Transia Plate Gluten - IUL Instruments GmbH

Transia Plate
Gluten Art. No: GL0301 - 1 plate
Intended use
Transia Plate Gluten is intended to be used for the
detection and quantification of wheat gliadin and related
prolamins from triticale, rye and barley in food products.
The assay does not cross-react with proteins from soya,
maize, rice and oats.
Gluten modified by chemical or enzymatic means are
detectable although the results are not quantifiable. The
gluten level of “toasted” samples such as breadcrumbs
is reduced in proportion to the degree of charring.
A tannin-binding additive (AK 0301) should be used for
chocolate, cocoa and coffee to ensure that the detection
of gluten is not inhibited.
Assay principle
The test is designed to be used either as a quantitative
or semi-quantitative assay.
The detection is based on a direct sandwich ELISA
(Enzyme Linked Immuno Sorbent Assay). The solid
support of the reaction is a microtiter plate coated with
anti-gliadin antibodies.
Materials
Kit components
• Microtiter plate: divisible strips 96 wells (12 strips x 8
wells): coated with anti-gliadin antibodies - 1 pc
• Vial 1: starch A (LOW) – the gluten concentration is
stated on the label - 1 x 6 g
• Vial 2: starch B (MEDIUM) – the gluten concentration
is stated on the label - 1 x 6 g
• Vial 3: starch C (HIGH) – the gluten concentration is
stated on the label - 1 x 6 g
• Vial 4: dilution buffer - concentrated 10X - 1 x 50 mL
• Vial 5: gliadin standard - lyophilised
• Vial 6: washing buffer - concentrated 20X - 1 x 60 mL
• Vial 7: concentrated conjugate – anti-gliadin antibody
conjugated to peroxidase - the concentration is stated
on the label - 1 x 0.8 mL
• Vial 8: substrate: hydrogen urea peroxide - 1 x 7 mL
• Vial 9: chromogen: TMB - 1 x 7 mL
• Vial 10: stop solution: H 2
SO 4
- ready to use - 1 x 7 mL
Equipment required but not provided
For sample and reagent preparation:
• Blender or homogeniser for 10-50 mL volumes e.g.
Ultra-turrax, Polytron, Ystral
• Magnetic stirrer
• Vortex
• Benchtop centrifuge – 2,500 rpm, suitable for 10 mL
or larger polypropylene or glass centrifuge tubes
• 5 mL and 10 mL (or larger) test tubes – use glass or
plastic with low protein-binding properties e.g.
polypropylen
• Scales and weighing vessels
• Micropipettes 100 -1000 µL
• Eppendorf multipipette with 5 and 2.5 mL combitips
• 5 mL and 10 mL graduated pipettes
• 500 mL and 2 L graduated cylinders
• 100 mL graduated cylinder
• Bottles for the diluted washing buffer (1.2 L) and the
dilution buffer (500 mL)
• 100 mL bottle for the 40% ethanol/water solution
For the preparation of additional washing buffer:
• Volumetric flask 1 L, glass flasks 1 L
• pH-meter
For the immunoenzymatic test:
• Vortex
• Wash bottle or automatic microplate washer
• Absorbent paper
• Micropipette 10-100 µL
• Microplate reader with 450 nm filter.
Material required but not provided
For sample preparation:
• Ethanol (analytical grade)
• Distilled water
For the preparation of additional washing buffer:
If the volume is insufficient for automatic washing systems,
the washing buffer 1X can be prepared by mixing
PBS and Tween (see Preparation of reagents).
• PBS: NaCl 3.8 g
Na 2
HPO 4
, 2H 2
O 0.36 g
KH 2
PO 4
0.11 g
H 2
O
1 L
• Tween 20 (SIGMA: ref P1379) 0.5 mL
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Gluten
Storage conditions
The kit components should be stored at 2-8°C.
Safety
Good laboratory practice should be employed when
using this kit. Safety clothing should be worn and skin
contact with reagents avoided. Do not ingest.
If any reagent comes into contact with eyes or skin,
rinse immediately with plenty of water. Do not pipette by
mouth.
Material and safety data sheets are available on request.
Test procedure
The test is designed to be used either as a quantitative
or semi-quantitative assay. The quantitative assay is
based on a standard curve with optical density (OD)
plotted versus gliadin concentration. In the semi-quantitative
assay reference starches are used to determine
if the gluten content of the sample is above or below
that of the reference starch. Both procedures are described
below.
The use of reference starches in the quantitative assay
is optional, but if used they will give an indication of the
efficiency of the extraction procedure.
Preparation of reagents
• Important: Allow the reagents to reach room temperature
(18-25°C). Remove them from the box at least
one hour before use.
• Have all the reagents and samples ready for use so
that the various materials can be added to the wells
without delay.
• Shake each vial manually or with a Vortex before use.
Preparation of the washing buffer 1X
Use 600 mL of diluted washing buffer to wash six strips.
The preparation could be done in advance or during the
first incubation step. See Assay procedure, step 3.
1. Dilute the washing buffer 20X (vial 6) in distilled
water 1:20 – 60 mL of washing buffer 20X and 1140
mL of distilled water.
2. Mix and fill the washing bottle.
3. Store the washing buffer 1X at 2-8°C for a maximum
of three months.
Preparation of additional washing buffer 1X
1. Dissolve the different components (see Material
required but not provided) in a one-litre beaker with
approximately 800 mL of distilled water and agitate.
2. Check the pH (7.2 ± 0.1) and complete to one litre in
a volumetric flask.
3. Transfer to a flask and label. Store at 2-8°C for a
maximum of three months.
Preparation of the 40% ethanol/water solution
Prepare a 40% ethanol/water solution by mixing 40 mL
of ethanol with 60 mL of distilled water. Note: measure
the ethanol and the water separately before mixing.
Store in a 100 mL bottle. Prepare fresh 40% ethanol/
water solution for each run.
Preparation of the dilution buffer 1X
Prepare only the volume necessary for the test, it is not
advisable to store dilution buffer 1X.
Dilute the dilution buffer 10X (vial 4) in distilled water
1:10 – 50 ml of dilution buffer 10X and 450 mL of distilled
water.
Mix gently.
Preparation of the reference starches for
a semi-quantitative assay
1. Weigh 1 g of each reference starch (vials 1-3) into
separate labelled containers.
2. Add 10 mL of 40% ethanol/water solution.
3. Homogenise for 30 seconds using homogeniser, or for
30 minutes using a magnetic stirrer.
4. Centrifuge at 2500 rpm for 10 minutes at room temperature,
18-25°C.
5. Collect the liquid extract of each reference starch and
dilute it as described below.
Dilute starch A 1/50
Add 100 µL of extract A to 4.9 mL of dilution buffer
1X.
Dilute starch B 1/250
First dilute the extract B 1/50, by adding 100 µL of
extract to 4.9 mL of dilution buffer 1X.
Then add 1 mL of dilution 1/50 to 4 mL of dilution
buffer 1X.
Dilute starch C 1/1000
First dilute the extract C 1/50, by adding 100 µL of
extract to 4.9 mL of dilution buffer 1X.
Then prepare a 1/250 dilution, by adding 1 mL of
dilution 1/50 to 4 mL of dilution buffer 1X.
Finally, add 1 mL of dilution 1/250 to 3 mL of dilution
buffer 1X.
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Preparation of the standards for
a quantitative assay
1. Reconstitute the gliadin standard by adding the
volume of 40% ethanol/water solution stated on the
vial label.
2. Gently mix by inversion or vortex until the powder is
completely dissolved. Store at 2-8°C for a maximum
of three months. The reconstituted concentrated
gliadin standard contains 500 µg gliadin /mL.
3. Prepare the following dilutions:
Dilution 1: 5 µg gliadin /mL
100 µL concentrated gliadin standard [500 µg /mL] +
9.9 mL dilution buffer 1X
Dilution 2: 0.80 µg gliadin /mL
400 µL dilution 1 + 2.1 mL dilution buffer 1X
Dilution 3: 0.20 µg gliadin /mL
400 µL dilution 2 + 1.2 mL dilution buffer 1X
Dilution 4: 0.16 µg gliadin /mL
400 µL dilution 2 + 1.6 mL dilution buffer 1X
Dilution 5 - 0.08 µg gliadin /mL
1 mL dilution 4 + 1 mL dilution buffer 1X
Dilution 6: 0.04 µg gliadin /mL
1 mL dilution 5 + 1 mL dilution buffer 1X
Dilution 7: 0.02 µg gliadin /mL
1 mL dilution 6 + 1 mL dilution buffer 1X
Dilution 8: 0.01 µg gliadin /mL
1 mL dilution 7 + 1 mL dilution buffer 1X
Dilutions 3 to 8 (0.2-0.01 µg gliadin /mL) are used as
standards in the assay. The standards must be prepared
for each run.
Dilution of conjugate
The concentration of the concentrated conjugate supplied
with the kit varies from batch to batch. The concentration
is always stated on the label of the vial (nX).
The dilution of the conjugate should not be prepared in
advance, it is done during the first incubation of the
assay. See Assay procedure, step 3.
Dilute the conjugate according to the concentrationfactor
given on the label on vial 7. For one plate, 12 mL
of diluted conjugate is prepared. If, for example, the
concentration is given to 40X on the vial label then mix
11.7 mL of dilution buffer 1X with 300 µL of conjugate
40X (vial 7).
Swirl gently to ensure mixing without generating foam.
Mixing of chromogen and substrate
• The mixture of chromogen and substrate can not be
prepared more than two hours in advance due to the
potential loss of activity. It is done during the
second incubation of the assay. See Assay
procedure, step 6.
• Mix 60 µL of substrate (vial 8) and 60 µL of chromogen
(vial 9) for each well used.
Preparation of samples
To avoid cross-contamination during extraction rinse the
blender or homogeniser with 40% ethanol/water solution
between each sample.
The extraction should be done the same day as the
immunoassay, store the extract at room temperature.
Extraction (general procedure)
1. Weigh 1 g of the sample into labelled containers.
2. Add 10 mL of 40% ethanol/water solution.
3. Homogenise for 30 seconds using an homogeniser, or
for 30 minutes using a magnetic stirrer.
4. Centrifuge at 2,500 rpm for 10 minutes at room temperature
(18-25°C).
5. Collect the liquid extract and dilute it as described
below (see Dilution of the extract).
Extraction – hard samples (cookies, noodles…)
1. Weigh 5 g of the sample.
2. Grind it into a powder.
3. Weigh 1 g of powder into a labelled container.
4. Add 10 mL of 40% ethanol/water solution.
5. Homogenise for 30 seconds using an homogeniser, or
for 30 minutes using a magnetic stirrer.
6. Centrifuge at 2,500 rpm for 10 minutes at room temperature
(18-25°C).
5. Collect the liquid extract and dilute it as described
below (see Dilution of the extract).
Extraction – meat, meat products
When the gluten is not evenly distributed a larger
sample is necessary. The ratio 1:10 (sample:extracting
solution) should be kept.
1. Weigh 10-20 g of the sample.
2. Add 100-200 mL of 40% ethanol/water solution.
3. Homogenise for 30 seconds using an homogeniser, or
for 30 minutes using a magnetic stirrer.
4. Centrifuge at 2,500 rpm for 10 minutes at room temperature
(18-25°C).
5. Collect the liquid extract and dilute it as described
below (see Dilution of the extract).
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Dilution of the liquid extract
The recommended dilutions for different types of food
are found in Table 1. In order to reduce the risk of
matrix interference a sample needs to be diluted at
least 1/50 before performing the assay.
The standard curve will give an accurate reading within
the range of the standards. If the sample has a concentration
outside this range it will need to be diluted further.
We recommend that you make at least two dilutions
for each sample.
For a semi-quantitative test and for unknown samples
prepare the dilutions 1/50, 1/250 and 1/1,000.
Note that this type of aqueous ethanol solution, such as
the liquid extract, is very difficult to pipette accurately.
After dispensing the extract into the dilution tube, rinse
the tip several times up and down with the buffer in the
dilution tube.
Dilution 1/50
Add 100 µL of the extract to 4.9 mL of the dilution buffer
1X.
Dilution 1/250
Add 1 mL of dilution 1/50 to 4 mL of the dilution buffer
1X.
Dilution 1/1,000
Add 1 mL of dilution 1/250 to 3 mL of the dilution buffer
1X.
Dilution 1/10,000
Add 1 mL of dilution 1/1,000 to 9 mL of the dilution
buffer 1X.
Dilution 1/20,000
Add 1 mL of dilution 1/10,000 to 1 mL of the dilution
buffer 1X.
TABLE 1
Sample Expected Dilution
gluten content
Starch quality control
or “gluten-free” products
e.g. dietary bread,
baking mix, baby food, 1/50
milk drink 0-0.02% to 1/1,000
Soup, processed meat,
analgesic, icing sugar
1/1,000 to
mix, pet food 0.1-2.0% 1/10,000
Biscuits, flour mixes,
bread crumb
preparations, noodles, 1/10,000
pasta >2.0% and higher
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Assay procedure
Important: Allow the reagents to reach room temperature (18-25°C). Remove them from the box
at least one hour before use. Shake the reagent vials before use.
Do not interchange reagents between kits with different batch numbers.
The washing step is very important. When washing, direct a strong beam at the bottom of the
well.
1. Attach the required number of strips to the plate: two wells for the negative control and two
wells for each dilution of sample extracts.
For a semi-quantitative assay add two wells for each reference starch.
For a quantitative assay add two wells for each standard. Note that only dilutions 3-8 are
used as standards.
Return the unused strips to the zip-lock bag containing dehydrating agent and close it tightly.
Write the position of the controls, standards and samples on the work sheet (ENR COM 113).
2. Distribute 100 µL of the negative control (use dilution buffer), standards
and/or reference starches and dilutions of sample extracts into the assigned detection wells.
3. Incubate at room temperature (18-25 o C) for 40 minutes. Note that the incubation starts
once the last well has been filled. Prepare the washing buffer and the conjugate solution - see
Preparation of reagents.
4. Empty the plate over a container and shake out the remaining contents by briskly flicking
your wrist. Rinse each well, keep the washing buffer in the plate for 5-10 seconds and then
remove it by inverting the plate over the container and shaking out the remaining contents.
Repeat the washing four times. At the end of the washing, drain the plate on absorbent paper,
tapping it firmly.
5. Add 100 µL of diluted conjugate to each well using a multipipette. Be careful not to touch the
wells with the tip. Mix the contents of the wells using a gentle circular motion.
6. Incubate at room temperature (18-25 o C) for 40 minutes. Note that the incubation starts
once the last well has been filled. Prepare the chromogen/substrate mixture - see Preparation
of reagents.
7. Perform the washing five times as described in step 4.
8. Add 100 µL of the substrate/chromogen mixture to all the wells, preferably using a
multipipette.
9. Incubate at room temperature (18-25 o C) for 30 minutes. Note that the incubation starts
once the first well has been filled. Mix the contents of the wells using a gentle circular motion.
10. Add 50 µL of stop solution (vial 10) to each well, following the same order used when the
substrate/chromogen mixture was added.
Mix the contents of the wells thoroughly to ensure complete colour conversion. The blue turns
to yellow.
11. Read the optical densities using a plate reader at 450 nm (blank on air).
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Result interpretation -
semi-quantitative assay
Validation of the test
The mean optical density (OD) of the negative controls
must be lower than 0.15. If not, the results are
not valid. Repeat the ELISA, taking extra care that all
the reagents are at room temperature and that the
washing step is performed correctly.
Gluten content
As defined by the Codex Alimentarius Commission in
1981 (Codex Standard for “Gluten-free” Foods, STAN
118-198, Food Agriculture Organisation/World Health
Organisation, Rome, Italy), products labelled glutenfree
may not contain more than 200 ppm (0.02%)
gluten.
Compare the mean OD of the sample with the mean
OD of the reference starches.
If the OD of the sample diluted 1/50 is lower than the
OD of starch A then the product may be labelled
“gluten-free”.
If the OD of the sample diluted 1/50 is higher than
the OD of starch A, but the OD of the sample diluted
1/250 is lower than the OD of starch B, then the
gluten level should be measured quantitatively to
determine if the product could be labelled “glutenfree”
or not.
If the OD of the sample diluted 1/250 is higher than
the OD of starch B, but the OD of the sample diluted
1/1,000 is lower than the OD of starch C, then the
label “gluten-free” is not acceptable.
If the OD of the sample diluted 1/1,000 is higher than
the OD of starch C then the label “gluten-free” is not
acceptable.
Result interpretation -
quantitative assay
Validation of the test
The mean OD of the negative controls must be lower
than 0.15.
The standards must meet the following requirements:
Dilution Standard OD
3 0.20 µg/mL ≥ 0.800
4 0.16 µg/mL ≥ 0.650
5 0.08 µg/mL ≥ 0.350
6 0.04 µg/mL ≥ 0.200
7 0.02 µg/mL ≥ 0.100
8 0.01 µg/mL ≥ 0.070
If the standards do not meet the requirements, repeat
the ELISA, taking extra care that all reagents are at
room temperature and that the washing step is performed
correctly.
Gluten content
Using a lin-lin graph paper, draw a standard curve
with the OD of the standards on the y-axis and the
concentration [µg/mL] on the x-axis. An example of a
standard curve is given in Appendix B.
Plot the mean OD of the chosen sample dilution on
the standard curve. Read the gliadin concentration
(G) of the sample dilution.
Note the gliadin concentration on the work sheet
(ENR COM 113) and calculate the gluten content by
using the following equation.
% gluten = G x D x 2 x 10 /10,000
G = gliadin concentration read from the standard
curve
D = dilution factor of the extract (e.g. 50, 250…)
Factor 2 ⇒ it is estimated that 50% of the protein in
gluten is in the form of gliadin.
Factor 10 ⇒ 10 mL of extraction solution is used for
1g of sample
Factor 10,000 ⇒ converts the concentration from
µg/g into %
See Appendix A for conversion from % to ppm.
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Gluten
Performance characteristics
Detection limit
The test is designed to detect down to 10 ppm (or
0.001%) gluten in the samples.
Limitation of standard curve –
- quantitative assay
The standard curve will give an accurate reading
within the range 0.01-0.2 µg gliadin/mL. If the sample
has a concentration outside this range it will need to
be diluted (see Preparation of samples).
Repeatability
The inter-assay coefficient of variation on standard
curves is 10%.
The inter-assay coefficient of variation on gluten
content, including sample preparation (extraction), is
10-30%, depending on the type of food.
Appendices
Appendix A - Units for gluten content
Appendix B - Example of standard curve
Work sheet ENR COM 113
This information is based on our present knowledge
and is intended to provide general notes on our
product and its use. It should not be interpreted as a
guarantee of the specific properties of the product
and its suitability for a particular application.
This kit is manufactured under licence by:
Diffchamb S.A.
8, rue Saint Jean de Dieu
69007 Lyon - France
Tel.: +33 (0)4 72 71 56 80
Fax: +33 (0)4 72 73 43 34
www.diffchamb.com
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Transia Plate
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Appendix A
Units for gluten content
Several different units are used to measure the gluten
content. The following is a simple key for converting
between different units.
mg gluten per kg sample =
= µg gluten per g sample=
= ppm
ppm = 10,000 x [%]
Example: 100 ppm gluten= 0.01% gluten
If the gliadin concentration is given, Factor 2 could be
used to estimate the gluten content.
Example: 50 ppm gliadin ⇒ 100 ppm gluten
Appendix B
Example of a standard curve
2,000
1,500
OD at 450 nm
1,000
0,500
0,000
0,00 0,05 0,10 0,15 0,20
Gliadin Concentration (µg/mL) ( NOT COM 111E 11/00
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