Resources
ELISA Troubleshooting Guide
Poor Precision
Pipetting errors?
Ensure pipettes are calibrated on a regular basis and that operators have been adequately trained in pipetting techniques
Incomplete washing of plate?
Ensure that plates are washed according to the IFU and that wash buffer has been correctly prepared
Interruption of assay set-up?
Assay set-up should be continuous. Ensure required reagents are prepared before starting set-up of the ELISA
No Colour Development or Low Absorbance Values
Omission of one or more reagents?
Check all reagents have been added
Kit reagents have expired?
Check expiry dates of all reagents. No individual reagent expiry should be less than the overall expiry of the kit
Reagents added in the wrong order?
Check reagents have been added in the correct order
Reagents incorrectly prepared or contaminated?
Follow reagent preparation as indicated in the IFU. Ensure that the reagents are not contaminated
The wrong volumes of reagents were used?
Re-run the assay, taking care to use correct volumes
The conjugate was not prepared correctly?
Follow instructions on the conjugate dilution sheet. Ensure conjugate is pipette diluted with the conjugate diluent and not sonicated
The reagents were not brought to the correct temperature before using?
Ensure reagents are brought up to the appropriate temperature before use
Laboratory temperature was too low?
Maintain the laboratory temperature between 20 - 25°C. Do not run assays under air conditioning vents or near cold windows
Inadequate incubation times and temperatures?
Refer to IFU for correct incubation times and temperatures for each step of the ELISA
The correct wash protocol was not followed?
Refer to IFU for wash protocol
Incorrect wavelength used to read the plate?
Ensure the wavelength specified in the IFU is used to read the plate (e.g. 450nm)
High Absorbance Values
The wrong volumes of reagents were used?
Re-run the assay, taking care to use correct volumes of reagents
The reagents were prepared incorrectly or contaminated?
Ensure that reagents are prepared according to instructions and that they are not contaminated
The conjugate was not prepared correctly?
Check instructions on the conjugate dilution sheet. Ensure conjugate is pipette dilutes with the conjugate diluent and not sonicated
Substrate solution has deteriorated or become contaminated?
Make sure the substrate is colourless before adding to the plate
Sonication of buffers?
It is not necessary to sonicate any of the buffers used, follow exact procedure documented in the IFU
Laboratory temperature was too high?
Maintain the laboratory temperature between 20 - 25°C. Do not run assays in direct sunlight or near sources of heat
Incubation temperatures too high or incubation times too long?
Refer to IFU for correct incubation temperatures and times
Plate inadequately washed?
Refer to IFU for detailed plate washing procedure
Use of a plate shaker?
A gentle tap on the side of the microtitre plate is adequate. Use of a plate shaker is not necessary
Plate reader was not functioning properly?
Ensure the plate reader is calibrated regularly
Poor Standard Curve
Standards were added in the wrong order?
Re-run the assay ensuring the standards are applied to the microtitre plate in the correct order
If appropriate, standards may not have been correctly prepared?
Prepare standards as instructed in the IFU
Standards and other reagents were not added to the microtitre plate simultaneously?
Ensure standards and other reagents are ready to use before starting the assay to avoid disruption in assay set-up
Inadequate incubation times?
Refer to the IFU for the correct incubation times for each step of the ELISA and ensure it is incubated in a dark environmen
Low Sample Absorbance
Concentration of analyte in the sample is too high to be detected?
Further dilute the sample so that it can be read from the existing standard curve
The sample may not have been adequately diluted?
Ensure samples are diluted according to instruction
