Aflatoxin M1 is the product of aflatoxin B1 transformation in the digestive apparatus of dairy animals. Once these animals are fed with B1 contaminated feed, liver enzymes transform it into aflatoxin M1. This toxin is found then in milk and dairy products and, even if less toxic than its precursor, is still mutagenic and carcinogenic. Due to aflatoxin M1 toxicity and the likelihood of its presence in milk and dairy products, the level of this toxin is strictly regulated from European and International laws. In the EU, the maximum tolerated level of aflatoxin M1 is 0.05 parts per billion (ppb), and a lower limit of 0.025 ppb is envisaged for so called baby-foods. In the US, the Food and Drug Administration (FDA) established a maximum level for aflatoxin M1 of 0.5 ppb; China –with GB 2761-2011- and the International CODEX ALIMENTARIUS adopted the same limit as well. Tecna presents here its portfolio of products dedicated to aflatoxin M1 screening in milk and dairy products. In order to fulfill international regulations new kits for extra EU markets have been recently introduced.

1. Tecna aflatoxin M1 screening tests
Complete port-folio of screening kits
for determination of Aflatoxin M1
in milk and dairy products

2. Tecna aflatoxin M1 screening tests
Aflatoxin M1
• Aflatoxin M1 is a mycotoxin that naturally occurs in milk and dairy products; it
is the product of aflatoxin B1 after it is metabolized by dairy animals.
• Aflatoxin M1 is less carcinogenic than its parental toxin, aflatoxin B1,
however it is still highly toxic for liver and kidneys in particular.
• 1-3% of the aflatoxin that is consumed within feed is excreted in milk within
24 hours from intake.
Clearance time ranges between 1 and 4 days.

3. Tecna aflatoxin M1 screening tests
Different options for different regulations
Different requirements can’t be adequately fulfilled by a single product. For this
reason Tecna implemented a complete port-folio of screening tests to cover
all the requirements.
The EU limit for aflatoxin
M1 in milk is 50 ppt.
• I’screen Afla M1 MILK
• I’screen Afla M1
The Codex Alimentarius
and the FDA fixed the
limit for aflatoxin M1 in
milk at 500 ppt
• Celer® Afla M1 500
• B ZERO AFLA M1 500

4. Tecna aflatoxin M1 screening tests
Product overview
• Assay principle: competitive enzyme immunoassay
• Applications: raw milk, milk powder
• Assay time: 75 minutes
• Calibration curve: 5 – 250 ppt
• Limit Of Detection
– Raw milk: 0.005 ppb = 5 ppt
– Milk powder: 0.05 ppb = 50 ppt
The leading
solution in
Italy and
Europe for the
screening of
aflatoxin M1 in
milk.
I’screen AFLA
M1 milk is
accreditable in
compliance
with ISO IEC
14675:2003
No sample
manipulation
required!

5. Tecna aflatoxin M1 screening tests
• Assay principle: competitive enzyme immunoassay
• Applications: raw milk, milk powder, cheese, yogurt
• Assay time: 75 minutes
• Calibration curve: 5 – 250 ppt
• Limit Of Detection
– Raw milk: 0.005 ppb = 5 ppt
– Milk powder: 0.05 ppb = 50 ppt
– Cheese: 0.037 ppb = 37 ppt
– yogurt: 0.025 ppb = 25 ppt
The leading
solution in
Italy and
Europe for the
screening of
aflatoxin M1 in
milk.
I’screen AFLA
M1 is
accreditable in
compliance
with ISO IEC
14675:2003
Product overview

6. Tecna aflatoxin M1 screening tests
Milk validation
• Each prototype developed by Tecna R&D department is subject to internal
validation before industrialization and production.
• Of 20 samples with < 5 ppt of aflatoxin M1 (HPLC), Tecna kits revealed no
positives: 100% specificity
• Of 20 samples spiked at 5 ppt of aflatoxin M1, no determination with Tecna
kits resulted < 5 ppt: 100% sensitivity
• Kit precision was set onto two Reference milk materials:
• 21.50 ppt: Intra-assay CV 3.7% - Inter-assay CV 8.0%
• 55.19 ppt: Intra-assay CV 2.8% - Inter-assay CV was 8.5%

7. Tecna aflatoxin M1 screening tests
I’screen AFLA M1 performances
10 blank low-fat and
10 blank whole
yogurt samples were
run with theoretical
cut off < 25 ppt.
Tecna kit revealed
no positives: 100%
specificity
The same 20
samples were
spiked at 40 ppt of
aflatoxin M1, no
determination with
Tecna kit resulted in
a false negative:
100% sensibility
To test the kit
accuracy a low fat
and a whole yogurt
samples samples
were randomly
selected and spiked
at 50, 100 and 250
ppt of aflatoxin M1.
The resulting mean
recovery was
81+11%
Yogurt
50 different incurred
cheese samples
were analysed by
HPLC and by
I’Screen AFLA M1
ELISA kit. The mean
recovery value was:
108+15%
A specific sample
procedure was
implemented for firm
cheese; the mean
recovery obtained by
analysing incurred
samples was
116+15%
Z-scores obtained in
two different
Proficiency tests for
cheese were - 0.21
and 0.17.
Cheese

8. Tecna aflatoxin M1 screening tests
It is important to monitor the kit performances. As a producer, Tecna performs a
strict quality control before releasing each batch of I’screen AFLA M1 milk, by
analysing at least three reference milk materials in three sessions, three
replicates each.
Quality control
Acceptance criteria
68.3% results must
not exceed 1 standard
deviation of the
reference sample
specs
Intra-assay CV < 15%
Inter-assay CV < 20%

9. Tecna aflatoxin M1 screening tests
In order to constantly monitor immunoassays performances, Tecna participates
every year to various proficiency tests from different suppliers.
Participation to proficiency tests
Since 2005, the
average z-scores
Tecna obtained with
its aflatoxin M1 kits
is 0.66 (n=35)

10. Tecna aflatoxin M1 screening tests
Product overview
• Assay principle: competitive enzyme immunoassay
• Applications: raw milk, milk powder
• Assay time: 20 minutes
• Calibration curve: 25 – 500 ppt (up to 100 – 2000 ppt)
• Limit Of Detection
– Raw milk: 0.025 ppb = 25 ppt
– Milk powder: 0.25 ppb = 250 ppt
A new FAST
screening
device for the
quantitative
detectin of
aflatoxin M1 in
milk
No sample
manipulation
required!

11. Tecna aflatoxin M1 screening tests
Product overview
• Assay principle: competitive enzyme immunoassay
• Applications: raw milk, milk powder
• Assay time: 20 minutes
• Calibration curve: 25 – 500 ppt (up to 50 – 1000 ppt)
• Limit Of Detection
– Raw milk: 0.025 ppb = 25 ppt
– Milk powder: 0.25 ppb = 250 ppt
A new FAST
screening
device for the
quantitative
detectin of
aflatoxin M1 in
milk.
B ZERO AFLA
M1 500 is the
master curve
version of
Celer® AFLA
M1 500
No sample
manipulation
required!
Only one
standard for
the
calibration
curve: more
analysis with
the same kit!

12. Tecna aflatoxin M1 screening tests
• Assay principle: competitive enzyme immunoassay
• Applications: raw milk, milk powder
• Assay time: 30 minutes
• Calibration curve: 10 – 200 ppt
• Limit Of Detection
– Raw milk: 0.01 ppb = 10 ppt
The first
master curve
ELISA kit for
the detection
of aflatoxin M1
in milk within
EU MRL.
No sample
manipulation
required!
Only one
standard for
the
calibration
curve: more
analysis with
the same kit!
Product overview

13. Tecna aflatoxin M1 screening tests
Focus on B ZERO AFLA M1
• The B ZERO line of Tecna kits is
dedicated to who implements few
analysis.
• The B ZERO AFLA M1 ELISA kit leads
to quantitative results by means of a
VIRTUAL MASTER CURVE – provided
within each batch – in 20 minutes only!
• At each analytical session, the analyst
can save the wells dedicated for each
calibrator; it is sufficient to run just the
ZERO STANDARD and the SAMPLES!
To analyze 3 samples, in a usual immunoassay at least 5
wells have to be used for calibrators.
In the B ZERO format, only 1 well has to be used to run the
ZERO standard.

14. Tecna aflatoxin M1 screening tests
Why Tecna?
• Tecna provides more information to check if the result of each assay is valid
or not.
• Tecna organizes theoretical and practical trainings to its customers.
• Tecna supports customers on the road to Quality Assurance of screening
results (ISO17025).
Visit us at
www.tecnalab.com!