Troubleshooting Guide for ELISA

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-HRPStreptavidin-HRP was not diluted properly Dilute the streptavidin -HRP as mentioned in the manual
Light exposure during substrate incubationThe 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 addedColor will continue to develop if stop solution is not added
Diluents came with the kit were not usedStandards/ 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 solutionsPrepare 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/ timeUse the recommended standard incubation conditions
Poor dilutionsPipetting 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 improperlyUse the recommended procedure to prepare the TMB substrate
HRP inhibitor in sample/ buffersCheck that the samples/ buffers do not have sodium azide as it will inhibit peroxidase reaction.
Vigorous washingIf the washing is done manually, pipette the wash buffer gently.
Dried wellsDo not allow the wells to dry out during the assay. Seal with the supplied adhesive cover during incubations
Improper plate reader settingsCheck the wavelength and read the plate again

Erratic duplicate OD values

Probable Cause:Solution/ Action
Insufficient washing of the plateFill 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 dilutionsPipetting error. Check pipetting technique and calculations. Use calibrated pipettes.
Improper mixing of samples/ buffersMix the samples well before pipetting Thoroughly mix the working solutions of detection antibody/ streptavidin-HRP
Contamination from other wellsDo 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/ bufferIf 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 wellsDo not allow the wells to dry out during the assay. Seal with the supplied adhesive cover during incubations

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