ELISA Technical Resources

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Troubleshooting guides

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ELISA Troubleshooting: High Background

The signal-to-noise ratio of your ELSIA assay is critical for the proper interpretation of results. High background signal can ruin the data of an entire assay. Before you redo your experiment, consult this guide for tips to help resolve the source of your high background results.

Problem Cause Solution
Antibody problems Excess antibody Lower antibody concentration
Run an optimization trial to determine most effective concentration
Cross reactivity Run negative control to determine cross reactivity
Cross-adsorb your antibody against cross-reactive targets
Use Boster antibodies guaranteed to bind only their specified targets
Non-specific antibody binding Use a different formulation of coating solution
Ensure wells are pre-processed
Use affinity purified antibody
Block with serum from same species as secondary antibody
Buffer/substrate problems Ineffective/contaminated blocking buffer Use higher blocking protein concentration
Increase blocking time
Mix fresh buffer
Excess substrate Reduce concentration of substrate
Optimize substrate concentration
Insufficient Tween 20 in buffers Use PBS containing 0.05% Tween
Suboptimal salt concentration in wash buffer Optimize salt concentration to reduce nonspecific interactions
Plate error Dirty or defective plates Wash plates before coating
Use Boster pre-coated ELISA plates
Unstopped color development Quench the color development reaction to avoid over-development

Keywords: ELISA, Troubleshooting, High Background, Saturated Background, High Signal to Noise

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