Trouble shooting guide

Sandwich ELISA – manual use

Premi®Test
Art. No. R3900/R3925

11/2023, Seite 1

TroubleshootingguideR-Biopharm • An der neuen Bergstraße 17, 64297 Darmstadt • E-mail: sales@r-biopharm.de • food.r-biopharm.com

Troubleshooting guidePremi®Test

Welcome to the Premi®Test troubleshooting guide! This guide will help you identify and resolve common
issues encountered during this analysis, ensuring accurate and reliable results. Covering sample preparation,
assay reagents, testing environment and equipment, each section presents observed issues, potential root
causes and recommended actions for resolution.
Our team is here to support you! If you need any additional assistance or have further questions whiletroubleshooting, please do not hesitate to contact us. We are dedicated to helping you achieve successful
outcomes in your analysis.

Test procedure
Procedure

Notes and advices

Step 1: Prepare the ampoules• Remove the required amount of ampoules from theframe.
• Be careful not to damage the foil of the remaining
ampoules.
• Use one additional ampoule for the negative control,which is a sample containing no antibiotics.

Step 2: Obtain meat juice

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Take approx. 2 cm3 of lean meat. Extract about 250 µL
of meat juice using the meat press delivered with thePremi®Test Starter Kit (Art. No. ZPT-2000).

Heat the meat press in an oven or under warm tap
water before usage.
Increase the pressure slowly and hold constant. Repeat
this step with new pieces of meat until approx. 200 µL
of meat juice is obtained.
• It might be necessary, to turn the meat press upside
down after some time.
• This technique works best with fresh red meat!
• Sometimes it is very difficult to get enough juice! Inthese cases it is recommended to freeze the samples
and then thaw them in a water bath at 65 °C.
• Do not use minced or ground meat!
• Rinse the meat press sufficiently with distilled waterfor cleaning.

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Step 3: Open the ampoule
Label the ampoules and open the ampoules with one
corner of the green frame.

It might be necessary to remove all of the pinched foilof the opened ampoule to allow proper rinsing.

Step 4: Transfer the meat juice in the ampoule

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Pipette 100 µL of juice slowly onto the agar in theampoule.

• Dip the pipette into the liquid and decrease thepressure on the bubble to allow the stem to fill withthe meat juice.
• Add the meat juice to the pipette by squeezing thesmaller upper bulb.
• By fully squeezing the top bulb, approx. 100 µL of themeat juice will be released.
• (Note: A small excess of liquid may remain in thelower bulb.)• Of course you can use a calibrated pipette from yourlab.
• Take care not to touch the agar!
• Use a new pipette for each sample!
• Make sure that the pipette tip is not blocked by small
pieces of meat in the juice!
• 100 µL is needed to get the right sensitivity.

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Step 5: Prediffusion
Allow to stand at room temperature (20 - 25 °C) for
20 min for prediffusion.

• Do not allow the samples to stand at temperatures
outside this range.
• Plug in the Premi®Test Incubator, so it can preheat to64 °C.

Step 6: Get the juice out of the ampoule
Flush the meat juice out of the ampoule by gently
washing twice with demineralized water. Carefully drain
the water from the ampoule.

• Do not try to remove the water from the ampoule bytapping: if agar comes off the ampoules false positive
results could be caused.
• Fill the ampoule with water and turn it upside down
to drain the water. Repeat this and then place theampoules upside down on a piece of paper so thatany remaining drops of water at the rim will beabsorbed.

Step 7: Close the ampoule

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Close the test ampoule with foil supplied with thePremi®Test to avoid evaporation.

It is essential to use the foil delivered with thePremi®Test!
This foil has specific characteristics: tiny holes in it
prevent water of condensation getting into the ampouleduring heating. If you do not use this foil, you could get
false positive results.

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Step 8: Start the incubation
Check the temperature of the incubator (64 °C).
Place the ampoules in the incubator.

• Did you remember to plug in the incubator at theprediffusion stage (see step 5)?
• Alternatively, you can incubate the ampoules in awater bath with a floating rack.

Step 9: Remove the ampoule

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Withdraw the ampoules from the incubator after approx. • At the moment the negative control of one species
2.5 h (as explained in the manual). Determine the color
changes into yellow, read all the samples of the sameof the lower 2/3 part of the solid agar.
species!
• If all samples are still purple or suspected, leave themin the incubator for another 5 min.
• After these 5 min, read again the color of the bottom
part of the contents of the ampoules.
• Repeat this operation until the negative control
changes its color.

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Step 10: Determine the color visually• Determine the color of the lower 2/3 of the contentsof the ampoule.
• A purple border can appear at the top of the contents.
• The top of the contents can also be colored by themeat juice.
• Be careful: color reading errors can lead to major
problems in the results.

Detection limit of the Premi®Test

Above

AroundUnder

11/2023, page 6• Above:
No color change = no growth of Bacillus = presence ofan antibiotic• Under:
Change of color = growth of Bacillus = absence of an
antibioticTroubleshooting guidePremi®Test

Frequently Asked Questions
FAQ

Note

What samples to take?
Samples should be representative (this depends on the Do not use:
type of animal), but economic considerations also play a • the tongue
role. It would be a waste to use the ham from a pig when • the kidney, except when kidney is to be tested (inthe diaphragm is sufficient. Do not take samples fromthat case use the recommended protocol for kidneymuscles that are too fatty or too tender.
samples)• the liver, except when liver is to be tested (in that caseExamples of suitable samples:
use the recommended protocol for liver samples)• pork: diaphragm• beef: lean meat
• poultry: filet
How much sample?
The minimum volume of a sample is 2 cm3.
Generally, a bigger volume is taken and frozen in afreezer. A part can be cut off for analysis. In some cases
it will be useful to take two samples and freeze themseparately.

• Never refreeze defrosted samples!
• Prevent surface contact between different samples.

How many samples per batch?
If the animals were given antibiotics collectively at the• Broilers or fish receive water treatment (groupfarm (e.g. through the feed in the case of broilers ortreatment), so testing 3 - 5 animals per batch gives apigs), 2 - 5 samples / animal per batch can be taken. The
good estimate of the antibiotic level in the flock.
heterogeneity within a group of animals is predictable,• Beef cattle is treated mainly individually, so here thebut animal to animal variation is not. Using 5 samples
test frequency should be higher (5 - 10 %).
per batch, the risk of not detecting a positive batch is low • Pigs can be treated individually or as a group. When
5 pigs per batch of 100 are tested, at least 3 tests
should be negative with the Premi®Test.
How to stock the samples?
For analyses within 48 h:Store sample in refrigerator (4 °C).

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For storage longer than 48 h:Freeze at least at -18 °C.
Preferably below -32 °C.

• Do not stock the samples at room temperature(20 - 25 °C), as this will increase bacterial growth in
the sample and / or formation of by-products. Thismay cause false positive results (due to formationof inhibiting compounds) or false negative results(due to enzyme production by bacteria e.g. Betalactamase).
• Avoid multiple freeze-thaw cycles! This may lead to adecrease in antibiotic stability.

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How to prepare frozen samples?
Option 1:
Thaw the samples at room temperature (20 - 25 °C).
Option 2:
Thaw the samples in a water bath at 65 °C.

• Do not use the liquid at the bottom of the flask orplastic bag!
• Watch out for water crystals in the frozen sample!
Water can give false positive results!
• Do not thaw the samples in the refrigerator for toolong!
• If you use a microwave oven, preferably heat thesample at 160 watt for 90 sec.

How to use the incubator?

11/2023, page 8• Please refer to the manual for more information.
• Plug in the Premi®Test incubator (220 or 110 Volt).
• It will take at least 5 min to reach a temperature of64 °C.
• Use the incubator in a room with a constanttemperature (10 - 35 °C) and do not place it near an
open window or in a draught.
• For best results, it is recommended not to insert new
cold vials during a test run as this may lower thetemperature.
• Premi®Test Incubator should be stored andtransported dry at a constant temperature between
5 - 35 °C. Avoid freezing and strong mechanicalshocks.

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How to prepare the negative control?
Recommendation

Premi®Test (Art. No. R3900/R3925)
Preliminary supplement
Preparation of negative control samples
Please see the instruction for use (Premi®Test, Art. No. R3900/R3925).

Notifications
For the use of Premi®Test, the negative control samples are obligatory as indication for the correct
incubation time to determine the results. Use only samples which verifiable contain no antibiotic residues.
Use only fresh samples for the preparation of negative control samples. Wash hands and clean working
surface before starting the procedure.

Reagents and equipment required(for this method, not mentioned in the instruction for use)
As described in the related instruction for use.

Procedure• Follow the instructions for the preparation of samples in accordance to the respective application note.
• Aliquot the samples in portions in a multiplicative volume of 100 μL (e.g. 5 x 100 μL = 500 μL).
• Make a test run with one of these samples in accordance to the respective application note.

Storage• Store the samples in a freezer at - 20 °C (- 4 °F) for a maximum of 6 months.
• The shelf life can be increased to 12 months when samples are stored at - 40 °C (- 40 °F).
• Do not freeze and thaw the negative control for a second time.

03/2022, page 1Disclaimer
This recommendation given is based on general experience, which has neither been verified nor validated by us. The recipient of thisrecommendation must verify and validate any recommendation given by himself/herself. R-Biopharm does not guarantee any specificoutcome or result.
R-Biopharm hereby disclaims any and all warranties and liabilities of any kind (including without limitation warranties on noninfringement of intellectual property rights of any third party) with respect to any and all information given in this recommendation.

R-Biopharm • An der neuen Bergstraße 17, 64297 Darmstadt, Germany • E-mail: sales@r-biopharm.de • food.r-biopharm.com• For a negative control, use meat from animals thatwere not treated with antibiotics. Test this meat first:
if the Premi®Test result is negative, you can press
more juice from this meat and use it for the negative
control.
• Store small quantities of meat juice (bags orcryotubes) in a freezer at -18 °C. These negative
controls can be stored for 6 months. The shelf life
increases to 12 months when these samples are
stored at -40 °C.
• It is advised to use a negative control of the species tobe tested in each analysis: when the negative changes
color, the samples should be read.
• Do not use water as negative control! Unlike meatjuice, water contains no nutrients, which means thespores of the Bacillus will develop less readily.

How to prepare the positive control?

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Instructions for the preparation of positive controls areavailable for:• macrolides• β-lactam antibiotics• sulfonamides• tetracyclines• Divide freshly prepared positive standards into smallportions (1 - 5 mL), e.g. in cryotubes.
• The positive control can be stored at -30 °C for 6months.
• Do not freeze and thaw a standard for a second time,since this will inactivate the antibiotic activity.
• Use the positive standard once every month.