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Troubleshooting Guides - ELISA

When trouble shooting, prepare all fresh buffers. Sterile filter buffers containing BSA and allow all reagents to reach room temperature prior to use.

Problem Possible Source                                  Suggestion
High Background/ 
Non-specific Color
Development       
Non-specific binding
of antibody
Modify blocking buffer used (e.g. substitute casein for BSA). Do not wash after blocking step; dump, blot & go directly to the next step.
  Plate not washed sufficiently Be sure all wells are filled with buffer during every wash step. Be sure washing apparatus is working properly. Wash 3-5x between steps. Do not add additional washes. After final wash, blot plate forcefully on paper towel to remove residual buffer. Be sure the correct amount of Tween was added to the wash solution (0.01- 0.1% recommended).
  Contaminated Buffers Prepare all buffers fresh (within 7 days of use) and sterile filter.
  Incubation temperature too high Incubate at room temperature (25ºC) throughout whole procedure.
  Incubation time too long Reduce incubation time.
  Contamination of the substrate with the conjugate or contamination of blank wells with positive control Change pipette tips between reagents and use separate reservoirs. Ideally, wash buffer should be aspirated (pouring/dumping may lead to cross contamination).
  Concentration of detection antibody or avidin-HRP is too high Check calculations or try further dilutions.
  Substrate exposed to light prior to use Keep substrate in the dark until ready to dispense into wells.
  Wrong filter used when taking readings Wavelength should be 405nm with a 650nm wavelength correction for ABTS, or 450nm with a 620nm wavelength correction for TMB, when using recommended plates.
  Read beyond time needed for development Read at 5 minute intervals in order to monitor blank
O.D. readings.
Weak/No Color Development A reagent or a step of the procedure omitted by mistake Check protocol and follow steps carefully.
  Detergent concentration in wash buffer too high Remove or decrease detergent (0.01-0.1% recommended).
  Plate washed inadequately Decrease number of washes between steps, especially if not using automated or semi- automated washer. 1-2 washes may suffice.
  Enzyme inhibitor present
in samples
Sodium azide inhibits peroxidase activity.
  Wrong incubation time or temperature Check and follow protocol recommendations. Place plates in an incubator during incubation periods to avoid temperature fluctuations (set to 25ºC).
  Wrong substrate volume added to wells Check pipette; use calibrated pipettes.
  Wrong enzyme-substrate
system used
Check manufacturer specifications:
avidin-HRP+ ABTS/ streptavidin + TMB.
  Conjugate or substrate inactive Test activity in another system and check expiration dates.
  Incorrect storage of components Store all components as recommended on data sheet.
  Wrong filter used when
taking readings
Wavelength should be 405nm with a 650nm wavelength correction for ABTS, or 450nm with a 620nm wavelength correction for TMB, when using recommended plates.
Poor Standard Curve Incorrect preparation of standard Reconstitute standard as suggested on data sheet. Dilute
only with recommended diluent buffer unless alternative buffer has been optimized.
  Dilutions made too early Prepare reagents immediately prior to use and add to
wells promptly.
  Capture Antibody did not bind Use ELISA-suitable plates (i.e. Nunc MaxiSorp Prod. # 439454); not tissue culture plates.
  Inefficient washing Be sure wash apparatus is working properly (i.e. distributing even volumes into each well). Be sure wells are empty after aspiration, yet be sure to fill wells in a timely manner.
  Pipetting error Dispense quickly and identically into the side of each well. Use calibrated pipettes.
  Read beyond time needed
for development
In general, reliable standard curves are obtained for ABTS kits when O.D. readings do not exceed 0.2 for blanks or 1.2 for the highest standard concentration and for TMB kits when O.D. readings do not exceed 0.15 for the blanks.
  Incorrect storage of components Store all components as recommended on data sheet. Do not allow reconstituted reagents to stay at room temperature for excess time.
Poor Precision Insufficient mixing of reagents Ensure adequate mixing of reagents before pipetting.
  Inefficient washing Be sure wash apparatus is working properly (i.e. distributing even volumes into each well). Be sure wells are empty after aspiration, yet be sure to fill wells in a timely manner.
  Pipetting error Dispense quickly and identically into the side of each well. Use calibrated pipettes.
  Reused materials Change pipette tips between samples, reservoirs between reagents, and plate sealers between incubation periods.
  Wells have been scratched by pipette tips or washing apparatus Use caution when dispensing and aspirating.
  Precipitate in samples Centrifuge vials prior to use.
  Unclean wells or debris Inspect wells and remove debris prior to use. Wipe bottom of plate to remove any debris or fingerprints prior to reading.
Edge Effects Evaporation Use plate sealers between each step.
  Uneven temperature Place plates in an incubator during incubation periods to avoid temperature fluctuations (set to 25ºC).
Do not stack plates.