How to Run an R&D Systems Quantikine® QuicKit™ ELISA
QuicKit ELISA Protocol
Accomplish more in your day without compromising quality. The Quantikine QuicKit ELISAs provide quick, accurate quantitation of proteins in serum, plasma, and cell culture supernates. Unlike traditional ELISA kits, QuicKit ELISAs have a fast, simplified protocol that only takes 90 minutes to results with just one wash step.
This video is a general step-by-step guide for running an R&D Systems QuicKit ELISA. R&D Systems QuicKit ELISAs offer a shortened protocol of under 90 minutes with a single wash step, for the detection of proteins in various sample types. QuicKit ELISAs use plates precoated with an anti-tag antibody. More ELISA information is available at R&DSystems.com/ELISA. In this video we are running a Human TNF-alpha QuicKit ELISA to determine human TNF-alpha concentrations in serum and cell culture supernates. This kit is for research use only and not for use in diagnostic procedures.
QuicKit 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 kit is validated for cell culture supernates, serum and plasma samples. We recommend that all samples are assayed immediately after they are collected, but samples can be frozen for future use.
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. This is also where you can consider running additional dilutions of your samples.
Follow the recommended sample dilution factor in the sample preparation section of your kit booklet. If a recommended dilution is not listed, samples can be run neat. Multiple dilutions are recommended for unknown samples. Keep in mind that many cytokines, such as TNF-alpha shown in this video, are expressed at very low abundance in serum and plasma from healthy individuals and will likely fall below the standard curve.
Wash Buffer Preparation
To prepare reagents, first bring all kit reagents to room temperature. To prepare your wash buffer, add 10 mL of wash buffer concentrate to 240 mL of deionized or distilled water in a graduated cylinder to yield 250 mL of wash buffer. Mix gently.Antibody Cocktail Preparation
Next, prepare your antibody cocktail. Reconstitute the Human TNF-alpha Capture Antibody Concentrate with 400 uL of the specified diluent. Gently mix to ensure complete reconstitution. Avoid vigorous mixing and let rest on benchtop for a minimum of 5 minutes.
Next, in a 15 mL conical tube, add 300 uL of the reconstituted capture antibody concentrate and 300 uL of the detection antibody concentrate to 5.4 mL of the specified diluent. This produces 6 mL of Antibody Cocktail. Avoid vigorous mixing.
Next, prepare your Human TNF-alpha Standard. Refer to the vial label for specific reconstitution volume. Reconstitute according to the kit booklet. This reconstitution produces a stock solution of 20 ng/mL. Gently mix the standard to ensure complete reconstitution and let it sit for a minimum of 15 minutes on the benchtop prior to making dilutions.
Standard Curve Preparation
It’s time to create the standard curve.
Pipette 50ul of the standard into the 2000 pg/mL tube. Then, add 450 uL of calibrator diluent into the same tube. This creates the high standard. Vortex gently to mix.
Check your kit booklet to ensure you have the right volume and diluent. Next, pipette 200 uL of the appropriate calibrator diluent into the remaining tubes. Use the high standard to produce a 7-point standard curve. 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.
Change pipette tips before transferring to the next tube. The 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.
In this assay, we’re using the kit-specific R&D Systems QC Controls, Catalog number QC259. Check your kit booklet for the analyte-specific controls. 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
Once you’ve prepared your samples and reagents as directed in the previous sections, it’s time to run your QuicKit 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.
First, add 50 µL of standard, control, or sample to each well. Then add 50 µL of Antibody Cocktail per well. It is important to follow this order and use consistent pipetting technique. Avoid contact with pipette tips and the solution in the well when adding the antibody cocktail. When all samples and solutions have been added to the plate, cover the plate with the adhesive strip provided and ensure it is completely sealed.
Incubate the plate for 1 hour at room temperature on the shaker at a point-one-two-inch orbit at 500 plus-or-minus 50 rpm.
In the last 5 minutes of your incubation, prepare your substrate solution. Mix together color reagents A and B in equal volumes. This substrate solution must be protected from light and used within 15 minutes. It should remain colorless until added to the plate. 100 uL of the mixture is required for each well.
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 for good performance. Washing with a multi-channel pipette is too gentle and is not recommended. We recommend adding a 30 second soak period after adding wash buffer. Wash for a total of three 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.
Next, add 100 µL of Substrate Solution to each well. Remember to protect the substrate solution from light.
Now incubate for 20 minutes at room temperature on the benchtop.
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.
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.