Two year survey on the occurrence and seasonal variation of aflatoxin M1 in milk and milk products in Serbia

The incidence of contamination of aflatoxin M1 (AFM1) in milk and milk products samples collected in Serbia was investigated by using the competitive enzyme linked immunosorbent assay (ELISA) technique. A total of 1438 samples composed of 678 raw milk, 438 heat treated milk and 322 milk product samples that were analyzed during the period of 2013–2014, including all seasons. The AFM1 levels exceeded the European Union maximum residue permitted amount (EU MRL) in 56.3% raw milk, 32.6% heat treated milk and 37.8% of milk product samples. Milk powders had the highest mean AFM1 concentration (0.847 μg/kg) of all types of milk products examined. Mean concentration of AFM1 in raw milk samples during the period of winter in Serbia was 0.358 μg/kg and did not significantly differ from the mean concentrations of 0.375 μg/kg during the spring. However, the AFM1 raw milk concentration in the summer (0.039 μg/kg) and autumn season (0.103 μg/kg) was significantly lower. Seasonal variation of AFM1 concentrations in heat treated milk samples followed the trend observed in raw milk. Mean raw milk AFM1 concentration has dropped down by 10 fold from 0.314 μg/kg in 2013 to 0.035 μg/kg in 2014. The fraction of raw milk samples exceeding the EU MRL has decreased from 62.3% to 11.5% by the end of 2014.     

Detection of aflatoxin M1 in milk products from China by ELISA using monoclonal antibodies

A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) method using monoclonal antibody for measuring aflatoxin M1 (AFM1) in milk and milk products has been described. One monoclonal antibody was isolated and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with AFM1 carboxymethyl oxime conjugated with bovine serum albumin (BSA). Cross-reactivities of the anti-AFM1 monoclonal antibody clone were 100, 13.9, 6.7 and <1% against AFM1, aflatoxin B1 (AFB1), aflatoxin G1 (AFG1) and deoxynivalenol (DON), respectively. Assays of milk samples mixed with AFM1 ranging in concentration from 0.1 to 3.2 ng/ml gave mean ELISA recovery of 98%. The limit of detection concentration of AFM1 was 0.04 ng/ml. AFM1 contamination was measured in 12 samples of raw milk, 15 samples of powdered milk, 104 samples of liquid milk and four cheese samples collected from different supermarkets in Northeast of China. Of 135 milk samples tested, 55 (41%) samples contained AFM1 at levels that ranged from 0.32–0.50 ng/ml, 24 (18%) samples contained 0.16–0.32 ng/ml, and 18 (13%) samples contained 0–0.16 ng/ml; in 38 (28%) samples AFM1 was not detected. The results indicate that the necessary precaution will have to be taken to minimize the AFM1 contamination in milk and milk products from Northeast of China.     

Aflatoxin M1 in raw,pasteurized and powdered milk available in the Syrian market

The incidence of contamination of aflatoxin M1 (AFM1) in milk samples collected from the Syrian market was investigated by using the competitive enzyme linked immunosorbent assay (ELISA) technique. A total of 126 samples composed of raw cow milk (74 samples), raw sheep milk (23), raw goat milk (11), pasteurized cow milk (10) and powdered milk (8) showed that 80% of tested samples were contaminated with various levels of AFM1 ranging from >20 to 765 ng/l. Percentages of AFM1-contaminated samples exceeding the American, Syrian and European tolerance limits were 22%, 38% and 52%, respectively.The range of contamination was relatively higher in pasteurized milk than in raw cow and sheep milk. 80% of AFM1-contaminated pasteurized cow milk samples exceeded the European tolerance limit with a range of contamination between 89 and 765 ng/l. Percentages of contaminated raw cow, sheep and goat milk exceeding the European tolerance limit were 59%, 24% and 14%, respectively.Milk powder was almost free of AFM1 contamination with only one sample containing a concentration lower than the European tolerance limit (12 ng/l).Extrapolation of aflatoxin B1 (AFB1) from AFM1 levels of contamination in milk samples indicates that contamination in dairy cattle feeds may range from 0.5 to 47.8 μg/kg.     

A survey on the Aflatoxin M1 occurrence and seasonal variation in buffalo and cow milk from Southern Italy

This study evaluates the aflatoxin M1 (AFM1) contamination in 804 samples of raw milk from cow and buffalo, collected randomly in Campania and Calabria regions of Southern Italy over a two years period.The competitive enzyme linked immunosorbent assay (ELISA) method was used to analyze AFM1 in the samples. AFM1 levels result above the CCβ value of 0.004 μg/kg in 51 (12.3%) cow milk samples and in 28 (7.2%) buffalo milk samples. Positive results from screening analysis were confirmed by high performance liquid chromatography with fluorimetric detection (HPLC-FLD) after a procedure of centrifugation, extraction and immunoaffinity column clean-up of milk. Only one cow milk sample exceeded the maximum limit (0.05 μg/kg) set by the European Regulation.The occurrence of AFM1 contamination was significantly (p < 0.05) higher in cold season, particularly fall, than in warm season, principally spring.Our results indicate that feedstuff used in the buffalo and cow farms were not highly contaminated with aflatoxins, determining a good quality of the analyzed milk. Therefore, the AFM1 contamination of the milk does not represent a serious public health problem in both regions in Southern Italy.     

Quantification of aflatoxin M1 in raw milk by a core-shell column on a conventional HPLC with large volume injection and step gradient elution

The immunoaffinity column cleanup method coupled with HPLC separation and fluorescence detection is still one of the most frequently used quantitative methods for routine analyses of AFM1. In this study, aflatoxin M1 (AFM1) was quantified with a chromatography column packed with 2.6 μm core-shell particles on a conventional HPLC. A large volume of solvent (100 μL) was injected into the highly efficient column without any noticeable reduction in separation performance with the help of stepwise gradient elution. The instrumental conditions were optimized by response surface analysis methodology (RSM) with a three-level three-factor Box–Behnken design.The use of core-shell columns on conventional HPLC for AFM1 analysis under the optimized instrumental conditions leads to increased analytical performance compared with traditional totally porous columns without the heavy costs associated ultra-high-pressure instruments. Moreover, the improved instrumental sensitivity enables simplified sample preparation by avoiding any solvent replacement. The method could be easily applied to enhance the sensitivity of HPLC-FLD for AFM1 analysis that is based on isocratic elution and is more widely used. The method was successfully applied to 40 raw milk samples collected in summer from 20 cattle ranches located in two different provinces in southwestern China.     

A study on the occurrence of aflatoxin M1 in milk consumed in Kosovo during 2009–2010

To assess public health hazards associated with the occurrence of AFM1 residues in milk a survey was carried out, during 2009–2010 in six different major regions of Kosovo (Prishtina, Prizren, Peja, Gjakova, Mitrovica and Gjilan). In the present study a total of 895 milk samples were collected, 656 raw milk and 39 UHT milk were analyzed in 2009, while in 2010 the research materials consisted of 170 raw milk, and 30 UHT milk. The occurrence and concentration range of AFM1 in the samples were investigated by competitive enzyme-linked immunosorbent assay (ELISA) method. From 895 samples examined, 25(2.8%) samples were contaminated with AFM1; none of contaminated samples did not exceed the maximum tolerable limit of the EC according to the European Union regulation limits of 50 ng/L. In 2009, 20 out of 695 samples (2.9%) were positive, and in 2010, 5 out of 200 samples examined (2.5%) were positive. In UHT milk, AFM1 was detected in two samples (2.6%). The positive incidence of AFM1 in milk samples collected in 2009 and in 2010 ranged from 5.2 to 26.6 ng/L and from 6.3 to 9.81 ng/L, respectively. In UHT milk levels of AFM1 were 7.2 and 9.9. In terms of regional distribution of positive samples during 2009 and 2010, Peja was the region with most of positive samples 12 or (63.2%), and 5 or (100%), respectively. Due to the unpredictability of climatic and environmental conditions, it is essential that the program of monitoring of AFM1 in milk in Kosovo should continue, to avoid an undetected problem, like occurred in other countries.     

Development of a one-step immunochromatographic assay with two cutoff values of aflatoxin M1

Aflatoxin M1 (AFM1) in milk has two standard limits, namely, the European standard set up at 0.05 ng/mL and the other one (including America, China, and other countries) at 0.5 ng/mL. A one-step immunochromatographic assay (ICA) with two cutoff values was developed to satisfy these two standard limits, wherein test zones including test 1 and 2 (T1 and T2) lines were formed by dispensing AFM1-BSA antigen onto a nitrocellulose membrane. The migrating AFM1 analyte in the sample and capture reagent immobilized on two test lines competed for the limited binding sites of AFM1 antibody, which conjugated with colloidal gold. Three red lines appeared at T1 and T2 lines and control (C) line when the sample was negative or the AFM1 analyte concentration was lower 0.05 ng/mL. However, only two red lines appeared at T1 and C lines when the AFM1 analyte concentration was 0.05 ng/mL or higher but lower 0.5 ng/mL. One red line appeared at C line when AFM1 analyte concentration was 0.5 ng/mL or higher. Results showed that the novel immunochromatographic assay was superior to traditional ICAs because of satisfying two standard limits at one step.     

Aflatoxin M1 in raw and UHT cow milk collected in Bosnia and Herzegovina and Croatia

Aflatoxin M1 (AFM1) concentrations were determined in raw and UHT cow milk samples collected in different regions of Bosnia and Herzegovina and Croatia during the autumn months of 2014. The mean AFM1 levels in the raw milk samples were (ng/kg): 6.22 in Bosnia and Herzegovina, 5.65 in Croatia. In all except one milk sample, AMF1 levels were below the LOQ value of 34.2 ng/kg (ELISA method). In four milk samples, AFM1 concentrations exceeded the EU MRL of 50 ng/kg. Samples were subjected to LC-MS/MS analysis which confirmed elevated values determined by ELISA. Elevated levels were in the range 56.6–132.6 ng/kg. Two positive milk samples from Bosnia and Herzegovina originated from Una Sana Canton, two from Croatia from eastern Croatia. The highest AFM1 levels of 132.6 ng/kg was measured in milk from eastern Croatia. In 214 samples of processed UHT milk from Bosnia and Herzegovina and Croatia, AFM1 ranged from 2.29 ng/kg to 21.4 ng/kg, all below the LOQ value. AFM1 exceeded the EU MRL value in only 0.62% of milk samples, indicating the sporadic use of contaminated feedstuff at farms in both countries.     

Influence of climate conditions on aflatoxin M1 contamination in raw milk from Minas Gerais State,Brazil

Aflatoxin M1 (AFM1) contamination in milk is a potential risk for animal and human health. The occurrence of AFM1 in raw milk from Minas Gerais State, Brazil, in different climate conditions was evaluated. A total of 129 milk samples were collected from dairy farms in three distinct periods (dry period, transition period and rainy period), and analyzed by enzyme-linked immunoabsorbent assay (ELISA) as screening test. Samples with AFM1 at concentrations above 0.05 μg/L were analyzed by liquid chromatography with fluorescence detection (HPLC-FD) as confirmatory method. All the analyzed samples showed contamination with AFM1. In the three periods, AFM1 was detected at concentrations below the permitted limit of 0.50 μg/L in milk, according to the Brazilian legislation, and 18 samples (13.95%) showed contamination with AFM1 above the permitted limit of 0.05 μg/L established by Codex Alimentarius and European Commission. Milk contamination with AFM1 was significantly affected by climatic conditions, and the highest values were verified in dry period. The AFM1 contamination was lower than the acceptable daily intake (ADI) estimated for Latin America, indicating that milk from this region is safe for human consumption. Control measures to monitor AFM1 in milk are mandatory in tropical climate countries especially in dry periods.     

A survey on the occurrence of aflatoxin M1 in raw and processed milk samples marketed in Lebanon

A survey was conducted to determine the occurrence of AFM1 in 77 cow and goat milk samples (38 raw milk, 25 pasteurized milk and 14 powder milk samples); obtained either from local small farms, or markets. The competitive enzyme – linked immunosorbent assay (ELISA) method was applied for this purpose positively detecting AFM1 in 64.9% of all tested milk samples. The revealed rates of AFM1 contamination were 73.6%, 68.0%, 35.7% for the raw, pasteurized and powder milk samples, respectively. The individual values, within each category of milk samples, ranged from 2.63 to 126 ng/l (average = 60 ng/l), 3.27–84.4 ng/l (average = 30.6 ng/l) and 9.18–16.5 ng/l (average = 13.7 ng/l) for the raw, pasteurized and powder milk samples, respectively. Of the positive samples, 29 were still below the permitted limit (50 ng/l) set by the European Commission whereas 21 exceeded the permissible limit. This work represents the data of the first survey on the occurrence of AFM1 in raw and processed milk marketed and consumed in Lebanon.     

Occurrence of aflatoxin M1 in pasteurized and UHT milks in China in 2014–2015

This survey was performed to assess the safety of milk in China, specifically by assessing the presence of aflatoxin M1 (AFM1) residues in pasteurized and ultra high temperature (UHT) milk. In 2014–2015, 193 samples of UHT milk were collected from different cities in China. In 2015, 38 samples of pasteurized milk were collected from different cities in China. AFM1 was detected using an enzyme-linked immunosorbent assay (ELISA). AFM1 positivity was defined as a concentration exceeding the detection limit of the assay (0.005 μg/kg). Other cut-offs that were used were the legal AFM1 limits in the European Union (EU) and China (0.05 and 0.5 μg/kg, respectively). In 2014 and 2015, 88.6% and 59.6% of UHT milk samples were AFM1-positive, respectively. The pasteurized milk samples were less frequently AFM1-positive (47.4%). In 11.9% of the 2014–2015 UHT milk samples, the AFM1 levels exceeded the EU limit. This is lower than the frequency we recorded in 2010 (20.3%). None of the pasteurized milk samples exceeded the EU limit in 2015. The UHT milk samples from the north of China were less likely to be contaminated than the samples from the south in both 2014 and 2015. None of the samples exceeded the Chinese legal limit.     

Levels of aflatoxin M1 in different types of milk collected in Serbia: Assessment of human and animal exposure

The level of aflatoxin M1 (AFM1) in 50 milk samples collected from February to June 2013 from Serbian market or domestically produced was determined using simple non-specific sample preparation method based on solid phase extraction (Oasis HLB, Waters) and ultra-high performance liquid chromatography with heated electrospray ionization triple quadrupole mass spectrometry (UHPLC/HESI-MS/MS). The range of detection was between < LOD and 1.44 μg/kg with mean value of 0.30 μg/kg. Thirty-eight samples (76%) exceed the maximum level of 0.05 μg/kg sets by EU. The highest level of 1.44 μg/kg was found in raw sample of domestically produced milk while the lowest one in organic produced milk. The evaluation of the exposure degree of AFM1 through the milk consumption by the average Serbian citizen was estimated at levels of 1.420, 0.769 and 0.503 ng/kg bw/day during February, April and May, respectively. Estimation of the corresponding concentration of AFB1 in feedstuffs was evaluated as 18.75 μg/kg. The calculated hazard index of 7.1, 3.8 and 2.5 for February, April and May, respectively, was higher than 1 indicated serious risk of AFM1 to Serbian consumers. This work presents the first insight in the occurrence of AFM1 in milk collected in Serbia as well as mycotoxin intake through milk consumption by Serbian adult population.     

Aflatoxin M1 contamination in raw milk from major milk-producing areas of China during four seasons of 2016

This survey was performed to determine the frequency with which raw milk from the major milk-producing areas of China was contaminated with aflatoxin M1 (AFM1) in 2016. In total, 5650 raw milk samples produced during the four seasons of 2016 were collected from the major milk-producing areas of China, including Hebei, Heilongjiang, Henan, Inner Mongolia, Shandong, and Xingjiang provinces. Contamination of AFM1 was detected in 267 of the 5650 raw milk samples in totally, with the incidence of 4.7%. Only 1.1% of raw milk samples exceeded the European Union legal limit (50 ng/L), and none of samples exceeded the Chinese and United States legal limit (500 ng/L). The incidence of AFM1 contamination in raw milk samples was much higher during winter (10.2%) than in spring, summer, or autumn (3.0%, 2.1%, and 4.4%, respectively) in China. Thus, it is particularly important to monitor AFM1 contamination in raw milk during the winter season. This comprehensive study will facilitate future risk analysis and the management of AFM1 contamination in raw milk in China.     

Seasonal effect on aflatoxin M1 contamination in raw and UHT milk from Croatia

Aflatoxin M1 contamination was examined in raw milk (3716) and UHT milk (706) samples collected from farms of eastern Croatia and markets of central Croatia from February to July 2013. A maximal mean AFM1 of 1135.0 ng/L was measured in raw milk in March. The AFM1 levels exceeded the European Union maximum residue permitted amount (EU MRL) in 45.9% raw and 36.2% UHT milk samples in February. In total, AFM1 levels exceeded the EU MRL values in 27.8% of raw and 9.64% of UHT milk samples. A slight decrease in the number of samples exceeding the EU MRLs was recorded in the period March to June. The results showed significant statistical differences between the mean AFM1 concentrations of raw and UHT milk samples collected during February, March, May and June (P < 0.05, all). Also, statistical differences in AFM1 concentrations were found between months for raw and UHT milk (P < 0.001, both). In conclusion, the frequency of control of feed and milk samples should increase and should strive to educate breeders and those involved in milk production about the harmful effect of aflatoxins to animal feed.     

Immunoaffinity pre-concentration combined with on-column visual detection as a tool for rapid aflatoxin M1 screening in milk

A non-instrumental rapid test was developed for the screening of aflatoxin M1 (AfM1) in milk at the 40 ng/L level. The method combines on one immunoaffinity gel layer, AfM1 pre-concentration and direct competitive immunoassay detection with visual evaluation. Aflatoxin B1-horse radish peroxidase (AfB1-HRP) and Sepharose 4B-immobilized anti-aflatoxin B1 monoclonal antibody with a 79% cross-reaction for AfM1 were used. The assay was performed in a standard column for solid phase extraction using a milk sample volume of 10 mL. Raw milk, pasteurized milk, milk powder, kefir and yogurt were analyzed with the developed test. It was shown that pasteurized milk could be analyzed without any sample preparation. The other types of milk were analyzed after centrifugation. For the assay procedure an aliquot of milk was flowed through the immunoaffinity gel layer in the tube by pushing the plunger of a syringe. Further, AfB1-HRP was applied and bound to free antibody binding sites. After addition of the chromogenic substrate the results were visually evaluated. Blue colour developed in case of negative milk samples while no colour appeared for positive samples. This method showed a false negative rate of only 2% and a high throughput (20–35 min for six samples).     

Incidence of aflatoxin M1 in human breast milk in Tehran, Iran

This study examined the exposure of infants to aflatoxin M1 (AFM1) and of lactating mothers to aflatoxin B1 (AFB1), using AFM1 in breast milk as a biomarker for exposure to AFB1. An enzyme-linked immunosorbent assay (ELISA) was modified for the analysis of AFM1 in breast milk samples from 160 women in Tehran, Iran. AFM1 was detected in 157 samples by average concentration of 8.2 ± 5.1 ng/kg (range 0.3–26.7 ng/kg).The concentration of AFM1 in one sample was higher than the maximum tolerance limit accepted by European Union and USA (25 ng/kg), but in 55 samples was higher than the maximum concentration recommended by Australia and Switzerland (10 ng/kg).Logistic regression Analysis failed to show significant correlation between AFM1 and gestational age, education, postnatal age, gender, nationality, clinical condition, the number of family member, the number of children, type and amount of dairy consumption, vegetable, fruits, oil and meat. But it was significant relation to the cereal consumption, also to the height at birth.     

A survey of aflatoxin M1 of raw cow milk in China during the four seasons from 2013 to 2015

In the present study, a total of 1550 raw cow milk samples were collected from Southern, Northern, Northeast, and Western regions of China during the four seasons from 2013 to 2015. Samples were analyzed for aflatoxin M1 (AFM1) using high performance liquid chromatography (HPLC). In 2013, AFM1 was detected in 21% of 366 raw cow milk samples with levels ranging between 0.01 and 0.24 μg/L. In 11.7% of samples, AFM1 levels were >0.05 μg/L, which is the legal limit in the European Union. The mean and median of positive samples were 0.069 ± 0.052 μg/L and 0.056 μg/L, respectively. In 2014, AFM1 was detected in 28.5% of 624 raw cow milk samples, with levels ranging from 0.01 to 0.25 μg/L. Of these samples, 7.7% had AFM1 levels exceeding 0.05 μg/L, with a mean of 0.042 ± 0.039 μg/L and median of 0.028 μg/L. AFMI was detected in 14.1% of 560 raw cow milk samples in 2015, with levels ranging from 0.01 to 0.144 μg/L. In 1.8% of these samples, AFM1 levels were above 0.05 μg/L, with a mean of 0.026 ± 0.024 μg/L and median of 0.017 μg/L. Our results demonstrate that AFM1 levels of the samples did not exceed the legal limit of 0.5 μg/L in China, the United States, and Codex Alimentarius Commission. Geographically, AFM1 contamination was more predominant in raw cow milk samples from Southern China than in those from other regions, with a higher number of samples containing AFM1 levels above 0.05 μg/L in 2013, 2014, and 2015. AFM1 levels were higher in autumn than in the other seasons during the entire study period. According to our survey, AFM1 contamination has been well-controlled in China during recent years; however, some samples still exceeded the European Union (EU) legal limit. Better prevention and management of aflatoxins in both feed and milk should be considered especially in Southern regions of China and in autumn.     

Assessment of aflatoxin M1 levels in pasteurized and UHT milk consumed in Prishtina,Kosovo

To assess public health hazards associated with the occurrence of AFM1 residues in pasteurized milk and UHT milk a survey was carried out, in Prishtina, capital city of Kosovo. In the present study, a total of 178 samples, 84 pasteurized milk and 94 UHT milk were collected during 6 months (July to December 2013). They were obtained from retail outlets in Prishtina city (Kosovo). The occurrence and concentration range of AFM1 in the samples were investigated by competitive enzyme-linked immunosorbent assay (ELISA) method. There was a high incidence of AFM1 (81.0%) in both pasteurized and UHT milk samples. Eighty three percent (83.3%) of the pasteurized milk samples and seventy eight percent (78.7%) of the UHT milk samples contained AFM1. The positive incidence of AFM1 in the pasteurized milk and the UHT milk samples ranged from 5.16 to 110 ng/L and from 5.02 to 62 ng/L, respectively. AFM1 levels in 18 (21.4%) pasteurized milk samples and 4 (4.2%) UHT milk samples exceeded the maximum tolerable limit of the EC according to the European Union regulation limits of 50 ng/L. AFM1 levels in the samples show that there is a presence of high AFM1 level that constitutes a human health risk in Kosovo. The results of this study imply that more emphasis should be given to the routine AFM1 inspection of milk and dairy products in the Prishtina region.     

Sensitive competitive direct enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip for detecting aflatoxin M1 in milk

Polyclonal antibodies specific to aflatoxin M1 were generated from rabbits immunized with AFM1-bovine serum albumin (BSA). By using these antibodies, this work establishes a rapid competitive direct enzyme-linked immunosorbent assay (cdELISA) and a gold nanoparticle immunochromatographic strip method for detecting AFM1 in milk and milk products. In the rapid cdELISA, AFM1 at a concentration of 0.014 ng/ml caused 50% inhibition (IC₅₀) of binding AFM1-horseradish peroxidase to the antibodies. Effective on-site detection of AFM1 was also developed based on a rapid and sensitive antibody-gold nanoparticle immunochromatographic strip method. This strip had a detection limit of 1.0 ng/ml for AFM1 in milk samples. Additionally, the whole analysis was completed within 10 min. Close examining 15 milk-based samples by cdELISA revealed that 6 were slightly contaminated with AFM1, with a mean concentration of 0.028 ng/ml. Moreover, all samples were negative in the immunochromatographic strip assays due to the contaminated levels were below the detection limit of the strip. Importantly, the proposed cdELISA and immunochromatographic strip methods are highly sensitive to the rapid detection of AFM1 in milk and milk products.     

Novel monoclonal antibody-based sensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for detecting aflatoxin M1 in milk

Monoclonal antibody (mAb) that is specific to AFM1 was generated from the hybridoma cell line, 10F3C10, which was obtained by the fusion of mouse NS1 myeloma cells with the spleen cells of mouse that had been immunized with AFM1-bovine serum albumin (BSA). The 10F3C10 mAb is belong to the immunoglobulin G1 isotype. Both competitive direct and indirect enzyme-linked immunosorbent assay (ELISA) was utilized to characterize the mAb for AFM1. The concentrations of AFM1, AFB1 and AFG1 that caused 50% inhibition (IC₅₀) of the binding of AFM1-horseradish peroxidase (AFM1-HRP) to the antibody were found to be 0.022, 0.310 and 2.12 ng/mL, respectively. The immunochromatographic strip (immunostrip) assay with mAb-gold nanoparticle conjugates as a detection marker exhibited a visual limit of detection of 0.1 ng/mL for AFM1 and the analysis took a total of 10 min. Closely examining 17 milk-based samples using cdELISA revealed that four were slightly contaminated with AFM1 at concentrations from 0.002 to 0.054 ng/mL. All milk samples were negative in the immunostrip test because the levels of contaminant were below the detection limit of the strip. Notably, the presented cdELISA and immunostrip methods are highly sensitive methods for detecting AFM1 in milk.