ELISA troubleshooting guide
Do you have a problem with your ELISA assay? The following table describes some of the most common problems that can occur when performing an ELISA and their 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 to see if your calculations are correct or use a higher concentration of detection reagent. | |
| Incorrect assay setup | Repeat the assay and make sure 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 by a 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. | |
| High background signal | Incorrect incubation temperature | Follow the kit manufacturers recommendation to optimize the incubation temperature |
| Incorrect incubation times | Follow the kit manufacturers recommendation to optimize the incubation temperature | |
| Insufficient washing | If pipetting manually invert the microplate on an absorbent cloth and allow to completely drain to remove any residual fluid. Increasing the number of washing cycles may also be useful. | |
| Substrate exposed to light | Perform substrate incubation in the dark | |
| Reactions have not stopped | Make sure you use stop solution as recommended in your protocol to avoid overdevelopment. | |
| Inconsistent replicates | Insufficient washing | If pipetting manually invert the microplate on an absorbent cloth and allow to completely drain to remove any residual fluid. Increasing the number of washing cycles may also be useful. |
| No plate-binding of capture antibody | Prepare both the coating and blocking steps as recommended in the protocol. Make sure you are using the correct ELISA plate. | |
| Incorrect plate sealing | Do not reuse sealer to avoid well-to-well contamination. Use a plate sealer during incubation. |
Are you having consistency problems in your ELISA assays?
Human error is an important source of problems in ELISA and automation may help solve them. If you are considering automating your ELISA please have a look at our workflow solutions.