Troubleshooting Guide: ELISA

This protocol is intended as a guide only.

Researchers can encounter a variety of problems when running an ELISA. Explore this page to understand the potential source of your problem and the steps we recommend for correcting the issue to help set you on a path for running a successful ELISA. For further assistance, please contact our technical service department.

High background
No signal
Too much signal - whole plate turned uniformly blue
Standard curve achieved but poor discrimination between points
Poor duplicates
Poor assay to assay reproducibility
No signal when a signal is expected, but standard curve looks fine
Samples are reading too high, but standard curve looks fine
Very Low Readings Across the Plate
Green color develops upon addition of stop solution when using streptavidin-HRP
Edge Effects
Drift

Problem: High Background

Possible Source Test or Action
Insufficient washing See washing procedure on page 4 of the ELISA Development Guide
Increase number of washes Add a 30 second soak step inbetween washes

Possible Source	Test or Action
Insufficient washing	See washing procedure on page 4 of the ELISA Development Guide
Increase number of washes Add a 30 second soak step inbetween washes

Problem: No signal when a signal is expected

Possible Source Test or Action
Reagents added in incorrect order, or incorrectly prepared Repeat assay Check calculations and make new buffers, standards, etc.

Review protocol
Contamination of HRP with Use fresh reagents
Not enough antibody used Increase concentration
Standard has gone bad (if there is a signal in the sample wells) Check that standard was handled according to directions.

Use new vial.
Buffer containing FCS used to reconstitute antibodies Requalify your reagents of choice
Capture antibody did not bind to plate Use an ELISA plate (not a tissue culture plate)

Dilute in PBS without additional protein
Buffers contaminated Make fresh buffers

Possible Source	Test or Action
Reagents added in incorrect order, or incorrectly prepared	Repeat assay Check calculations and make new buffers, standards, etc. Review protocol
Contamination of HRP with	Use fresh reagents
Not enough antibody used	Increase concentration
Standard has gone bad (if there is a signal in the sample wells)	Check that standard was handled according to directions. Use new vial.
Buffer containing FCS used to reconstitute antibodies	Requalify your reagents of choice
Capture antibody did not bind to plate	Use an ELISA plate (not a tissue culture plate) Dilute in PBS without additional protein
Buffers contaminated	Make fresh buffers

Problem: Too much signal - whole plate turned uniformly blue

Possible Source Test or Action
Insufficient washing/washing step skipped - unbound peroxidase remaining See washing procedure on page 4 of the ELISA Development Guide
Substrate Solution mixed too early and turned blue Substrate Solution should be mixed and used immediately
Too much streptavidin-HRP Check dilution, titrate if necessary
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
Buffers contaminated with metals or HRP Make fresh buffers

Possible Source	Test or Action
Insufficient washing/washing step skipped - unbound peroxidase remaining	See washing procedure on page 4 of the ELISA Development Guide
Substrate Solution mixed too early and turned blue	Substrate Solution should be mixed and used immediately
Too much streptavidin-HRP	Check dilution, titrate if necessary
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
Buffers contaminated with metals or HRP	Make fresh buffers

Problem: Standard curve achieved but poor discrimination between points (low or flat curve)

Possible Source Test or Action
Not enough streptavidin-HRP Check dilution, titrate if necessary
Capture antibody did not bind well to plate Use an ELISA plate (not a tissue culture plate)
Dilute in PBS without additional protein
Not enough detection antibody Check dilution, titrate if necessary
Plate not developed long enough Increase Substrate Solution incubation time

Use recommended brand of Substrate Solution
Incorrect procedure Go back to General ELISA Protocol; eliminate modifications, if any
Improper calculation of standard curve dilutions Check calculations, make new standard curve

Possible Source	Test or Action
Not enough streptavidin-HRP	Check dilution, titrate if necessary
Capture antibody did not bind well to plate	Use an ELISA plate (not a tissue culture plate) Dilute in PBS without additional protein
Not enough detection antibody	Check dilution, titrate if necessary
Plate not developed long enough	Increase Substrate Solution incubation time Use recommended brand of Substrate Solution
Incorrect procedure	Go back to General ELISA Protocol; eliminate modifications, if any
Improper calculation of standard curve dilutions	Check calculations, make new standard curve

Problem: Poor Duplicates

Possible Source Test or Action
Insufficient washing See washing procedures on page 4 of the ELISA Development Guide

If using an automatic plate washer, check that all ports are clean and free of obstructions, add a 30 second soak step and rotate plate halfway through the wash
Uneven plate coating due to procedural error or poor plate quality (can bind unevenly) Dilute in PBS without additional protein

Check coating and blocking volumes, times and method of reagent addition. Check plate used

Use an ELISA plate (not a tissue culture plate)
Plate sealer reused Use a fresh plate sealer for each step
No plate sealers used Use plate sealers
Buffers contaminated Make fresh buffers

Possible Source	Test or Action
Insufficient washing	See washing procedures on page 4 of the ELISA Development Guide If using an automatic plate washer, check that all ports are clean and free of obstructions, add a 30 second soak step and rotate plate halfway through the wash
Uneven plate coating due to procedural error or poor plate quality (can bind unevenly)	Dilute in PBS without additional protein Check coating and blocking volumes, times and method of reagent addition. Check plate used Use an ELISA plate (not a tissue culture plate)
Plate sealer reused	Use a fresh plate sealer for each step
No plate sealers used	Use plate sealers
Buffers contaminated	Make fresh buffers

Problem: Poor assay to assay reproducibility

Possible Source Test or Action
Insufficient washing See washing procedure on page 4 of the ELISA Development Guide

If using an automatic plate washer, check that all ports are clean and free of obstructions
Variations in incubation temperature Adhere to recommended incubation temperature

Avoid incubating plates in areas where enviromental conditions vary
Variations in protocol Adhere to the same protocol from run to run
Plate sealer reused, resulting in presence of residual HRP which will turn the TMB blue Use fresh plate sealer for each step
Improper calculation of standard curve dilutions Check calculations, make new standard curve

Use internal controls
Buffers contaminated Make fresh buffers

Possible Source	Test or Action
Insufficient washing	See washing procedure on page 4 of the ELISA Development Guide If using an automatic plate washer, check that all ports are clean and free of obstructions
Variations in incubation temperature	Adhere to recommended incubation temperature Avoid incubating plates in areas where enviromental conditions vary
Variations in protocol	Adhere to the same protocol from run to run
Plate sealer reused, resulting in presence of residual HRP which will turn the TMB blue	Use fresh plate sealer for each step
Improper calculation of standard curve dilutions	Check calculations, make new standard curve Use internal controls
Buffers contaminated	Make fresh buffers

Problem: No signal when a signal is expected, but standard curve looks fine

Possible Source Test or Action
No cytokine in sample Use internal controls

Repeat experiment, reconsider experimental parameters
Sample matrix is masking detection Dilute samples at least 1:2 in appropriate diluent, or preferably, do a series of dilutions to look at recovery

Possible Source	Test or Action
No cytokine in sample	Use internal controls Repeat experiment, reconsider experimental parameters
Sample matrix is masking detection	Dilute samples at least 1:2 in appropriate diluent, or preferably, do a series of dilutions to look at recovery

Problem: Samples are reading too high, but standard curve looks fine

Possible Source Test or Action
Samples contain cytokine levels above assay range Dilute samples and run again

Possible Source	Test or Action
Samples contain cytokine levels above assay range	Dilute samples and run again

Problem: Very low readings across the plate

Possible Source Test or Action
Incorrect wavelengths Check filters/reader
Insufficient development time Increase development time
Coated plates are old and have gone bad Coat new plates
Capture antibody did not bind to the plate Use an ELISA plate (not a tissue culture plate)

Dilute in PBS without additional protein
Buffer containing FCS used to reconstitute antibodies Requalify your reagents of choice

Possible Source	Test or Action
Incorrect wavelengths	Check filters/reader
Insufficient development time	Increase development time
Coated plates are old and have gone bad	Coat new plates
Capture antibody did not bind to the plate	Use an ELISA plate (not a tissue culture plate) Dilute in PBS without additional protein
Buffer containing FCS used to reconstitute antibodies	Requalify your reagents of choice

Problem: Green color develops upon addition of stop solution when using streptavidin-HRP

Possible Source Test or Action
Reagents not mixed well enough in wells Tap plate

Possible Source	Test or Action
Reagents not mixed well enough in wells	Tap plate

Problem: Edge Effects

Possible Source Test or Action
Uneven temperatures around work surface Avoid incubating plates in areas where environmental conditions vary
Use plate sealers

Possible Source	Test or Action
Uneven temperatures around work surface	Avoid incubating plates in areas where environmental conditions vary
Use plate sealers

Problem: Drift

Possible Source Test or Action
Interrupted assay set-up Assay set-up should be continuous - have all standards and samples prepared appropriately before commencement of the assay
Reagents not at room temperature Ensure that all reagents are at room temperature before pipetting into the wells unless otherwise instructed in the antibody inserts

Possible Source	Test or Action
Interrupted assay set-up	Assay set-up should be continuous - have all standards and samples prepared appropriately before commencement of the assay
Reagents not at room temperature	Ensure that all reagents are at room temperature before pipetting into the wells unless otherwise instructed in the antibody inserts