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.

2.

Reaction not stopped

Colour will keep developing if the substrate reaction is not stopped.

3.

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.

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.

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.

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.

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.

Always make fresh buffers.

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.

Use fresh plate sealer and reagent reservoir for each step

26.

Buffers contaminated with metals or HRP.

Make fresh buffers

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

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

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.

Clean the bottom of the plate carefully before re-reading the plates

38.

Bubbles in wells

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

43.

Improper calculations of standard curve

Check calculations, make new standard curve & use internal controls

44.

Buffers contaminated

45.

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

46.

47.

Check the matrix ingredients for cross reacting components (e.g. interleukin modified tissue culture medium).

48.

49.

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.

Perform dilutions to determine optimal working concentration.

52.

Check that the incubation temperature did not exceed 37°C

53.

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.

Prepare fresh buffers

57.

Ensure the reagent has been diluted properly or decrease the recommended concentration of detection reagent

58.

59.

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.

Increase dilution factor of sample or decrease concentration of substrate

64.

Clean the bottom of the plate with a wipe

65.

66.

67.

Add substrate solution and continue

68.

69.

Incubation time too short

70.

71.

72.

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

75.

Sample prepared incorrectly

76.

Insufficient antibody

Try different concentrations/dilutions of antibodies

77.

78.

Verify the wavelength and read the plate again

79.

80.

Wells dried out

81.

Prepare substrate solution immediately before use. Ensure the stock solution has not expired and is not contaminated. Allow longer incubation.

82.

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

83.

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.

85.

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.

Re-evaluate reagents used.

87.

Reconstitute standard according to the protocol provide and follow storage instructions

88.

Check for pipetting errors and correct the reagent volume

89.

90.

Wells not completely aspirated

Completely aspirate between steps, use plate wash where possible

91.

Plates stacked during incubation

Keep plates separated

92.

93.

94.

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.

Try plotting use different scales, e.g. log-log, 5 parameter logistic curve fit

97.

Pipetting error

98.

Ensure that you are using and ELISA plate, not a tissue culture plate.