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]

TITLE: Neogen® Training Series: Food Allergens - Allergen Extraction Method for Veratox® (non-gliadin)

[A lab technician pours water from a large bottle into a smaller bottle sittng on a table.]

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

[Image of the printed instructions included.]

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.

[A lab table is shown with items from or needed for the testing kit. Each item is highlighted when mentioned.]

SUPERIMPOSE: Allergen Extraction Kit #8429

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]

TITLE: Step 1: Reagent Preparation

[A lab technician opens a foil pouch and pours the contents into a bottle of water, closes the bottle and gently rocks the bottle back and forth.]

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

SUPERIMPOSE: For Veratox Mustard, please use the mustard allergen extraction buffer packet included in the kit

Mix.

[The lab technician opens a shaker water bath machine and places the bottle of water solution inside.]

SUPERIMPOSE: Veratox Crustacea only heat to 30°C

Pre-heat the extraction solution to 60 degrees Celsius.

[A lab technician weighs a plastic bottle and places ground powder into the bottle. Then the technician adds a scoop of additive.]

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.

[The lab technician measures water in a graduated cylinder and pours that into the plastic bottle.]

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

[The lab technician places the plastic bottle in a shaker water bath machine.]

SUPERIMPOSE: For Crustacea assay, shake at 30°C for 30 minutes

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

[The lab technician removes the plastic bottle and places it on the table next to the shaker water bath machine.]

SUPERIMPOSE: Reference respective kit insert

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.

[The lab technician pipettes fluid from the plastic bottle into a small tube on the table and caps the tube with a lid.]

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]

TITLE: Neogen Training Series: Food Allergens - Veratox® Assay Procedure

[A lab technician uses a 6-channel pipettor to put fluid into a microwell array.]

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

[Image of the printed instructions included.]

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.

[A lab table is shown with items from or needed for the testing kit. Each item is highlighted when mentioned.]

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

TITLE: Step 1: Wash Buffer Preparation

[A lab technician standing at a table pours solution from a small bottle into a larger bottle.]

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

[A lab technician measures water in a graduated cyclinder and pours the measured water into the small bottle, swirls the bottle and pours the solution into the 1-liter bottle. The technician then pours the remaining water into the 1-liter bottle and performs the actions as described.]

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.

TITLE: Step 2: Veratox Assay

[A lab technician pulls a line of microwells from a foil packet and begins counting from one end before breaking off the required number of microwells and placing them in a microwell holder. The tecnician repeats these actions.]

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.

SUPERIMPOSE: Use the tab to identify the first well or mark side of the well with a permanent marker

SUPERIMPOSE: Reseal the foil pack

[The technician picks up the foil pack and presses closed the opened end.]

[A technician at a lab table performs the following instructions making sure to close each bottle after opening and extracting the contents.]

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.

SUPERIMPOSE: 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 microwell holder back and forth on a flat surface without splashing.

SUPERIMPOSE: Reference your kit instructions for mixing times.

and allow wells to incubate at room temperature.

[A lab technician throws out the red marked microwells and picks up the microwell holder. The technician pours the microwell's contents into the trash bin. The technician pours water over the microwells from a bottle and then pours the water out of the microwells into the trash bin.]

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.

[A lab technician places the microwells and holder upside down on a paper towel and wraps the paper towel around the microwell holder. Lifting up the microwell holder wrapped in the paper towel, the technician taps it downward several times on the table.]

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.

[The lab technician pours fluid from a bottle into a trough-like reagent boat.]

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

[A lab technician uses a 12-channel pipettor with only 7 pipettor tips to place fluid into the emptied microwells and slides the microwell holder back and forth on the table.]

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.

[A lab technician picks up the microwell holder. The technician pours the microwell's contents into the trash bin. The technician pours water over the microwells from a bottle and then pours the water out of the microwells into the trash bin.]

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

[The lab technician pours fluid from a bottle into a trough-like reagent boat.]

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

[A lab technician uses a 12-channel pipettor with only 7 pipettor tips to place fluid into the emptied microwells and slides the microwell holder back and forth on the table.]

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 microwell holder back and forth on a flat surface.

Incubate for 10 minutes at room temperature.

[The lab technician pours fluid from a bottle into a trough-like reagent boat.]

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

[A lab technician uses a 12-channel pipettor with only 7 pipettor tips to place fluid into the emptied microwells and slides the microwell holder back and forth on the table.]

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.

[A lab technician removes the microwells from the holder and dries the bottom of the microwells. The technician places the microwells into a machine labelled "Stat Fax 4700".]

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.

TITLE: 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

[A lab technician performs the following instructions.]

Narrator: 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.

[The read-out screen of the Stat Fax machine is shown and the three buttons are highlighted as they are mentioned in turn.]

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

[The read-out screen of the Stat Fax machine is shown and the button labelled "# Wells" is highlighted.]

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

[The technician performs the following button presses and then the reader pulls the microwells into it.]

Select limit wells and press 7 and enter.

The reader will begin reading and calculating your results.

[A close up of the Stat Fax's paper readout is shown as the results print out.]

SUPERIMPOSE: If correlation coefficient is <0.98, results should be considered invalid

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.

TITLE: 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