V.1/Aug/2017
Rapid Test Pro for Egg (Cat.# M2231)
Rapid Test Pro for Casein (Cat.# M2232)
Rapid Test Pro for Gluten (Cat.# M2233)
Rapid Test Pro for Buckwheat (Cat.# M2234)
Rapid Test Pro for Peanut (Cat.# M2235)
The Quick Detection for Protein of Allergic Ingredients in Foods and Food-processing Equipment
10 Test Sticks
For Research or Laboratory Use Only.
Not for Use in Diagnostic Procedures.
Please read full descriptions in this manual before use.
Manufactured by:
Morinaga Institute of Biological Science, Inc. (MIoBS) 2-1-16 Sachiura, Kanazawa-ku, Yokohama 236-0003, Japan
E-mail: [email protected]
Website: http://www.miobs-e.com
Intended Use Rapid Test Pro quickly determines whether there are residual proteins of allergic ingredients in foods,
including unprocessed and processed food products. The kit can also be used to detect proteins of
allergic ingredients in swabs and Clean-in-place rinse water (TABLE 1). The kits use antibodies that are
highly specific for particular proteins of allergic ingredients. The novel extraction solution that is included
in the kit enhances the yield of extraction and recovery of proteins from test samples even from highly
processed foods, resulting in more reliable results. Follow the instructions closely to obtain the best
results.
TABLE 1: Performance characteristics
Sample Food Swab Rinse water
Sample preparation time 5-30 min 5-30 min 0-30 min
Chromatographic run time 15 min (25 min for Rapid Test Pro for Buckwheat)
Detection limit*
(Recommended
Method)
Test solution 25 ng/mL in Test Solution
Corresponding value
for test sample
5 μg protein
/g food (5 ppm)
250 ng/mL in
Sample Extract
5 μg /mL in
rinse water
* Detection limit is shown for each protein concentration as follows:
Egg (whole egg protein), Casein (whole milk protein), Gluten (whole wheat protein),
Peanut (whole peanut protein), Buckwheat (whole buckwheat protein).
For Rapid Test Pro for Gluten, the detection limit of gluten in foods is 4 μg gluten/g food using the known
conversion factor of 0.85 ng gluten per ng total whole wheat protein. (Reference 1-4)
Kit Storage 1. Store the kit at 2-8˚C (35-46˚F), and DO NOT FREEZE.
2. Do not use the kit after the expiration date indicated on the box.
Kit Components Kit components are listed in TABLE 2.
TABLE 2: Kit components
Component Amount
Rapid Test Pro
Extraction Solution 10 packs (19 mL/pack)
Diluent ① 1 bottle (12 mL)
Diluent ② 1 bottle (1.2 mL)
Test stick 10 packs (1 stick/pack)
Pipette (L) 10
Pipette (S) 20
Polypropylene tube (L), 50 mL volume 10
Polypropylene tube (S), 1.5 mL volume 10
Cotton swab 10 packs
Paper tube rack 1 piece
Materials required but not provided
● Homogenizer/blender
● Balance
● Vortex mixer
● Water bath
● Centrifuge (for 3000 x g)
● Paper filter
● pH test strip
● Heat-resistant glove
Warnings and Precautions 1. Wear suitable protective clothing, goggles and gloves when handling the kit.
2. All procedures should be performed under contamination-free conditions to obtain reliable
results. Make sure to avoid cross-contaminations via equipments, devices, tubes, containers,
pipette tips, etc., and the use of disposable materials is recommended.
Note 1: The extensive denaturation of proteins during processing can cause the proteins to become non-reactive to
the antibody and/or insoluble, which may result in false-negative results. Therefore, there is a possibility that
such processed foods may still contain denatured proteins of allergic ingredients even though the test results
show negative.
Sample Extraction
(A) Food sample
I. Recommended Extraction Method
1. Grind and mix the test food sample to homogeneity with a contamination-free homogenizer/
blender.
2. Place 1.0 g of the homogenized sample and 1 pack of Extraction Solution in a Polypropylene
tube (L), cap tightly and vortex for 30 seconds.
3. Place the capped tube in 100°C (212F) water for 10 minutes.
4. Place the tube under running water to cool down to ambient temperature. This will take about
10 minutes.
5. Vortex the tube for 30 seconds.
6. Place the tube for few minutes to collect the supernatant as Sample Extract.
Note 2: Check the pH with a pH test strip, and neutralize (pH 6-8) with HCl or NaOH as step 7 if necessary.
Note 3: Centrifuge the tube at 3,000×g for 20 minutes at 20-30°C (68-86F), and collect the supernatant as Sample
Extract if it is difficult to obtain the supernatant by a pipette in step 6.
II. Simplified Extraction Method
In some raw foods or low processed foods, the heating (procedures 3 to 5) can be omitted from the
Recommended Extraction Method.
Note 4: The Simplified Extraction Method is less efficient than the Recommended Extraction Method for protein
extraction and prevention of non-specific reactions. In some samples, the Simplified Extraction Method
may indicate false-negative and/or false-positive results. The Recommended Extraction Method should be
used to test highly processed foods (e.g. cookies). Details are reviewed in our website.
Note 5: Cap the Polypropylene tube (L) tightly so as not to leak out while heating.
(B) Surface swab test sample
I. Recommended Extraction Method
1. Moisten a swab with tap water and thoroughly wipe-off the specified surface area.
2. Place the swab into polypropylene tube (L) containing 1 pack of Extraction Solution,
immerse the swab into the solution, cap the tube, and vortex for 30 seconds to extract
proteins from swab into the Extraction Solution.
3. Place the capped tube in 100°C (212F) water for 10 minutes.
4. Place the tube under running water to cool down to ambient temperature. This will take about
10 minutes.
5. Vortex the tube for 30 seconds. The resulting solution is referred to as Sample Extract.
Note 6: Filter the supernatant with filter paper if necessary in step 5.
Note 7: Check the pH with a pH test strip, and neutralize (pH 6-8) with HCl or NaOH as step 6 if necessary.
Note 8: Cap the Polypropylene tube (L) tightly so as not to leak out while heating.
Extraction Solution
Food sample
Moistened swab
Extraction Solution
2. 3. 4.
5.
1. 3. 2.
4. 5.
(19 mL)
(1.0 g)
(19 mL)
II. Simplified Extraction Method
In some test samples, the heating (procedures 3 to 5) can be omitted from the Recommended
Extraction.
Note 9: Using these methods has a higher possibility of getting a false negative than when using the
Recommended Extraction Method, because the extraction efficiency and reactivity may decrease
depending on the test samples. The Recommended Extraction Method should be used for swab samples
collected from the production line of highly processed foods (e.g. cookies). Details are reviewed in our
website.
(C) Rinse water sample
I. Recommended Extraction Method
Follow the Recommended Extraction Method for food samples. Use 1 mL of rinse water instead of 1 g
of food sample.
II. Simplified Extraction Method
In some samples, follow the Simplified Extraction Method for food samples. Use 1 mL of rinse water
instead of 1 g of food sample.
Note 10: Using these methods has a higher possibility of getting a false negative than when using the
Recommended Extraction Method, because the extraction efficiency and reactivity may decrease
depending on the test samples. The Recommended Extraction Method should be used for rinse water
collected from the production line of highly processed foods (e.g. cookies). Details are reviewed in our
website.
Chromatography Procedures 1. Dispense 900 µL of Diluent ① with Pipette (L) and 100 µL of Diluent ② with Pipette (S) into
polypropylene tube (S) as Diluting Solution and discard 100 µL of it using the same Pipette
(S).
2. Add 100 µL of Sample Extract with a new Pipette (S) to the polypropylene tube (S) containing
900 µL of Diluting Solution and mix. The resulting solution is referred to as Test Solution.
3. Place the test stick horizontally and add 200 µL of Test Solution to the sample application slot
with the same Pipette (S) as used in step 2.
4. Let it stand for 15 min (25 min for Rapid Test Pro for Buckwheat) at room temperature.
Strictly observe the reaction time to achieve correct results.
5. Interpret the results according to the criteria described below.
Note 11: False-negative results may occur if the Test Solution contains proteins of allergic ingredients at very high
concentrations, due to the “high-dose hook effect” that is well known in one-step immunoassay systems. If
the test sample is suspected to contain a high protein content, it is recommended to perform the test using a
Test Solution that is further diluted than 10-fold. To achieve this, increase the dilution of the Sample
Extract by adding more Extraction Solution, and maintain the dilution (10-fold) with Diluting Solution
constant.
Note 12: Do not use the same pipette for dispensing Diluent ① and Diluent ② in step 1.
Fig. Test Stick
Fig. Pipette (L) Fig. Pipette (S)
Judgment criteria
A: A red-purple line in the test window together with a red color in the confirmation window indicates a
POSITIVE result.
B: No line in the test window together with a red color in the confirmation window indicates a NEGATIVE
result.
C: No color in the confirmation window indicates that the test was unsuccessful due to chromatographic
failure. Repeat the test with a new stick.
Samples with high viscosity may result in the unsuccessful chromatography.
Reference
1. T. B. Osborne (1924), The Vegetable Proteins, Longmans and Co., London, UK
2. John Holme (1966), “A review of Wheat Flour Proteins and their functional properties”, The Bakers
Digest, Vol.40, pp.38-42
3. S. Shibata and T. Nakae (Ed.) (1990), Komugikoseihinno chisiki [Knowledge of wheat products].
Japan, Saiwai Shobo
4. Nihon mugirui kenkyuukai (Ed.) (1964), Komugiko-sono genryou to kakouhin [Wheat-material and
processing] Japan, Yuni Atoo
Warranty Morinaga Institute of Biological Science, Inc. makes no warranty of any kind, either expressed or implied,
except that the materials from which its products are made are of standard quality. Buyer assumes all risk
and liability resulting from the use of this product. There is no warranty of merchantability of the product,
or of the fitness of the product for any purpose. Morinaga Institute of Biological Science, Inc. agrees to
replace any defective product, but expressly disclaims liability for damages, including special or
consequential damage, or expenses arising directly or indirectly from the use of this product.
B Negative
Red
sign Red-purple
line
A Positive
C Unsuccessful
Diluent ①
Pipette (L) 1-1.
Diluent ②
Pipette (S)
1-2. 1-3.
900 µL 100 µL 100 µL
100 µL 100 µL
2. Case: food or rinse sample 2. Case: swab sample
3.
200 µL
Test sample
A-I A-II B-I, C-I B-II, C-II
Recommended
Extraction Method
Simplified Extraction
Method
Recommended
Extraction Method
Simplified Extraction
Method
Detection limit25 ng/mL
(in Test Solution)
25 ng/mL
(in Test Solution)
25 ng/mL
(in Test Solution)
25 ng/mL
(in Test Solution)
For swab test sample For swab test sample
250 ng/mL
(in Sample Extract)
250 ng/mL
(in Sample Extract)
For Rinse water For Rinse water
5 µg/mL
(in rinse water)
5 µg/mL
(in rinse water)
Reagent preparation
Extraction ~
▼ ▼ ▼ ▼
▼ ▼
Boil in a water bath
for 10 minutes
(100℃, 212°F)
Boil in a water bath
for 10 minutes
(100℃, 212°F)
▼ ▼
Allow the sample to cool
and vortex for 30
seconds
Allow the sample to cool
and vortex for 30
seconds
▼ ▼
Centrifuge and filter if
necessaryFilter if necessary
▼ ▼ ▼ ▼
Preparation of the
Test Solution
Test and Results
Food sample Swab test sample and Rinse water
Test method
Corresponding value
for test sample
(Calculated value)
5 μg/g of food 5 μg/g of food
Note: Using the Simplified Extraction Method (A-II, B-II and C-II) has a higher possibility of getting a
false negative than when using the Recommended Extraction Method (A-I, B-I and C-I), because
the extraction efficiency and reactivity may decrease depending on the test samples.
Extraction Solution and Diluting Solution
Mix and homogenize the food sample Swab the specified area / Sample the rinse water
Preparation of the
Sample Extract
Add 19 mL of Extraction Solution to 1 g of the
homogenized sample and vortex for 30 seconds
For swab test sample,
Place the swab into 19 mL of Extraction Solution
and vortex for 30 seconds
For rinse water,
Add 19 mL of Extraction Solution to 1 mL of rinse
water and vortex for 30 seconds
Add 200 µL of Test Solution to the test stick
▼
Check results after 15 minutes
(25 minutes for Buckwheat kit)
Sample Extract Sample Extract
▼ ▼
Dilute the Sample Extract 10-fold with Diluting Solution to prepare the Test Solution
▼