (Figure 1). The equation of the trendline in Figure 1 is as follows:
(1)y=0.016x2-1.940x+91.34.Figure 1
Calibration curve of standard solutions of AFM1 with concentrations of 5, 10, 20, 40, and 80 ppt by ELISA analysis.


## 3. Statistical Analysis

Data were analysed using Excel 2007 and results reported asmean±SD. The calibration curve and trendline equation prepared using Excel 2007.


## 4. Results and Discussion

The standard solutions of concentration from 5 to 80 ppt AFM1 were used to find calibration/standard curve. The results showed the linearity of the standard curve over the range studied. Figure 1 gives the calibration curve of standard solutions of AFM1 with concentrations of 5, 10, 20, 40, and 80 ppt by ELISA analysis.Analytical results showed that the incidence of AFM1 contamination in pasteurized milk samples was low. Although 97.6% of the samples were contaminated with AFM1, the toxin concentration was lower than Iranian national standard and FDA limit (500 ppt) and only in three (1.6%) of the samples AFM1 concentration was greater than the maximum tolerance limit (50 ppt) accepted by European Union and Codex Alimentarius Commission. Table 2 shows the distribution and percentage of AFM1 contamination in pasteurized milk samples. The minimum and maximum contamination level of AFM1 was found to be 6.4 and 71.4 ppt, respectively. The mean±SD AFM1 level in the analyzed samples of pasteurized milk was 23±16 ppt.Table 2
Aflatoxin M1 distribution and percentage of pasteurized milk samples.
AFM1 levels ppt in positive samples
<10
10–30
30–50
50–70
>70*
N
1
5
27
5
3
1
%2
12.3
65.6
12.3
7.4
2.4
N
1 number of contaminated samples.
%2 Percentage of AFM1 positive samples.
*71.3 ppt in the contaminated sample.The mean AFM1 concentrations in milk in European, Latin American, and Far Eastern diets have been reported by the Joint FAO/WHO Expert Committee on Food Additives [7] to be 23, 22, and 360 ng/L, respectively. Thus, the observed mean AFM1 concentration in Mashad milk samples was as high as the European and Latin American and much lower than those reported for the Far Eastern diets.On the other hand, several studies have been done to determine AFM1 contamination of milk in Iran (Table 3). The incidence of AFM1 observed in the present study was lower than the incidence of AFM1 reported by other authors [8–17], yet, in all studies, the averages of toxin concentrations are below 100 ppt. The variations may be attributed to differences in region, season, and especially analysis method.Table 3
The incidence of milk contamination in Iran in other studies.
Location
Reference
Method ofdetection
Samplesize
Percent ofcontamination
Percent ofcontamination >50 ppt
AFM1  concentration (ppt)
Mashad (north east of Iran)
Current study
ELISA
42
97.6
1.6
23.2
Mashad (north east of Iran)
Mohamad Sani and Nikpooyan, 2012 [8]
HPLC
60
100
1.6
16.16
Mashad (north east of Iran)
Mohamadi Sani et al., 2010 [9]
ELISA
196
100
80.6
77.9
Five states of Iran
Tajkarimi et al., 2007 [10]
HPLC
98
100
37.7
39
Tehran (capital of Iran)
Heshmati and Milani, 2010 [17]
ELISA
210
55.2
33.3
58
14 states of Iran
Tajkarimi et al., 2008 [13]
HPLC
319
54
23
57
Shiraz (south of Iran)
Alborzi et al., 2006 [12]
ELISA
624
100
17.8
n.r*
Ahwaz (south of Iran)
Rahimi et al., 2010 [16]
ELISA
311
42.1
12.5
43.3
Sarab (north west of Iran)
Kamkar 2005 [11]
TLC
111
76.6
40
61.4
Central part of Iran
Fallah 2010 [14]
ELISA
225
67.1
33.1
49.9
Ardabil  (north west of Iran)
Nemati et al., 2010 [15]
ELISA
90
100
33
n.r*
*Not reported.Based on the above results, especially later studies in Mashad, the present situation is hopeful and might represent the possibility of altering standard limit of AFM1 concentration in milk in Iran. We suggest reduction of the limit as low as 100 ppt for raw milk.



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*Source: 121926-2012-09-11.xml*