Veratox® Food Allergens Training

August 25, 2021

This video demonstrates the most accurate and efficient method of collecting and extracting a sample for analysis.

 

Video Transcript

[Music]

[NEOGEN® Training Series: Food Allergens]

[Allergen Extraction Method for Veratox® (non-gliadin)]

This video demonstrates the most accurate and efficient method of collecting and extracting a sample for analysis. This video serves only as a companion to the written materials supplied with the food allergen test kits. Please read and follow the written instructions in their entirety.

This demonstration makes use of NEOGEN's Allergen extraction kit in the sample extraction process.

The kit contains 250 milliliter disposable extraction bottles and transfer pipettes.

In addition to the kit, you'll need the following materials for the extraction step:

  • a shaker water bath capable of heating 30 or 60 degrees Celsius
  • graduated cylinder to measure 125 milliliters
  • a scale to weigh a 5 gram sample
  • one liter bottle to prepare extraction solution
  • deionized or distilled water
  • a timer
  • PBS or other extraction powder from respective Veratox allergen kit

[Music]

Prepare extraction solutions by adding contents of the PBS foil pouch to 1 liter of distilled or deionized water..

[For Veratox Mustard, please use the mustard allergen extraction buffer packet included in the kit]

Mix.

Pre-heat the extraction solution to 60 degrees Celsius.

While heating the extraction solution, gather a representative sample and grind to a fine particle size.

Measure 5 milliliters or weigh 5 grams of sample in a new 250 milliliter bottle.

Add one level scoop of the appropriate extraction additive to the sample.

Pour 125 milliliters of the 60 degrees Celsius extraction solution into the sample bottle.

Extract by shaking in a 60 degrees Celsius water bath at 150 RPM for 15 minutes.

Remove from the water bath and let material settle for five minutes.

Some samples may require filtration to achieve a supernatant free of suspended material.

Remove approximately 1 milliliter of the supernatant and transfer it to a collection tube to allow sample to cool.

Your sample is now ready to be tested.

Samples should be analyzed within 2 to 3 hours using the appropriate Veratox allergen test kit.

[Music]

 

[NEOGEN Training Series: Food Allergens]

[Veratox® Assay Procedure]

This video demonstrates the Veratox assay for food allergens to review sample preparation please refer to our other videos.

This video serves only as a companion to the written materials supplied with the Veratox for food allergen and gliadin test kits.

Please read and follow the written instructions in their entirety.

Each Veratox kit for allergens contains:

  • a foil pouch containing clear, antibody-coated wells and red-marked transfer wells
  • up to six yellow-labeled vials containing the range of controls
  • blue-labeled bottle containing conjugate
  • green-labeled bottle containing substrate
  • red-labeled the bottle containing red stop
  • small foil pouches containing PBS powder
  • wash buffer concentrate
  • extraction additive with scoop

In addition to NEOGEN's test kit, materials recommended but not provided are:

  • microwell strip or plate reader
  • timer
  • one liter bottle to prepare wash buffer
  • deionized or distilled water
  • graduated cylinder
  • wash bottle
  • pipette tips
  • adjustable single and 12-channel pipettor
  • microwell holder
  • reagent boats

[Step 1: Wash Buffer Preparation]

Prepare wash buffer solution by pouring the 40 milliliters of wash buffer concentrate into a 1 liter bottle.

Measure 960 milliliters of distilled or deionized water.

Pour some water into the wash buffer bottle to rinse and add to the 1 liter bottle.

Pour remaining water into the bottle, cap and mix.

Transfer wash buffer to a squeezable wash bottle.

[Step 2: Veratox Assay]

Remove one well for each sample to be tested plus one well for each of the controls for this example we will have two samples and five controls for a total of seven wells.

Break them off and place in the well holder.

Do this for both the red-marked transfer wells and clear, antibody-coated wells.

[Use the tab to identify the first well or mark side of the well with a permanent marker]

Using a new pipette tip for each, transfer 150 microliters of each control and sample extracts to the appropriate red-marked transfer wells.

Do not use more than 2 full strips or 24 wells at one time.

[Change pipette tips in between pipetting to avoid cross-contamination]

Place tips on the 12-channel pipetter and transfer 100 microliters of the reagents from the red-marked transfer wells to the antibody-coated wells.

Be sure to use proper pipe heading techniques including priming the tips and always using new tips.

Set timer for 10 minutes.

Mix for 10 to 30 seconds by sliding the microwave holder back and forth on a flat surface without splashing.

[Reference your kit instructions for mixing times.]

and allow wells to incubate at room temperature.

The red-marked wells can be discarded at this time.

After the 10-minute incubation, empty the well's contents into a waste receptacle or sink.

Fill each antibody well with the wash buffer solution and dump out.

Repeat the washing 5 to 10 times depending on the assay, then turn the wells upside down and tap out on a paper towel until remaining wash solution is gone.

Pour the needed volume of conjugate from the blue-labeled bottle into a clean reagent boat.

Using the 12-channel pipetter transfer, 100 microliters of the conjugate into all the wells and mix by sliding the microwell holder back and forth on a flat surface.

Incubate for 10 minutes at room temperature.

Wash all wells with the wash buffer solution as demonstrated prior.

Pour the needed volume of substrate solution from the green-labeled bottle into a clean reagent boat.

Place new tips on the 12-channel pipetter and transfer 100 microliters of substrate into each well.

Set timer for 10 minutes.

Mix by sliding the microwave holder back and forth on a flat surface.

Incubate for 10 minutes at room temperature.

Prepare for the next step by pouring the needed volume of red stop solution from the red label bottle into a clean reagent boat.

At the completion of the incubation, do not wash the wells.

Using new tips on the 12-channel pipe header transfer 100 microliters of red stop solution into each well and mix by sliding the microwell holder back and forth on a flat surface.

Make sure there is no layering or bubbling in the wells.

Remove from the well holder.

Wipe the bottom of the microwells with a dry cloth or towel and transfer to the far-right slot of the tray holder of NEOGEN's Stat Fax microwell strip reader.

Results should be read within 20 minutes after the addition of red stop solution.

[Step 3: Using the NEOGEN Stat Fax reader]

[If you are not using the NEOGEN Stat Fax reader that is pre-programmed with our assays, you will need an equivalent strip or plate reader with a 650nm filter and NEOGEN Veratox Software]

Using the 4700 reader, position the carrier to the left so strip A is in the center of the track.

Use the run test key to access the user's test menu.

The instrument will display the pre-programmed tests.

Press arrow up or down or advance page to locate the correct test or, if you know the test number, press "By #", enter the test number and press "Enter".

For this example, we are running the Veratox for egg allergen.

Confirm the test selection or press "No" to select a different test.

After selecting the test there will be three options: accept the test, limit the number of wells, or quit the test.

Limiting the wells is not required but helpful when running only a few wells as the reader will read all 12 positions unless limited.

Select limit wells and press 7 and enter.

The reader will begin reading and calculating your results.

If the correlation coefficient is less than 0.98, the message "Invalid – R less than 0.98" will be printed at the end of the run.

Results should be considered invalid if this message appears.

[If you have questions about these procedures, please call NEOGEN's Technical Sales staff at 1-800-234-5333]

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Category: Science Insights