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

ProblemPossible CausesSolution
Weak or no signalIncorrect reagent storageDouble 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 reagentsDo not use reagents that are past the expiration date.
 Incorrect dilutions preparedCheck if your calculations are correctly. Use a higher concentration of detection reagent.
 Incorrect assay setupRepeat assay and ensure reagents have been prepared according to the protocol and are added in the correct order.
 Incorrect plate reader settingsMake sure your ELISA reader settings reflect the recommended wavelength/filters.
 Not enough antibody usedIncrease 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 signalIncorrect incubation temperatureFollow your kit manufacturers recommendation or optimise the incubation temperature
 Incorrect incubation timesFollow your kit manufacturers recommendation or optimise the incubation time
 Insufficient washingIf 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 lightPerform substrate incubation in the dark
 Reactions not stoppedEnsure you use stop soltion as recommended in your protocol to avoid overdevelopment.
Inconsistent replicatesInsufficient washingIf 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 antibodyPrepare both, coating and blocking steps as recommended in the protocol. Make sure you´re using an ELISA plate.
 Incorrect plate sealingDon´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.

 

ELISA Workflow Solutions