ELISA troubleshooting guide
Do you have a problem with your ELISA? The following table describes some of the most common problems that can occur when performing an ELISA and possible solutions.
ELISA problems and solutions
| Problem | Possible Causes | Solution |
|---|---|---|
| Weak or no signal | Incorrect reagent storage | Double check if you have followed the recommendations of your kit manufacturer. |
Antibody stored at 4°C for several weeks or subjected to repeated freeze/thaw cycles | Use fresh aliquot of antibody. | |
| Expired reagents | Do not use reagents that are past the expiration date. | |
| Incorrect dilutions prepared | Check if your calculations are correctly. Use a higher concentration of detection reagent. | |
| Incorrect assay setup | Repeat assay and ensure reagents have been prepared according to the protocol and are added in the correct order. | |
| Incorrect plate reader settings | Make sure your microplate reader settings reflect the recommended wavelength/filters. | |
| Not enough antibody used | Increase the concentration of your primary and/or secondary antibody | |
| Wells damaged with pipette or washing tips. | Be careful when dispensing and aspirating into and out of wells when pipetting manually. Recalibrate your automated plate washers so that the tips do not touch the bottom of the wells if required. | |
| High background signal | Incorrect incubation temperature | Follow your kit manufacturers recommendation or optimise the incubation temperature |
| Incorrect incubation times | Follow your kit manufacturers recommendation or optimise the incubation time | |
| Insufficient washing | If pipetting manually invert plate on absorbent tissue and allow to completely drain, to remove any residual fluid. Increasing the number of washing cycles may be useful, too. | |
| Substrate exposed to light | Perform substrate incubation in the dark | |
| Reactions not stopped | Ensure you use stop soltion as recommended in your protocol to avoid overdevelopment. | |
| Inconsistent replicates | Insufficient washing | If pipetting manually invert plate on absorbent tissue and allow to completely drain, to remove any residual fluid. Increasing the number of washing cycles may be useful, too. |
| No plate-binding of capture antibody | Prepare both, coating and blocking steps as recommended in the protocol. Make sure you´re using an ELISA plate. | |
| Incorrect plate sealing | Don´t reuse sealer to avoid well-to-well contamination. Use plate sealer during incubation. |
Are you having consistency problems in your ELISA assays?
Human error is an important source of problems in ELISA and other assays, and automation may help solve them. If you are considering automating your ELISA, have a look to our workflow solutions.