This video is a general step-by-step guide to running an R&D Systems DuoSet™ ELISA. Please refer to your assay insert and Certificate of Analysis for specific instructions for your assay. If you have any questions or concerns about running your DuoSet ELISA please contact R&D Systems Technical Service at techsupport@bio-techne.com.
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R&D Systems™ DuoSet® ELISA Development Systems
DuoSet ELISA Protocol
This video is a general step-by-step guide to running an R&D systems DuoSet™ ELISA. This is a development assay which provides the components for performing sandwich ELISAs and has been validated for cell culture supernate samples. Additional assay optimization and validation is recommended for testing complex matrices such as serum and plasma. Read the assay insert in its entirety before using this product and refer to the lot specific certificate of analysis for component concentrations as they may vary.
Reagent Preparation
Begin by preparing the reagents.
- Fully equilibrate all reagent buffers and lyophilized vials to room temperature. Buffers and other materials required are listed on the assay insert. Recommended reagents can be found in the assay insert and on the Supplemental products tab of the products web page for this assay video.
We will be using the recommended DuoSet Ancillary Reagent Kit (Catalog # DY008). This kit provides enough components to run 596 well assay plates.
Antibody Reconstitution
- Reconstitute both lyophilized antibodies. Avoid vigorous mixing of the vials. Allow the capture antibody to set for a minimum of 15 minutes before coating your plates or preparing single-use aliquots for storage. Store the detection antibody overnight at 4 degrees Celsius. Before aliquoting, refer to the certificate of analysis for storage temperatures.
Plate Preparation
- Design your plate layout to determine how many wells you will need to coat for the assay plate. Our assay plate will be set-up like this, with the standard curve in duplicate in the first two strips, the samples in duplicate in strips 3, 4, 5, & 6 and the remaining wells as blanks.
- Now prepare the plate. Careful and precise plate preparation is essential to good assay performance.
- Dilute the capture antibody to the working concentration in PBS after gentle mixing. Immediately coat a 96-well, high protein-binding microplate with precisely 100 microliters per well.
- Seal the plate and incubate overnight at room temperature.
- On day two, ensure that all reagents are equilibrated to room temperature. Aspirate or decant each well from the coated plate and wash by filling each well fully with wash buffer using a squirt bottle, manifold dispenser, or auto washer. Washing with a multi-channel pipette is too gentle and lacks the force to thoroughly flush the wells.
- Thoroughly decant by blotting the inverted plate against clean paper towels. Repeat this process for a total of three washes.
Plate Blocking
- Next, block the coated plate by adding three hundred microliters of reagent diluent to each well. Incubate at room temperature for a minimum of one hour.
- During this incubation, prepare your samples for testing. Samples may be diluted in reagent diluent and should have a volume sufficient to add 100 microliters to each well. It is recommended that all samples are assayed in duplicate.
Standard Curve
The standard curve should be prepared last. So that it may be added to the plate with minimal delay, a seven-point standard curve using twofold serial dilutions is outlined in the assay insert.
- Prepare 1000 microliters of high standard per plate and place it in the first tube.
- 500 microliters of reagent diluent is added to the remaining six standard curve tubes.
- Serial dilute the standard curve by transferring 500 microliters from the 1000 picogram per milliliter standard to the 500 picograms per milliliter tube. Gently mix, then transfer 500 microliters from the 500 picograms per milliliter tube to the 250 picogram per milliliter tube and so on, until the curve is complete.
Assay Procedure
- Next, repeat the wash step and then add 100 microliters of sample or standard per well. Cover with an adhesive strip and incubate for two hours at room temperature on the benchtop.
- During this sample incubation, dilute the biotinylated detection antibody to the working concentration.
- Next, wash the plate just as you did in the plate preparation section.
- Add 100 microliters of the diluted biotinylated detection antibody to each well. Cover with a new adhesive strip and incubate for two hours at room temperature on the benchtop.
- During this detection antibody incubation, dilute the streptavidin-HRP in reagent diluent as specified on the vial label.
- Next, repeat the wash step.
- Add 100 microliters of the diluted streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature on the bench top. Avoid placing the plate in direct light.
- In the last five minutes of the streptavidin-HRP incubation, prepare the substrate solution, a 1:1 mix of hydrogen peroxide and tetramethyl benzidine.
- Repeat the wash step.
- Add 100 microliters of substrate solution to each well. Incubate for 20 to 30 minutes at room temperature on the bench top. Avoid placing the plate in direct light.
- Finally, add 50 microliters of stop solution by quickly dispensing it into each well. Gently tap the plate to ensure thorough mixing. Read the optical density of each well immediately using a microplate reader set to 450 nanometers.
Evaluating Results
- If wavelength correction is available, set it to 540 or 570 nanometers. This subtraction will correct for optical imperfections in the plate.
- Average the duplicate readings for each standard control and sample and subtract the average zero standard optical density.
- Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic curve fit.
This concludes our video for running an R&D Systems DuoSet ELISA. For more helpful protocols, subscribe to our YouTube channel. For more information on our R&D Systems ELISAs, visit R&DSystems.com/ELISA or contact us.