2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:51 PM Page 51
ELISA Troubleshooting Guide
POSSIBLE PROBLEM POSSIBLE CAUSE OR EXPERIMENTAL VARIABLES POSSIBLE CORRECTIVE ACTION
Blank well absorbance too high Contaminated reagents or contaminated well Check the storage condition. Repeat with new
reagents from the same lot if possible.
Blank wells with sample and conjugates Use only buffer in blank wells.
Positive standards values Expired reagents or reagents deterioration Check the expiration date and storage conditions.
lower than the stated range
Used components from different lots or Use only components from the same lot and do not
manufacturers substitute components from different kits or from
different suppliers.
Incubation time too short or incubation Follow instruction and check incubation
temperature too low conditions and equipment.
Incorrect readings Read the results within 5 minutes after the
Dilution error reaction. Check the reader optical pathway
and wavelength.
Signal suppression by serum matrix Prepare the standard curve in 50% serum (diluted in
sample diluent). Also, use 50% serum as the blank.
To get an accurate backfitted concentration of the
unknown sample, dilute the serum sample
(unknown) to 50% in sample diluent as well.
Poor Precision Incomplete washing and/or aspiration of the wells Ensure that wash apparatus is working correctly.
If washing by multi-channel pipettor, be sure that
all channel uptakes are the same.
Unequal mixing of reagents Ensure adequate mixing.
Edge Effects Experiment performed under uneven Ensure that laboratory temperature is in the 20-26°C
temperature. range. If possible, perform experiment in a controlled
temperature incubator set at 24°C.
Plate was not covered during incubation steps. Ensure that the plate is properly covered during
incubation periods.
Drift Effect Uneven air flow in the laboratory where Ensure that experiment is done under an enclosed
the experiment was carried area with constant temperature.
TMB substrate was not at room temperature Ensure that TMB substrate is properly equilibrated
to room temperature. To achieve proper
equilibration, incubate the substrate (initially
stored at 4°C) for at least 30 minutes at room temp
in dark prior to use.
Long time intervals between addition Ensure that reagents were added to all the wells in a
of reagents to the wells short period of time.
Poor Standard Curve Improper dilution of reagents, or Ensure correct dilution steps were taken and that
contamination of blank buffer appropriate buffer is used as blank.
Unequal volume of reagents used Check pipette function and calibration. Ensure that
correct pipetting technique is used.
Incomplete washing of the wells Ensure that wash apparatus is working correctly. If
washing by multi-channel pipettor, be sure that all
channel uptakes are equivalent.
Plate defects or contamination Check plate for scratches and fingerprints.
Improper curve fitting Perform 4-Parameter (logistic-log-model)
curve fitting.
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