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 ELISAs 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 ELISAs.

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.

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.
2. Reaction not stopped Colour will keep developing if the substrate reaction is not stopped.
3. Plate left too long before reading on the plate reader Colour will keep developing (though at a slower rate if stop solution has been added)
4. Contaminants from laboratory glassware Ensure reagents are fresh and prepared in clean glassware
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.
6. Wells are insufficiently washed Wash wells as per protocol recommendations.
7. Too much detection reagent added Ensure the reagent has been diluted properly or decrease the recommended concentration of detection reagent.
8. Blocking buffer ineffective (e.g. detection reagent binds blocker; wells not completely blocked) Try different blocking reagent and/or add blocking reagent to wash buffer.
9. Salt concentration of incubation/wash buffers Increasing salt concentrations may reduce non-specific and/or weak off-target interactions.
10. High antibody concentration Try different dilutions for optimal results.
11. Precipitate formed in wells upon substrate addition Increase dilution factor of sample or decrease concentration of substrate.
12. Dirty plate Clean the bottom of the plate
13. Incorrect standard curve dilutions
prepared
Check your pipetting technique. Calibration of pipettes might be required.
14. Longer incubation times than
recommended
Make sure your incubation times are correct and adhere to the protocol provided with the technical manual
15. Plate sealers or reagent reservoirs reused, resulting in presence of residual HRP. This will turn the TMB blue non-specifically

 

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
16. Contamination of buffers Always make fresh buffers

 

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.
18. Samples contain analyte concentrations higher than the highest standard point Samples may require further dilution
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.
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.
21. Incorrect dilutions prepared Check pipetting technique—see below—and double-check calculations.
22. Longer incubation times than
recommended
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.
23. Substrate solution mixed too early and turned blue Substrate solution should be mixed and used immediately
24. too much streptavidin-HRP Check dilution, titrate if necessary
25. 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
26. Buffers contaminated with metals or HRP Make fresh buffers

High Variation

27. Multichannel pipette errors Calibrate the pipettes
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
29. Non-homogenous samples Thoroughly mix samples before pipetting
30. Samples may have high particular matter Remove the particulate matter by centrifugation
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
32. Cross well contamination When reusing plate sealers check that no reagent has touched the sealer. Care should be taken when using the same pipette tips used for reagent additions. Ensure that pipette tips do not touch the reagents on the plate.
33. Plates stacked during the incubations Stacking of plates does not allow even distribution of temperature across the wells of the plates. Avoid stacking.
34. Pipette inconsistent Ensure pipettes are working correctly and are calibrated. Ensure pipette tips are pushed on far enough to create a good seal. Take particular care when diluting down the plate and watch to make sure the pipette tips are all picking up and releasing the correct amount of liquid.
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.
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.
37. Bottom of the plate is dirty Clean the bottom of the plate carefully before re-reading the plates
38. Bubbles in wells Ensure no bubbles are present prior to reading the plate
39. Edge effects Ensure the plate and all reagents are at room temperature
40. Storage Ensure reagents and samples are stored are correct temperature
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.
42. Variations in protocols Adhere to the protocol that comes with your  assay
43. Improper calculations of standard curve Check calculations, make new standard curve & use internal controls
44. Buffers contaminated Use fresh buffers
45. Well bottom scrapped 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

Background is high

46. Background wells were contaminated Avoid cross-well contamination by using the sealer appropriately. Use multichannel pipettes without touching the reagents on the plate.
47. Matrix used has endogenous analyte or interference Check the matrix ingredients for cross reacting components (e.g. interleukin modified tissue culture medium).
48. Insufficient washes Increase number of washes. Increase soaking time between washes prior to addition of substrate solution.
49. Cross-Reactivity Detection antibody cross-reacting with coating antibody. Run appropriate controls.
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
51.. Concentration of conjugated second antibody too high Perform dilutions to determine optimal working concentration.
52.. Incorrect assay temperature Check that the incubation temperature did not exceed 37°C
53. Inadequate washing 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.
54. Contaminating enzymes present in sample Test sample with substrate alone to check for contaminating enzyme activity.
55. Wells are insufficiently washed Wash wells are per protocol recommendations.
56. Contaminated wash buffer Prepare fresh buffers
57. Too much detection reagent Ensure the reagent has been diluted properly or decrease the recommended concentration of detection reagent
58. Blocking buffer ineffective Try different blocking buffer reagent and/or add blocking reagent to wash buffer
59. Salt concentrations of incubation/wash buffers Increasing salt concentrations may reduce non-specific and/or weak off target interactions.
60. Waiting too long to read plate after addition of stop solution Read plate immediately after adding stop solution
61. High antibody concentration Try different dilutions of optimal results
62. substrate incubation is carried out in light Substrate incubations should be carried out in the dark or as recommended by manufacturer.
63. Precipitate formed in wells upon substrate addition Increase dilution factor of sample or decrease concentration of substrate
64. Dirty plate Clean the bottom of the plate with a wipe

No Signal

65. Incorrect or no detection antibody was added add appropriate detection antibody and continue
66. Avidin-HRP was not added Add avidin-HRP according to protocol and continue
67. substrate solution was not added add substrate solution and continue
68. wash buffer contains azide Avoid sodium azide in the wash buffer
69. Incubation time too short Incubate samples overnight at 4’C or follow manufacturers guidelines
70. Target present below detection limits of assay Decrease dilution factor or concentrate samples
71. Incompatible sample type Detection may be reduced or absent in untested samples types. Include a sample that the assay is known to detect the positive control
72. Recognition of epitope impeded by absorption plate 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
73. Assay buffer incompatibility Ensure assay buffer is compatible with the target of interest (e.g. enzymatic activity retained, protein interactions retained.)
74. Not enough detection reagent Increase concentration of amount of detection reagent following manufacturer guidelines
75. Sample prepared incorrectly Ensure proper sample preparation/dilution. Samples may be incompatible with microtiter plate assay format
76. Insufficient antibody Try different concentrations/dilutions of antibodies
77. Incubation temperature is too low Ensure the incubations are carried out at the correct temperature. All reagents including plate should be at room temperature or as recommended by the manufacturer before proceeding.
78. Incorrect wavelength Verify the wavelength and read the plate again
79. Plate washing is too vigorous Check the correct pressure in the automatic plate washer. Pipette wash buffer gently if washes are done manually.
80. Wells dried out Do not allow wells to become dry once the assay has started. Cover the plate using sealing film or tape for all incubations.
81. Slow colour development of enzymatic reactions Prepare substrate solution immediately before use. Ensure the stock solution has not expired and is not contaminated. Allow longer incubation.
82. Uneven Colour

 

Ensure all wells are washed correctly, use a ELISA plate washer where possible
83. 

 

Reagents not at room temperature

 

All reagents should at room temperature from the start of the assay. Room temperature should be reached following 15–20 minutes on the bench.
84. Expired Reagents Ensure all reagents used are within date
85.

 

 

 

 

Assay format not sensitive enough 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
86. Buffer containing FCS used to reconstitute antibodies Re-evaluate reagents used.

     Poor standard curve

87. standard was incompletely reconstituted or was incorrectly stored Reconstitute standard according to the protocol provide and follow storage instructions
88. Reagents were added to the wells at incorrect concentrations Check for pipetting errors and correct the reagent volume
89. incubations done at incorrect temperature Follow protocol for storage, incubation and agitation
90. Wells not completely aspirated Completely aspirate between steps, use plate wash where possible
91. Plates stacked during incubation Keep plates separated
92. Poor dilution series Check dilution steps according to protocol
93. Reagents poorly mixed Make sure to mix reagents thoroughly
94. Poor or variable adsorption of reagents to plate 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
95. Standard degraded Check that standard was stored correctly
96. Curve doesn’t fit scale Try plotting use different scales, e.g. log-log, 5 parameter logistic curve fit
97. Pipetting error Check pipettes and calibrate
98. Capture antibody didn’t bind to the plate Ensure that you are using and ELISA plate, not a tissue culture plate.
99. Not enough detection antibody Check dilution, titrate if necessary
100.

 

Incorrect calculation of standard curve dilution Check your calculations and make a new curve.
101. Mixing or Substituting reagents from different kits Avoid this as it can affect the quality of your assay