Troubleshooting Guide: ELISA Development

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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


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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

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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

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