Running a Forensic RTU ELISA Kit

May 06, 2021

This video will provide step-by-step instructions on running a Neogen® Forensic RTU ELISA kit.

Video Transcript

[Music]

TITLE: Forensic RTU ELISAs - Screening for drugs of abuse in forensic matrices

Narrator: This video will provide step-by-step instructions for running a Neogen Forensic RTU kit.

While some kit components and protocols will vary slightly, this demonstration will help you visualize each step of the procedure.

Setup

When you are ready to run your samples, remove the Neogen kit from the refrigerator. If any kit components, like controls, are stored frozen, you will need to take them out at this time so that they can thaw completely before beginning the run.

Neogen Forensic RTU kits include all of the reagents you will need to perform this assay as well as a protocol outlining step-by-step instructions for the procedure. Also included is a certificate of analysis which includes important lot specific data like lot numbers expiration dates and typical data.

Materials you will need but are not provided include:

  • deionized water
  • precision pipettes ranging from 10 microliters to 1,000 microliters
  • disposable tips for those pipettes
  • a graduated cylinder to mix the wash buffer
  • a plate cover
  • glassware
  • a microplate reader with a 450 nanometer filter and
  • optionally, a microplate shaker.

Sample Treatment

Some sample types require a dilution to mitigate any matrix effects. These dilutions must be prepared prior to running.

Locate the sample treatment section of the product insert to determine which sample dilution is appropriate for your sample type. If your sample type is not listed contact technical support for more information.

Once you have identified the appropriate dilution prepare your dilutions in clean test tubes using the dilution buffer provided with the kit. For example, if you are running blood samples and they require a one to five dilution, you can add 400 microliters of EIA buffer and 100 microliters of blood for a total volume of 500 microliters. Keep in mind you should not dilute the Neogen controls or calibrators that are included with the kit, however, if you have in-house controls made in the same matrix as your samples you will want to dilute those.

Additional Preparation

The antibody-coated plate included in the kit has removable strips letting the users run less than a full plate if needed.

Do not break a strip of eight wells as this will negatively affect your data.

To determine how many strips will be needed add the number of samples and controls that will be tested. Remember, it is recommended that controls and samples are assayed in duplicate. You may find it helpful to create a plate map to track which samples are in each well.

If you are only running a partial plate, gently pop out any unneeded strip of wells and place them back in the bag with the desiccant bag so that they may be used at a later time.

Unused kit materials must be stored at 4 degrees Celsius to ensure their stability.

Some kit components may require different storage temperatures so be careful to check the kit insert for storage conditions.

Gently mix the ready-to-use conjugate solution by inverting the bottle a minimum of 15 times or by placing the solution on a rocker for 5 minutes.

Do not vortex or shake this solution vigorously as this will compromise the integrity of the conjugate and, thus, your results.

Running the Assay

Using a clean tip, add the specified volume of sample, calibrators, and or controls into the appropriate wells.

If your kit comes with calibrators, run the calibrators that match your sample type.

For best results, prime the pipette tip before adding the sample into the bottom corner of each well at a 45 degree angle.

Next, add the specified volume of the ready-to-use drug enzyme conjugate to each strip using an 8 or 12 multi-channel pipette for rapid addition.

This step is time sensitive, so pipette as quickly and as accurately as possible.

It is important to not allow the pipette tip to touch the inside of the well or any of the reagent already inside the well.

Doing so may result in cross-contamination and inaccurate results.

Once the conjugate has been added to all wells, gently shake the plate for a minute or use a microplate shaker if one is available.

Cover the plate and allow it to incubate at room temperature for 45 minutes.

During this 45 minute conjugate incubation or prior to the run, dilute the concentrated wash buffer 10-fold with deionized water and mix the wash buffer solution thoroughly.

The prepared wash buffer will be stable for 5 days at room temperature or 7 days at 4 degrees Celsius.

Once 45 minutes has elapsed, dump the liquids from the wells, tap the plate clean on a lint-free towel to remove any remaining liquid in the wells.

Wash the plate by adding 300 microliters of the prepared wash buffer to each strip using an 8 or 12 multi-channel pipet.

Once the wash buffer has been added to all of the strips, dump the liquid and tap the plate clean on a clean, lint-free towel.

Repeat this step for a total of 3 washings.

After completing the third wash manually, wipe the outside bottom of the wells with a towel to remove any liquid on the bottom or the sides of the wells.

If you are using automation to wash the plate, wash the plate a total of 5 times.

After the wash step is complete, add the specified volume of K-blue substrate to each well using an 8 or 12 multi-channel pipette for rapid addition and best results.

Cover the plate and allow the substrate to incubate for 30 minutes.

When 30 minutes has elapsed use a multi-channel pipette to add acid stop to each well to stop the reaction.

Mix the plate gently for a few seconds and read the plate at 450 nanometers.

Results are inversely proportional.

The absence of drug in the sample will result in strong color development, whereas the presence of drug will result in decreased or no color development.

Please feel free to contact Neogen at any time should you have any questions about the best use of this or any of Neogen's products.

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Category: Solution Spotlights