Troubleshooting Guide: ELISA
Problem: High Background
| Possible Source | Test or Action |
|---|---|
| Insufficient washing | Increase number of washes; Add a 30 second soak step inbetween washes |
Problem: No signal when a signal is expected
| Possible Source | Test or Action |
|---|---|
| Reagents added in incorrect order, or incorrectly prepared | Repeat assay; Check calculations and make new buffers, standards, etc. Review protocol |
| HRP is Contaminationed | Use fresh reagents |
| Not enough antibody used | Increase concentration |
| Standard has gone bad (if there is a signal in the sample wells) | Check that standard was handled according to directions. Use new vial. |
| Buffer containing FCS used to reconstitute antibodies | Requalify your reagents of choice |
| Capture antibody did not bind to plate | Use an ELISA plate (not a tissue culture plate) Dilute in PBS without additional protein |
| Buffers contaminated | Make fresh buffers |
Problem: Too much signal - whole plate turned uniformly blue
| Possible Source | Test or Action |
|---|---|
| Insufficient washing/washing step skipped - unbound peroxidase remaining | Ensure thorough washing between steps. |
| Substrate Solution mixed too early and turned blue | Substrate Solution should be mixed and used immediately |
| Too much streptavidin-HRP | Check dilution, titrate if necessary |
| Plate sealers or reagent reservoirs reused, resulting in presence of residual HRP. This will turn the TMB blue non-specifically | Use fresh fresh plate sealer and reagent reservoir for each step |
| Buffers contaminated with metals or HRP | Make fresh buffers |
Problem: Standard curve achieved but poor discrimination between points (low or flat curve)
| Possible Source | Test or Action |
|---|---|
| Not enough streptavidin-HRP | Check dilution, titrate if necessary |
| Capture antibody did not bind well to plate | Use an ELISA plate (not a tissue culture plate) Dilute in PBS without additional protein |
| Not enough detection antibody | Check dilution, titrate if necessary |
| Plate not developed long enough | Increase Substrate Solution incubation time Use recommended brand of Substrate Solution |
| Incorrect procedure | Go back to General ELISA Protocol; eliminate modifications, if any |
| Improper calculation of standard curve dilutions | Check calculations, make new standard curve |
Problem: Poor Duplicates
| Possible Source | Test or Action |
|---|---|
| Insufficient washing | If using an automatic plate washer, check that all ports are clean and free of obstructions, add a 30 second soak step and rotate plate halfway through the wash |
| Uneven plate coating due to procedural error or poor plate quality (can bind unevenly) | Dilute in PBS without additional protein Check coating and blocking volumes, times and method of reagent addition. Check plate used Use an ELISA plate (not a tissue culture plate) |
| Plate sealer reused | Use a fresh plate sealer for each step |
| No plate sealers used | Use plate sealers |
| Buffers contaminated | Make fresh buffers |
Problem: Poor assay to assay reproducibility
| Possible Source | Test or Action |
|---|---|
| Insufficient washing | If using an automatic plate washer, check that all ports are clean and free of obstructions |
| Variations in incubation temperature | Adhere to recommended incubation temperature Avoid incubating plates in areas where enviromental conditions vary |
| Variations in protocol | Adhere to the same protocol from run to run |
| Plate sealer reused, resulting in presence of residual HRP which will turn the TMB blue | Use fresh plate sealer for each step |
| Improper calculation of standard curve dilutions | Check calculations, make new standard curve Use internal controls |
| Buffers contaminated | Make fresh buffers |
Problem: No signal when a signal is expected, but standard curve looks fine
| Possible Source | Test or Action |
|---|---|
| No cytokine in sample | Use internal controls Repeat experiment, reconsider experimental parameters |
| Sample matrix is masking detection | Dilute samples at least 1:2 in appropriate diluent, or preferably, do a series of dilutions to look at recovery |
Problem: Samples are reading too high, but standard curve looks fine
| Possible Source | Test or Action |
|---|---|
| Samples contain cytokine levels above assay range | Dilute samples and run again |
Problem: Very Low Readings Across the Plate
| Possible Source | Test or Action |
|---|---|
| Incorrect wavelengths | Check filters/reader |
| Insufficient development time | Increase development time |
| Coated plates are old and have gone bad | Coat new plates |
| Capture antibody did not bind to the plate | Use an ELISA plate (not a tissue culture plate) Dilute in PBS without additional protein |
| Buffer containing FCS used to reconstitute antibodies | Requalify your reagents of choice |
Problem: Green color develops upon addition of stop solution when using streptavidin-HRP
| Possible Source | Test or Action |
|---|---|
| Reagents not mixed well enough in wells | Tap plate |
Problem: Edge Effects
| Possible Source | Test or Action |
|---|---|
| Uneven temperatures around work surface | Avoid incubating plates in areas where environmental conditions vary |
| Use plate sealers |
Problem: Drift
| Possible Source | Test or Action |
|---|---|
| Interrupted assay set-up | Assay set-up should be continuous - have all standards and samples prepared appropriately before commencement of the assay |
| Reagents not at room temperature | Ensure that all reagents are at room temperature before pipetting into the wells unless otherwise instructed in the antibody inserts |