ELISA troubleshooting
ELISA troubleshooting
Hundreds of variables influence ELISA data. Below is a collection of general assay running tips and troubleshooting strategies that you may find helpful when trying to resolve ELISA issues. For Q-View Data Analysis tips, please see https://www.quansysbio.com/data-analysis-q-view.
When running a Q-Plex™ Array:
Do’s
- DO be exact when calibrating shaker speed, being off by even 100 RPM can affect results
- DO dilute all sample types at least 1:2 (50%) with sample diluent (except cell culture media, which can be tested NEAT)
- DO load all standards and samples into the microplate within 10 minutes of each other
- DO be exact with incubation times, particularly the SHRP incubation
- DO be exact when mixing Substrate A and B, being off by even 50 µL can affect results, and mix thoroughly
Don’ts
- DON’T allow the plate to dry out between steps, particularly between washing SHRP and adding substrate
- DON’T allow the SHRP, substrate, or IR dye to be exposed to UV light, as this may degrade it
- DON’T analyze from a color or jpeg image; save grayscale images using a lossless image file type such as Tiff or RAW
Q-Plex Experimental Troubleshooting
| NP40 | No interference at 1% |
| Tween | No interference at 1% |
| Triton | No interference at 1% |
| Citrate | No interference at 20% |
| EDTA | No interference at 20mM |
| Water | No interference at 55M |
| Heparin | No interference at 30 mg/ml |
| Urea | No interference at 1 M |
| DMEM | No interference at 100% |
| HAMS | No interference at 100% |
| RPMI | No interference at 100% |
| SFM4 | No interference at 100% |
Image Troubleshooting
| Q-Plex Array Microplate | Spotted and blocked 96-well polystyrene microtiter plate | Ready for use. | If the plate contains un-wetted wells to be used later, return the plate to 4°C in its sealed foil pouch. Good until kit expiration. |
| Wash Buffer Concentrate (20X) | Liquid, 50 mL/vial of a concentrated solution of buffered surfactant | Place 50 mL of the 20X concentrate into 950 mL deionized water, mix thoroughly. | 4°C until kit expiration. |
| Calibrator | Lyophilized, recombinant calibrators in a buffered protein base with preservatives, see the Product Card for high points | When ready to begin the assay, reconstitute in Sample Diluent according to the Product Card which accompanies the kit, mix gently until fully reconstituted. | Differences Between ELISA Kits ExplainedDiscard unused reconstituted calibrator. Do not freeze/thaw. Good for 6 hrs on ice.* |
| Sample Diluent | Liquid, 6 mL/vial of a buffered protein solution with heterophilic antibody and rheumatoid factor blockers, and preservatives | Ready for use. | 4°C until kit expiration |
| Detection Mix | Liquid, 6 mL/vial of biotinylated antibodies in a buffered protein solution with preservatives | Ready for use. | 4°C until kit expiration |
| Streptavidin-HRP 1X | Liquid, 6 mL/vial of streptavidin-conjugated horse radish peroxidase | Ready for use. Do not expose to UV light. | Do not expose to UV light. 4°C until kit expiration. |
| Substrate A | Liquid, 3 mL/vial of stabilized hydrogen peroxide | Do not expose to UV light. Do not cool after mixing. During the assay, mix 3 mL of Substrate A with 3 mL of Substrate B, and mix gently. | Do not expose to UV light. Do not cool after mixing. Store mixed substrate solution at room temperature for up to 1 week. Store unmixed solution at 4°C until kit expiration. |
| Substrate B | Liquid, 3 mL/vial of stabilized signal enhancer | Do not expose to UV light. Do not cool after mixing. During the assay, mix 3 mL of Substrate A with 3 mL of Substrate B, and mix gently. | Do not expose to UV light. Do not cool after mixing. Store mixed substrate solution at room temperature for up to 1 week. Store unmixed solution at 4°C until kit expiration. |
| Plate Seals (3) | Adhesive strips | None | Non-perishable |
We take great care to ensure that our products are suitable for use with all valid samples. If you have any questions about troubleshooting, or about our products or services, please contact us at 888-QUANSYS (782-6797) or info@quansysbio.com