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: info@miobs.com

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

▼