• Elisa troubleshooting

  • High Background

    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

     

    Poor Standard Curve

    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.

     

     

     

     

     

    No Signal

    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

    Erratic duplicate OD values

    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

     

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