101 ELISA Troubleshooting Tips
101 ELISA Troubleshooting Tips
Our 101 ELISA troubleshooting tips guide is designed to help you improve and troubleshoot the common problems that researchers have with their ELISA kits when performing assays. Optimising your ELISA and removing common mistakes that are made can dramatically improve your results and the sensitivity of your ELISA assays. In this ELISA troubleshooting guide we have detailed the common areas where researchers encounter problems with their ELISA.
ELISA Troubleshooting areas
High Signal:
High Signal can occur for numbers reasons including insufficient plate washing, not stopping the reaction and adding too much detection reagent. If you have a high signal this can results in a lot of false positives and incorrect data.
Out of Range:
Sometimes this can happen based on your samples, insufficient washing or incorrect dilutions prepared. This can result in a loss of data due to negative or no results.
High Variation:
High variation can be due to sample preparation mistakes, pipette errors and inconsistencies, insufficient plate agitation among other problems. Data with high variation can skew the real results and cause inconsistencies in your data.
Background is high
High background may result from inadequate washing steps, cross reactivity of samples or contamination. Again high background may result in false positive/negative data and affect your results.
No Signal
No signal in your ELISA assay may result from numerous sample and assay problems including wash buffer contains azide, target below detection of assay or avidin-HRP was not added. No signal may mean no results from precious samples, have a read through the reasons below to avoid these problems.
Poor Standard Curve
A poor standard curve will prove unpublishable results if not prepared correctly. Reasons may included reagents are poorly mixed, the standard has degraded or pipetting errors.
ELISA troubleshooting for High Signal
1. |
TMB Substrate Solution was contaminated |
Use fresh TMB substrate solution which should be clear and colorless prior to addition to wells. Use a clean V bottom container prior to pipetting substrate solution into wells. Use a clean V bottom container prior to pipetting substrate solution into wells. |
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2. |
Reaction not stopped |
Colour will keep developing if the substrate reaction is not stopped. |
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3. |
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Colour will keep developing (though at a slower rate if stop solution has been added) |
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4. |
Contaminants from laboratory glassware |
Ensure reagents are fresh and prepared in clean glassware |
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5. |
Substrate incubation carried out in the light |
Substrate incubation should be carried out in the dark. Ensure substrate is not exposed to light—store in a dark place. Limit exposure to light while running assay. |
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6. |
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Wash wells as per protocol recommendations. |
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7. |
Too much detection reagent added |
Ensure the reagent has been diluted properly or decrease the recommended concentration of detection reagent. |
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8. |
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Try different blocking reagent and/or add blocking reagent to wash buffer. |
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9. |
Salt concentration of incubation/wash buffers |
Increasing salt concentrations may reduce non-specific and/or weak off-target interactions. |
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10. |
High antibody concentration |
Try different dilutions for optimal results. |
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11. |
Precipitate formed in wells upon substrate addition |
Increase dilution factor of sample or decrease concentration of substrate. |
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12. |
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Clean the bottom of the plate. |
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13. |
Incorrect standard curve dilutions |
Check your pipetting technique. Calibration of pipettes might be required. |
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14. |
Longer incubation times than |
Make sure your incubation times are correct and adhere to the protocol provided with the technical manual. |
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15. |
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Reuse of plate sealers may lead to the presence of residual HRP, leading to non- specific colour change of TMB. To avoid this use fresh plate sealer and reagent reservoir for each step. |
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16. |
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Always make fresh buffers. |
ELISA Troubleshooting for out of Range
17. |
Samples contain no or below detectable levels of analyte |
If samples are below detectable levels, it may be possible to use high sample volume. Check with technical support for appropriate protocol modifications. |
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18. |
Samples contain analyte concentrations higher than the highest standard point |
Samples may require further dilution |
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19. |
Insufficient washing |
Use appropriate washing procedure—see below. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid. |
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20. |
Plate sealers not used or reused |
During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other. |
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21 |
Incorrect dilutions prepared |
Check pipetting technique—see below—and double-check calculations. |
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22. |
Longer incubation times than |
Manufactured kits have optimized protocols. Make sure to follow recommended incubation times. If developing ELISA using antibody pairs you may need to optimize the assay. See ELISA Development and Optimization for more information. |
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23. |
Substrate solution mixed too early and turned blue |
Substrate solution should be mixed and used immediately |
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24. |
Too much streptavidin-HRP |
Check dilution, titrate if necessary |
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25. |
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Use fresh plate sealer and reagent reservoir for each step |
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26. |
Buffers contaminated with metals or HRP. |
Make fresh buffers |
ELISA Troubleshooting for High Variation
27. |
Multichannel pipette errors |
Calibrate the pipettes |
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28. |
Plate washing was not adequate or uniform |
Make sure pipette tips are tightly secured. Confirm all reagents are removed complete in all wash steps |
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29. |
Non-homogenous samples |
Thoroughly mix samples before pipetting |
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30. |
Samples may have high particular matter |
Remove the particulate matter by centrifugation |
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31. |
Insufficient plate agitation |
The plate should be agitated during all incubation steps using an ELISA plate shaker at a speed where solutions in wells are within constant motion without splashing |
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32. |
Cross well contamination |
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33. |
Plates stacked during the incubations |
Stacking of plates does not allow even distribution of temperature across the wells of the plates. Avoid stacking. |
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34. |
Pipette inconsistent |
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35. |
Antibody dilutions/reagents are not well mixed |
To ensure a consistent concentration across all wells, ensure all reagents and samples are mixed before pipetting onto the plates. |
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36. |
Well allowed to dry out |
Ensure lids are left on the plates at all times when incubating. Place a humidifying water tray (bottled clean/sterile water) in the bottom of the incubator. |
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37. |
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Clean the bottom of the plate carefully before re-reading the plates |
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38. |
Bubbles in wells |
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39. |
Edge effects |
Ensure the plate and all reagents are at room temperature |
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40. |
Storage |
Ensure reagents and samples are stored are correct temperature |
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41. |
Capture antibody didn’t bind to the plate |
Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps. |
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42. |
Variations in protocols |
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43. |
Improper calculations of standard curve |
Check calculations, make new standard curve & use internal controls |
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44. |
Buffers contaminated |
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45. |
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Avoid contact with the bottom of the well during pipetting. Aim the pipette tip to the side of the well to avoid disrupting the bottom |
ELISA Troubleshooting for Background is high
46. |
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47. |
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Check the matrix ingredients for cross reacting components (e.g. interleukin modified tissue culture medium). |
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48. |
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49. |
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Detection antibody cross-reacting with coating antibody. Run appropriate controls. |
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50. |
Non-specific binding of antibodies |
Ensure a block step is included and a suitable blocking buffer is being used. We recommend using 5 to 10% serum from the same species of the secondary antibody, or bovine serum. Ensure wells are pre-processed to prevent nonspecific attachment Use an affinity purified antibody, preferably pre-absorbed |
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51. |
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Perform dilutions to determine optimal working concentration. |
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52. |
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Check that the incubation temperature did not exceed 37°C |
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53. |
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Ensure all wells are filling with wash buffer and are being aspirated completely. Use an automated plate washer if available. Increase number of washes. Add 30 second soak step in-between washes. |
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54. |
Contaminating enzymes present in sample |
Test sample with substrate alone to check for contaminating enzyme activity. |
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55. |
Wells are insufficiently washed |
Wash wells are per protocol recommendations. |
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56. |
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Prepare fresh buffers |
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57. |
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Ensure the reagent has been diluted properly or decrease the recommended concentration of detection reagent |
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58. |
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59. |
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Increasing salt concentrations may reduce non-specific and/or weak off target interactions. |
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60. |
Waiting too long to read plate after addition of stop solution |
Read plate immediately after adding stop solution |
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61. |
High antibody concentration |
Try different dilutions of optimal results |
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62. |
Substrate incubation is carried out in light |
Substrate incubations should be carried out in the dark or as recommended by manufacturer. |
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63. |
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Increase dilution factor of sample or decrease concentration of substrate |
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64. |
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Clean the bottom of the plate with a wipe |
ELISA Troubleshooting for No Signal
65. |
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66. |
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67. |
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Add substrate solution and continue |
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68. |
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69. |
Incubation time too short |
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70. |
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71. |
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72. |
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To enhance the detection of a peptide by direct or indirect ELISA, conjugate peptide to a large carrier protein before coating onto a microtiter plate |
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73. |
Assay buffer incompatibility |
Ensure assay buffer is compatible with the target of interest (e.g. enzymatic activity retained, protein interactions retained.) |
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74. |
Not enough detection reagent |
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75. |
Sample prepared incorrectly |
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76. |
Insufficient antibody |
Try different concentrations/dilutions of antibodies |
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77. |
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78. |
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Verify the wavelength and read the plate again |
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79. |
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80. |
Wells dried out |
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81. |
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Prepare substrate solution immediately before use. Ensure the stock solution has not expired and is not contaminated. Allow longer incubation. |
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82. |
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Ensure all wells are washed correctly, use a ELISA plate washer where possible |
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83. |
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All reagents should at room temperature from the start of the assay. Room temperature should be reached following 15–20 minutes on the bench. |
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84. |
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85. |
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Switch to a more sensitive assay type (e.g. direct ELISA to sandwich ELISA). Lengthen incubation times or increase temperature. Or change the detection method |
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86. |
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Re-evaluate reagents used. |
ELISA Troubleshooting for Poor standard curve
87. |
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Reconstitute standard according to the protocol provide and follow storage instructions |
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88. |
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Check for pipetting errors and correct the reagent volume |
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89. |
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90. |
Wells not completely aspirated |
Completely aspirate between steps, use plate wash where possible |
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91. |
Plates stacked during incubation |
Keep plates separated |
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92. |
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93. |
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94. |
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Check choice of coating buffer, usually PBS with a pH of 7.4 or carbonate bicarbonate buffer pH 9.6. Try extending this incubation time or consider using different plates |
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95. |
Standard degraded |
Check that standard was stored correctly |
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96. |
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Try plotting use different scales, e.g. log-log, 5 parameter logistic curve fit |
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97. |
Pipetting error |
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98. |
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Ensure that you are using and ELISA plate, not a tissue culture plate. |
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99. |
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Check dilution, titrate if necessary |
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100. |
Incorrect calculation of standard curve dilution |
Check your calculations and make a new curve. |
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101. |
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Avoid this as it can affect the quality of your assay |