How to Run an R&D Systems Quantikine ELISA

R&D Systems Quantikine^™ ELISA Kits are complete, fully validated, ready-to-run ELISAs that are designed to measure proteins in complex sample types. This video is a general step-by-step guide to running a Quantikine ELISA. If you have any questions or concerns about running your Quantikine ELISA please contact R&D Systems Technical Service at techsupport@bio-techne.com. Please refer to your kit booklet and Certificate of Analysis for specific instructions for your assay.

Important Notes

There are a few important things to keep in mind before we begin.

• First, some proteins are detectable in saliva, so it may be important to wear a facemask to prevent contamination. Refer to the precautions section in your kit booklet for specifics.
• Also, be sure to wear personal protective equipment and refer to the Safety Data Sheet on our website prior to use.
• Finally, protocols vary by kit and sample, so read your kit booklet carefully before you begin and during each step for specific instructions.
• This protocol requires proper pipetting to yield the best results. There are two different methods of pipetting, forward and reverse.
  • Forward pipetting involves filling up the tip with the exact volume required and dispensing the complete volume. Consistency is key.
  • Reverse pipetting starts by depressing your plunger all the way to the second stop and filling up the tip with more than the required volume, then slowly dispensing the measured volume to the first stop. You can repeat this process for replicates, but discard the remaining volume and change tips when switching solutions.
  • Pre-wetting tips and reverse pipetting are recommended when working with viscus solutions to minimize splashing and bubbles when volume permits.

We recommend changing your pipette tips between standard concentrations, control levels and each sample. The first step in the process is to collect samples. This kit is validated for cell culture supernate, serum and plasma samples. Citrate plasma has not been validated for use in this assay. We recommend that all samples are assayed immediately after they are collected, but samples can be frozen for future use. Refer to your kit booklet for sample-specific collection and storage instructions.

Sample Preparation

This leptin receptor assay requires a minimum 5-fold dilution for serum samples. Check the sample preparation section of your kit booklet for the suggested dilution factor, if applicable.

Use a separate tube for each sample. We’ve labeled our tubes s1 and s2.

1. Add 50 uL of sample to their respective tubes. Then add 200 uL of Calibrator Diluent RD6Q to each tube. This produces a 5-fold dilution.
2. Vortex gently to mix.
3. Refer to your kit booklet for specific dilution instructions. Sometimes a sample will read higher than the highest standard. Therefore, an additional dilution to a lower concentration is recommended.

Plate Setup

4. If you haven’t done so already, take some time to think about how you’ll setup your plate. In our plate the standard curve will be setup in duplicate in strips one and two, and our controls and samples in duplicate in strips three and four. Here’s where you can consider running additional dilutions of your samples.

Wash Buffer Preparation

5. To prepare reagents, first bring all kit reagents to room temperature. To prepare your wash buffer, add 20 mL of wash buffer concentrate to 480 mL of deionized or distilled water in a graduated cylinder to yield 500 mL of wash buffer. Mix gently.
6. Next, prepare your Human Leptin Receptor Standard. Refer to the label on your vial for specific reconstitution volume. Reconstitute with deionized or distilled water. The reconstitution produces a stock solution of 200 ng/mL.
7. Gently mix the standard to ensure complete reconstitution and let it sit for a minimum of 15 minutes prior to making dilutions.

Standard Curve

8. It’s time to create the standard curve. For serum samples, pipette 900 mL of calibrator diluent RD6Q into the 20 ng/mL tube.
9. Then, pipette 100ml of the standard into the same tube. This creates a solution with a concentration of 20ng/ml. Vortex gently to mix.
10. Check your kit booklet to ensure you have the right volume and diluent.
11. Next, pipette 300 uL of the appropriate calibrator diluent into the remaining tubes.
12. Use the stock solution to produce a dilution series and be sure to mix each tube thoroughly by very briefly vortexing. If you don’t have a vortexer you can lightly tap the side of the vial or pipette up and down. Try to minimize foaming and bubbles.
13. The 20 ng/mL standard serves as the high standard and the appropriate calibrator diluent serves as the blank. Blank wells contain zero standard and are treated identically to assay wells. They serve as the non-specific binding control for all the assay reagents.

Preparing Controls

• In this assay, we’re using the kit-specific R&D Systems QC Controls (Catalog # QC112). These should be reconstituted according to their lot-specific certificate of analysis and analyzed as-is, without dilution. If you’re not using R&D Systems QC Controls, we recommend formulating your own control.

Loading the Plate

15. Once you’ve prepared your samples and reagents as directed in the previous sections, it’s time to run your Quantikine ELISA. We recommend that all samples, controls, and standards be assayed in duplicate. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal. We recommend labeling the plate strips. Remember this is a general guide, so refer to your kit booklet for specific reagents and volumes.
16. The next step in the assay is to add 100 μL of Assay Diluent RD1W to each well.
17. Then add 50 μL of standard, control, or sample per well. Be consistent. It’s recommended to gently touch the side of the well when pipetting. When all samples have been added to the plate, cover the plate with the adhesive strip provided and ensure it is completely sealed.
18. For this kit, incubate the plate for 2 hours at room temperature on the shaker at a point-one-two-inch orbit at 500 plus-or-minus 50 rpm.
19. Next, Aspirate each well and wash by filling each well with 400 uL of Wash Buffer using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential to good performance. Wash for a total of four times and after the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. When washing, we recommend adding a 30 second soak period after adding wash buffer.
20. Next, add 200 μL of Human Leptin Receptor Conjugate to each well.
21. Cover the plate with a new adhesive strip and incubate for 2 hours at room temperature on the shaker.
22. In the last 5 minutes of your conjugate incubation, prepare your substrate solution.

Substrate Solution

23. Mix together color reagents A and B in equal volumes. Substrate solution must be protected from light and used within 15 minutes. It should remain colorless until added to the plate. 200 uL of the mixture is required for each well.
24. When your plate is done incubating, repeat the wash process for a total of four washes.
25. Next, add 200 μL of Substrate Solution to each well. Remember to protect the substrate solution from light. Now incubate. For Serum or Plasma Samples incubate for 30 minutes at room temperature on the benchtop. Be sure to check your kit booklet for specific incubation times.

Stop Solution

26. Next add 50 μL of Stop Solution to each well. Stop solution should be added to the plate in the same order as the substrate solution. It is important to quickly dispense the stop solution into the well at a 45-degree angle so the color in the wells changes completely from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing or place the plate back on the shaker. As a last resort, you can use a pipette tip to individually mix each well, but this may result in loss of volume and could impact your results.

Collecting Results

27. Now you can collect your results. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. To evaluate your data, the average of the blank wells should first be subtracted from all wells. If wavelength correction is available, set to 540 or 570 nm. This will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate due to the slight imperfections in the plastic of the 96-well ELISA plate. Plot your standard curve and read controls and unknowns off the standard curve. The blank wells should not be incorporated into the standard curve.

This concludes our video guide for running an R&D Systems Quantikine ELISA. For more helpful protocols, subscribe to our YouTube channel. For more information about R&D Systems Quantikine ELISAs, visit R&DSystems.com/ELISA or contact us.