Tips

ELISA TIPS

We are dedicated to helping our customers achieve exceptional results. While the information below can never be an exclusive solution to every problem you may encounter; it is our hope that you will find the information useful and even beneficial in troubleshooting any problems you may have. Of course, if you find you are still having problems you may submit them to us using this ELISA Troubleshooting Form.

SYMPTOMS

> Elevated background
> Elevated sample/standard
> Weak color
> Poor precision
> No signal
> Low readings across plate


ELEVATED BACKGROUND
ISSUERECOMMENDATION
Lack of antigenCheck protein expression by in situ hybridization.

Insufficient washingWash well by completely removing liquid and leaving no residue.

Evaporation of sample from wellsBe sure to cover plate tightly with parafilm during all incubations.

Improperly set up blanksFollow protocol when designating blank wells. Blank wells contain only TMB and stop solution. Blank results should be subtracted from the reading for all other wells.

Reaction was not stoppedEnsure the use of fresh stop solution.

Metals in buffer (whole plate turned blue)Make sure all buffers contain no metals.

TMB exposed to light prior to experimentEnsure the use of using new TMB only in the dark.


ELEVATED SAMPLE/STANDARD
ISSUERECOMMENDATION
Standards diluted incorrectlyFollow protocol for correct dilutions and dilute using the appropriate buffer.

Incorrect HRP dilutionFollow protocol for correct dilutions and dilute using the appropriate buffer.


WEAK COLOR
ISSUERECOMMENDATION
Substrates not brought up to room temperature prior to useSubstrates need to be at 25°C prior to use.

SAV HRP made at beginning of protocolSAV HRP will lose its potency. Make no sooner than fifteen minutes prior to use.



POOR PRECISION
ISSUERECOMMENDATION
Wells scrappedDo not scrape bottom of well with pipette tip.

Incorrect amounts of liquid added to wellsGenerally more than 100uL and less than 300uL should be added to each well.

"Edge effect"This happens due to uneven temperature of the plate: from the outer edge wells to the wells in the center of the plate.



NO SIGNAL
ISSUERECOMMENDATION
Capture antibody did not bindEnsure to use ELISA plates and not tissue culture plates.



LOW READINGS ACROSS PLATE
ISSUERECOMMENDATION
Plate expiredNothing is able bind to a microtiter plate, it is best to coat new plates.

Insufficient development timeAllow the plate to develop longer based on optimization.

Insufficient antibodyIncrease antibody concentration.

Insufficient incubation Increase incubation time.

Insufficient temperature too lowIncubation temperature may require optimization for the best results.

Bottom of the plate is dirtyClean bottom of the plate to allow the machine to read properly.