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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.
|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 NoiseClick for more troubleshooting tips