Probable Cause:

Solution/ Action

High incubation temperature:

Incubate at room temperature (25 ^oC) throughout the procedure

Insufficient washing of the plate:

Fill the wells with wash buffer and aspirate completely for the next wash

Increase the number of washes

Add soak time (20-30 seconds) in between the washes

Use automated plate washer, if available and check that all the channels are operating properly

Concentrated streptavidin-HRP

Streptavidin-HRP was not diluted properly

Dilute the streptavidin-HRP as mentioned in the manual

Light exposure during substrate incubation

The TMB substrate is light sensitive and turns to blue color in the presence of light. The incubation must be carried out in dark.

Stop solution not added

Color will continue to develop if stop solution is not added

Diluents came with the kit were not used

Standards/ sample, detection antibody and streptavidin-HRP must be diluted in the respective buffers came with the kit. Do not use buffers from other kits

Contaminated solutions

Prepare fresh working solutions

Probable Cause:

Solution/ Action

Improper standard reconstitution:

Spin the vial briefly before opening

Reconstitute the standard as mentioned in the manual. After reconstitution, leave it atleast for 10 minutes at room temperature

Do not store and reuse diluted standards

Curve fitting problem:

Log transform the values on both axes

Use 4-PL/ 5-PL curve fitting programs

Incubation temperature/ time

Use the recommended standard incubation conditions

Poor dilutions

Pipetting error. Check pipetting technique and calculations.

Use calibrated pipettes.

Probable Cause:

Solution/ Action

Omission of reagent(s):

Read the manual entirely. Check that all the reagents are added in the correct order as stated in the manual

Incorrect detection antibody was used:

Use the detection antibody came with the kit

Chromogen solutions were mixed improperly

Use the recommended procedure to prepare the TMB substrate

HRP inhibitor in sample/ buffers

Check that the samples/ buffers do not have sodium azide as it will inhibit peroxidase reaction.

Vigorous washing

If the washing is done manually, pipette the wash buffer gently.

Dried wells

Do not allow the wells to dry out during the assay. Seal with the supplied adhesive cover during incubations

Improper plate reader settings

Check the wavelength and read the plate again

Probable Cause:

Solution/ Action

Insufficient washing of the plate

Fill the wells with wash buffer and aspirate completely for the next wash

Increase number of washes

Add soak time (20-30 seconds) in between the washes

Use automated plate washer, is available and check that all the channels are functioning properly

Poor dilutions

Pipetting error. Check pipetting technique and calculations.

Use calibrated pipettes.

Improper mixing of samples/ buffers

Mix the samples well before pipetting

Thoroughly mix the working solutions of detection antibody/ streptavidin-HRP

Contamination from other wells

Do not reuse the adhesive covers from previous assay setups

Change pipette tips during reagent addition. If same pipette tip is being used to dispense reagents, care should be taken, not to touch the solution in the well

Precipitates in the samples/ buffer

If precipitates are visible in wash buffer concentrate, keep it at 37 ^oC for 10-15 minutes until no precipitates are visible

Centrifuge the samples to remove particulate matter

Dried wells

Do not allow the wells to dry out during the assay. Seal with the supplied adhesive cover during incubations