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-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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