Text X358.2IE v1703

XEMATest FROG

Rapid immunochromatographic test for qualitative determination of common frog antigen in foods, kitchen and production facilities 
Version of the test I (1606)
Amphibiae is a very abundant group of animals (more than 5000 species), and frogs comprise about 85% of this quantity. 
Consumption and handling of the materials of amphibian origin is strictly prohibited by some religious confessions. Some amphibians are eaten, however presence of amphibian antigens in food or washouts from surfaces in food manufacturing, storage, transportation chain surfaces, retail and public catering may be considered a warning sign of poor hygiene.
Frog is known to be potentially allergenic and has been the object of some case reports of occupational and food allergy.
Incidental contamination of meat products or food processing facilities by amphibian tissues is a marker of poor production discipline and general hygienic conditions. The components of amphibian body may bring serious diseases and endanger public safety. 
XEMAtest Frog detects one of two major allergens of frog muscular tissue (meat): parvalbumin (allergen Ran e 1 and Ran e 2). This protein is homologous to the counterpart protein of fish which is considered as major fish allergen.

XEMATest FROG is based on immunochromatographic principle (lateral flow). The target antigen is bound by specific antibodies attached to colored microparticles. Then this complex migrates to the test line where binds to another specific antibody to form a colored line indicating positive result.

The test is applicable for qualitative detection or semiquantitative measurement of target antigens in the samples of complex foods and surface swabs. The qualitative detection is usually done for screening and the semiquantitative measurement is applied for monitoring or comparative studies.

TEST SENSITIVITY AND SPECIFICITY
XEMATest FROG utilizes a combination of monoclonal antibodies which enables to detect an antigen common for all tested amphibian species tested. 
The test does NOT detect the antigens in fish, reptiles, lamprey, crustaceans (shrimp, prawn, lobster etc) and mollusks. The scattered false positive reactions may occur with some mollusks (eg conch) which potentially uptake amphibian derived antigens during their life cycle.
A common frog antigen is not present in the row, and only appears in the late development stage of the tadpole.
A sensitivity of XEMATest FROG (LOD, limit of detection) in the extract prepared according to present instruction is at least 25 ppm by wet weight of frog muscle tissue homogenate of common water frog (Pelophylax esculentus) in model extract, and the range (ROD, range of detection) is 25-50,000 ppm. The sensitivity of the test is highly variable among species due to variability of the homology to the reference frog species; the sensitivity also decreases with heating of the food (cooking) and in fat-rich environment (e.g. in presence of oil).

PLEASE NOTE: the sensitivity is calculated for the target antigenic material content in solid material extracted by the method described below, (solid/liquid ratio 1:10 wt/vol). The sensitivity of the test may be upgraded by lowering of the solid/liquid ratio, however this may result in thick liquid which will not penetrate into the test strip. The LOD data for liquid samples are 5x lower with standard procedure, ie with regular liquid specimens the test reaches higher sensitivity.

IMPORTANT NOTE: this test is designed for ultrasensitive detection of frog antigens. When using in apparent frog homogenate (minced frog), the test may show paradoxical negative result due to antigen overload (“prozone effect”). To solve this situation, dilute the sample 5–10 fold by Species extraction buffer and repeat the test.

More details of test performance (sensitivity, specificity, impact of matrix and processing) are available on request or online at www.xematest.com
If the visual test gives unclear results, we recommend to recheck the sample by the quantitative laboratory methods, e.g. ELISA (Xema, Cat# K358), or PCR. 

CONTENTS
-	2 test strips individually packed into sealed pouches;
-	2 Specimen collection tubes;
-	1 vial of Specimen extraction buffer, 10 ml;
-	Instruction for use.

SPECIMEN HANDLING 
The specimens should be brought to temperature range +18...+35 °C before use; testing of colder specimens diminishes the sensitivity of the assay; testing of hot specimens is NOT possible!
The test is designed to detect the target antigen in different types of the material, please select the relevant section of the text below.

TEST PROCEDURE FOR SOLIDS
1.	Allow the test strips to reach room temperature for 5-10 minutes before opening the pouches. 
2.	Put a small piece of tested material (0,1-0,5 g) into a Specimen collection tube. If the balances are available, record the actual weight in grams. 
3.	If the accurate volume measurement tool (pipette) is available, calculate required Specimen extraction buffer volume as 10x actual weight (for example, if the weight is 0,23 g, add 2,3 ml of the buffer). Otherwise, add not more than half of tube volume of Specimen extraction buffer. Adjust the sensitivity of the assay if the weight/volume ratio is different from 1:10.
4.	Screw the cap securely onto the tube and shake vigorously for 15 – 30 seconds or use the vortex mixer. Allow to settle for 2 minutes. 
5.	If the liquid is thick and cloudy, it may not penetrate into the test strip, and testing fails. To avoid it, allow to settle for longer period (up to 6 hours). The extract may be also filtered by textile material or centrifuged.
6.	Open the pouch, taking care not to damage the test strip. Dip the test strip into the liquid layer, do not touch the solid settled down on the bottom.
7.	Allow the strip to remain in the solution for 5 – 10 seconds. Remove the test strip and place onto a clean horizontal surface; do not touch or move the test strip for 10 minutes. Read the test result. 

CAUTION: ENSURE THE TEST STRIP IS DIPPED AS SHOWN IN A FIGURE. THE DIRECTION AND THE DEPTH OF IMMERSION MUST BE AS SHOWN FOR CORRECT OPERATION. 

TEST PROCEDURE FOR LIQUIDS
The limitation for liquid specimens is their viscosity and turbidity (presence of particulate matter). If the specimen is viscous and cannot reach the test zone of the test strip, it should be diluted by Specimen extraction buffer right in the Specimen collection tube or separately. In this case, the sensitivity of the test should be adjusted by dilution factor. Cloudy specimens should be allowed to settle, filtered through textile material, or centrifuged.
1.	Allow test strips to reach room temperature for 5-10 minutes before opening the pouches. 
2.	Collect 1 ml of liquid specimen into a Specimen collection tube (by first marker on the tube wall or using a pipette).
3.	Add an equal volume of Specimen extraction buffer, screw the cap securely onto the tube and mix by shaking or use the vortex mixer.  If the liquid is cloudy, allow to settle, then follow the instructions from point 5 of test procedure for solid materials outlined above
TEST PROCEDURE FOR SURFACE SWABS 
1.	Allow test strips to reach room temperature for 5-10 minutes before opening the pouches.
2.	Add 1 ml of Specimen extraction buffer to the tube into a Specimen collection tube (by first marker on the tube wall or using a pipette)
3.	 Place the swabbing tool (for example, cotton tip) into the tube, rinse the swabbing tool in the buffer
4. Squeeze off any excessive liquid off the swabbing tool against the tube wall
5. Swab the tested surface with special attention to suspected spots. 
6. Put the swab back into the tube containing the rest of the buffer and shake vigorously for 15 – 30 seconds or use the vortex mixer.
7. Remove the swab from the tube and follow the instructions from point 5 of test procedure for solid materials outlined above.

INTERPRETATION OF THE RESULTS
Test is considered POSITIVE if TWO colored lines appear in the test zone.

Test is considered NEGATIVE if only ONE colored line is clearly visible.
 
If NO colored line is formed, the test is INVALID. 

Try to repeat it with another test strip, check the correct specimen handling and test procedure, expiry date and storage conditions.

PRECAUTIONS
*The test strips should be stored at +10...+30°C
*Use the test within 10 minutes after opening the pouch because the test strips are very sensitive to moisture
*Do not touch the reaction membrane
*Do not use the test strip if its pouch is torn, or test strip is broken or damaged
*The test strips are disposable; do not use them repeatedly
*Do not use the test strips beyond the expiration date

MANUFACTURER: 
Xema group of companies
www.xematest.com