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Enzyme immunoassay for the detection of aflatoxin B1

(code MA222/MA224)
B ZERO AFLA B1 is an ELISA kit prepared for an or samples are mixed and then transferred into the anti-
immunoenzymatic assay for the quantitative analysis of aflatoxin B1microtiter plate. During the first incubation, free
aflatoxin B1. aflatoxin in the sample and enzyme-labelled aflatoxin
The kit contains the procedure and the materials sufficient for compete for the anti-aflatoxin antibody binding sites on the
96 determinations (code MA224) or 48 determinations (code solid phase. Any unbound enzyme conjugate and aflatoxin
MA222) including standards. molecule are then removed in a washing step. The bound
A microtiter plate photometer or a strip photometer is enzyme activity is determined adding a fixed amount of a
required. chromogenic substrate. The enzyme converts the colourless
chromogen into a blue product. The addition of the stop
Analysable samples reagent leads to a colour change from blue to yellow. The
Cereals, high moisture corn, silage and mash, feed, nuts, dried absorbance is measured with a microplate reader at 450 nm.
fruits, DDGS, cottonseed. The colour development is inversely proportional to the
aflatoxin B1 concentration in the sample.
Sample preparation
- Cereals, high moisture corn, silage and mash, feed, nuts, 2 PROVIDED REAGENTS
dried fruits, DDGS: grinding and homogenization, extraction Premixing microtiter plate: non-coated wells, blank.
in methanol-water, filtration Code MA224: 96 wells (12 strips of 8 wells).
- Cottonseed: grinding and homogenization, extraction in Code MA222: 48 wells (6 strips of 8 wells).
methanol-water, filtration, purification on EasypurAFLA Microtiter plate: coated with anti-aflatoxin antibody, in an
aluminium bag with a desiccant bag.
Assay time: 15 minutes (sample preparation not included). Code MA224: 96 wells (12 strips of 8 wells)
Code: MA222: 48 wells (6 strips of 8 wells).
Detection limit As the strips are breakable, the wells can be used individually.
1 ppb For this purpose, it is sufficient to get out the wells from the
frame and to break the joint.
Specificity Std 0: 1 amber plastic vials containing 0 ppb of aflatoxin B1.
ATTENTION: only the standard zero is provided. B/B0
Compound Cross-reactivity % values of calibration curve (1-40 ppb) are reported in the kit
certificate of conformity.
Aflatoxin B1 100 Code MA224: 3 ml, code MA222: 1,5 ml.
Enzyme conjugate: 1 amber plastic, code MA224: 14 ml, code
Aflatoxin B2 5±1 MA222: 8 ml.
Washing-buffer 10x: 1 plastic bottle containing 50 ml.
Aflatoxin G1 19 ± 1 Developing solution: 1 amber plastic bottle, code MA224:
15ml, code MA222: 8 ml.
Aflatoxin G2 <1 Stop solution: 1 glass vial, code MA224: 9 ml, code MA222:
6 ml. White cap.

1 TEST PRINCIPLE 3 REQUIRED BUT NOT PROVIDED MATERIALS

The assay is performed in plastic microwells that have been - Distilled water
coated with anti-aflatoxin B1antibody. In the premixing wells - Methanol
the enzyme labelled aflatoxin and the zero standard solution
1
_________________________________________________________
Tecna S.r.l. - Area Science Park - Padriciano 99, Trieste, Italy V.5 – 01/14
tel: +39 040 3755341; fax +39 040 3755343
e-mail: export@tecnalab.com web: http://www.tecnalab.com
_______________________________________________________________________________________________________________________________________________
- NaCl (for cereals, high moisture corn, silage and mash,
feed, nuts, dried fruits, cottonseed).
- Equipment
- Balance
- Mill (grinding)
- Shaker or vortex, or blender (like “Osterizer”)
- Filter paper (Whatman 1)
- EasypurAFLA (code AC010; for cottonseed)
- Centrifuge for 15 ml tubes (optional; for cottonseed)
- 20-200 µl micropipettes with tips
- 50-300 µl multichannel micropipette with tips
- Microtiter plate or strip reader equipped with a 450 nm
filter.
4 WARNING AND PRECAUTIONS FOR THE USERS
- The product is for in vitro diagnostic use only.
- Some reagents contain preservative. The stop solution
contains sulphuric acid and is corrosive. The zero
standard solution is toxic and inflammable because of
methanol.
- Handle the reagents with caution, avoiding contact with
skin, eyes and mucous membranes.
- Safety data sheet are available on Tecna’s web site.
5 HANDLING AND STORAGE INSTRUCTIONS
- Store the kit at +2/+8 °C and do not freeze components.
- Reseal the unused strips of the anti-aflatoxin B1microtiter
plate in the bag together with the desiccant bag provided.
- Do not use components after the expiration date.
- Do not intermix components from different kit lots.
- Do not use photocopies of the instruction booklet. Keep
always the instruction booklet that is included inside the
kit.
6 SAMPLES PREPARATION
6.1 Cereals and feed
- Mix carefully the sample to be analysed in order to make
it homogeneous.
- Finely grind the sample.
- Weigh 50 g of ground sample and add 10 g of NaCl. Add
250 ml of a solution of 70% methanol in distilled water.
Alternatively: weigh 5 g of ground sample and add 1 g
of NaCl. Add 25 ml of a solution of 70% methanol in
distilled water.
- Blend or shake thoroughly for 3 minutes.
- Filter the sample (Whatman 1) and collect the filtrate.
If the sample is dosed >40 ppb, dilute the extract 5 times
(1+4) in methanol 70%, in order to obtain a dosage range 5-
200 ppb.
It is suggested to weigh 50 gr in order to have a more
representative analysis of the sample.
6.1.1 High moisture corn
- Extract samples according to point 6.1 procedure.
- To relate result to dry matter, take into account the
sample moisture percentage.
_________________________________________________________
Tecna S.r.l. - Area Science Park - Padriciano 99, Trieste, Italy
tel: +39 040 3755341; fax +39 040 3755343
e-mail: export@tecnalab.com web: http://www.tecnalab.com
6.1.2 Silage and mash
- Extract samples according to point 6.1 procedure. Adjust
extracts pH to 6.5-7.5 with 5M NaOH (1-10 µl).
- If dry samples are analysed, it is suggested to dry them at
temperatures not higher than 60°C.
6.2 Nuts
- Mix carefully the sample to be analysed in order to make
it homogeneous.
- Finely grind the sample.
- Weigh 50 g of ground sample and add 10 g of NaCl. Add
250 ml of a solution of 60% methanol in distilled water.
Alternatively: weigh 5 g of ground sample and add 1 g
of NaCl. Add 25 ml of a solution of 60% methanol in
distilled water.
- Blend or shake thoroughly for 3 minutes.
- Filter the sample (Whatman 1) and collect the filtrate.
It is suggested to weigh 50 gr in order to have a better
representative analysis of the sample.
6.3 Dried fruits
- Finely mince the sample.
- Weigh 5 g of minced sample.
- Add 0,5 g of NaCl.
- Add 25 ml of a solution of MeOH 80% in distilled water
and mix thoroughly for 3’ using a high-speed blender or
for 15 minutes using a low speed shaker.
- Filtrate the sample (Whatman 1) or centrifuge at 3500xg
for 5’; recover the supernatant /filtrate.
6.4 DDGS
- Mix carefully the sample to be analysed in order to make
it homogeneous.
- Finely grind the sample.
- Weigh 50 g of ground sample and add 250 ml of a
solution of 70% methanol in distilled water.
Alternatively: weigh 5 g of ground sample and add 25 ml
of a solution of 70% methanol in distilled water.
- Blend or shake thoroughly for 15 minutes.
- Filter the sample (Whatman 1) and collect the filtrate.
- If the sample is dosed >40 ppb, dilute the extract 5 times
(1+4) in methanol 70%, in order to obtain a dosage range
5-200 ppb.
6.5 Cottonseed
- Mix carefully the sample to be analysed in order to make
it homogeneous.
- Finely grind the sample.
- Weigh 50 g of ground sample and add 10 g of NaCl. Add
250 ml of a solution of 70% methanol in distilled water.
ATTENTION: for a more representative analysis of the
sample, it is suggested to do not extract lower amounts.
- Blend or shake thoroughly for 3 minutes.
- Filter the sample (Whatman 1) and collect the filtrate. If
filtration turns out to be difficult, let the sample settle and
take the supernatant.
- Add 2 ml of filtrate/supernatant to an EasypurAFLA tube
(code AC010).
- Mix vigorously for 30 seconds. Take care to resuspend
the gel completely.
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- Centrifuge 5 minutes at 1500xg or let the gel settle for 10 2. Add 100 µl of enzyme conjugate in each
minutes. The clear supernatant is ready for analysis; it is premixing well.
suggested to transfer it into a 1.5 ml tube in order to avoid
the gel resuspension during the transferring of the extract 3. Add 50 µl of standard 0 and samples into the
into the microtiter plate. corresponding premixing wells. The
standard/sample contain high percentage of
7 WORKING SOLUTIONS PREPARATION methanol: take care to rinse the tip pipetting up
Std 0: ready to use.
Enzyme conjugate: ready to use.
and down the solutions before adding to the
Washing buffer: dilute the concentrate 1:10 (1+9) with wells.
distilled water; ATTENTION: in presence of crystals, bring 4. Using the micropipette, mix the content of each
the solution at room temperature and stir in order to solve premixing well (pipette up and down three
them completely.
times) and immediately transfer 100 µl into the
The diluted washing buffer is stable at room temperature for
24 hours and at +2/+8°C for two weeks. corresponding anti-aflatoxin B1 antibody coated
Developing solution: ready to use; this solution is light microwell.
sensitive: keep away from direct light; ATTENTION: use new tips for each well to
Stop solution: ready to use. Attention: it contains 2 M avoid cross-contamination.
sulphuric acid. Handle with care and in case of contact wash
5. Incubate 10 minutes at room temperature;
thoroughly with tap water.
Do not prolong the first incubation time and do
8 ASSAY PROCEDURE
not shake during incubation .
8.1 Preliminary comments 6. Washing sequence
- Bring all reagents to room temperature before use, and - At the end of incubation, pour the liquid out
keep them at room temperature at least for an hour. from the wells.
- Return all reagents to +2/+8 °C immediately after use. - Fill completely all the wells with washing
- Do not change the assay procedure, in particular:
- do not prolong the first incubation time;
buffer 1x using a squeeze bottle. Pour the
- do not incubate the plate at a temperature higher than liquid out from the wells. Repeat the washing
25°C or lower than 18°C; sequence for a total of three times.
- do not shake the plate during the incubations; - Remove the remaining droplets by tapping
- use for dispensing accurate and precise micropipettes the microplate upside down vigorously
with suitable tips.
- Once started, complete all the steps without interruption.
against absorbent paper.
- The reproducibility of ELISA results depends largely Do not allow the wells to dry out.
upon the efficiency and uniformity of microwells 7. Developing
washing; always keep to the described procedure. − Add 100 µl of development solution to each
- Use a single disposable tip for the zero standard and each
well and mix thoroughly with rotatory motion
sample to avoid cross-contamination.
- Do not allow tips to contact the liquid already in the for few seconds;
microwells. 8. Incubate for 5 minutes at room temperature.
- Avoid direct sunlight during all incubations. It is 9. Add 50 µl of stop solution to each well
recommended to cover the microtiter plate without using micropipette and mix thoroughly with rotatory
sealing tapes.
motion for few seconds.
8.2 Assay procedure 10. Measure the absorbance at 450 nm. Read within
1. Predispose the assay layout, taking into account 15 minutes.
that one well is required for zero standard and 9 RESULTS CALCULATION
each sample; remove the wells not to be used − Divide the absorbance value of each sample by the
from the anti-aflatoxin B1 microtiter plate and absorbance of the Standard 0 (B0) and multiply by 100;
replace them in the pouch with the desiccant gel the Maximum Binding (B0) is thus made equal to 100%
and reseal the pouch very well using the clump and the absorbance values are quoted as percentage:
provided.
Prepare an equal number of premixing wells. sample absorbance B
---------------------------------------- X 100 = ----
ATTENTION: it is suggested to carry out no Standard 0 (B0) absorbance B0
more than 16 determinations in each assay
(standard 0 included). - Enter the B/B0 provided for each standard (1; 5; 20; 40
ppb) in the kit lot conformity certificate in a semi-
3
_________________________________________________________
Tecna S.r.l. - Area Science Park - Padriciano 99, Trieste, Italy
tel: +39 040 3755341; fax +39 040 3755343
e-mail: export@tecnalab.com web: http://www.tecnalab.com
_______________________________________________________________________________________________________________________________________________

logarithmic system of coordinates against the aflatoxin B1

standard concentration and draw the standard curve.
- Take the B/B0 value for each sample and interpolate it to
the corresponding concentration in the calibration curve.
Standards concentration (ppb) already considers the
sample dilution factor.
Please note: For the calibration, use the “point to point”
curve; Excel spreadsheets are available on Tecna website
www.tecnalab.com and can be downloaded directly from the
bottom of the product page upon registration.

10 RESULTS EVALUATION
After results elaboration, it is necessary to verify the assay
performance. The verification is performed by comparison of
obtained data with those given in kit specifications (paragraph
11). If the values are out from the specifications given, it is
advised to control the expiry date of the kit, the wavelength of
absorbance recording, as well as the procedure employed. If
operation errors do not emerge, contact our technical
assistance. WARNING: substitution will be possible just in
case of rendered kit. The kit must be conserved in its integral
version and at the temperature indicated in this booklet.

11 KIT SPECIFICATIONS
11.1 Assay specification
Bo absorbance > 0,7 OD450nm

11.2 Assay performance

Matrix Cut off LOQ

ppb ppb
Maize <1 2
Hazelnut <1 1
Pistachio nut <1 1
Raisins <1 2
Figs <1 2

12 BIBLIOGRAPHY
G. Rosar, L. Persic, V. Bassani, F. Gon, C. Ranieri, F. Diana.
Effects of "master curve calibration" on the performances of
some ELISAs for food contaminants. Poster presentation at
6th International Symposium on Recent Advances in Food
Analysis, 2013, November 5-8, Prague, Czech Republic.

13 LIABILITY
Samples evaluated as positive using the kit have to be re-
tested with a confirmation method.
Tecna shall not be liable for any damages to the customer
caused by the improper use of the kit and for any action
undertaken as a consequence of results.
Tecna shall not be liable for the unsafe use of the kit out of the
current European safety regulations.

4
_________________________________________________________
Tecna S.r.l. - Area Science Park - Padriciano 99, Trieste, Italy
tel: +39 040 3755341; fax +39 040 3755343
e-mail: export@tecnalab.com web: http://www.tecnalab.com