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Require technical support on an ELISA you’re running? See our troubleshooting guide for common errors and how to solve them.
- High background
- No signal
- Too much signal – whole plate turned uniformly blue
- Standard curve achieved but poor discrimination between points
- Poor duplicates
- Poor assay to assay reproducibility
- No signal when a signal is expected, but standard curve looks fine
- Samples are reading too high, but standard curve looks fine
- Very Low Readings Across the Plate
- Green color develops upon addition of stop solution when using Streptavidin-HRP
- Edge Effects
- Drift
Problem: High Background
| Possible Source | Test or Action |
|---|---|
| Insufficient washing | See washing procedure on page 4 of the ELISA Development Guide |
| Increase number of washes Add a 30 second soak step inbetween washes |
Learn more about what causes high background and how to prevent it on our blog here.
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 |
| Contamination of HRP with | 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 |
Learn more about how to best reconstitute and prepare lyophilized reagents on our blog here.
Problem: Too much signal – whole plate turned uniformly blue
| Possible Source | Test or Action |
|---|---|
| Insufficient washing/washing step skipped - unbound peroxidase remaining | See washing procedure on page 4 of the ELISA Development Guide |
| 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 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 | See washing procedures on page 4 of the ELISA Development Guide 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 | See washing procedure on page 4 of the ELISA Development Guide 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 |
Learn more about what how to best prepare your samples for ELISA here.
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 |
For any inquiries or assistance, our team of accomplished experts is ready to provide the guidance and support required to propel your scientific endeavors forward. If a product does not perform in your experiment as described on our website or datasheet using the recommended protocol, please contact your local distributor or the Krishgen team within 12 months of product receipt.
Contact Tech Support
The Krishgen tech support team strives to provide swift responses and resolution to your queries. Please share as much information about your assay run, including raw data, to enable our technical experts to consult and troubleshoot.