Dear Earthling,

If you are having trouble with saturated signals in your ELISA data results, check out this table for Boster’s possible solutions to your problem:

Possible Causes Possible Solutions
High sample concentration Use higher sample dilutions (Determine the optimal dilutions by titration assay)
Excessive substrate Decrease concentration or amount of substrate: Follow manufacturer guidelines (The substrate provided with the ELISA kit might require further dilution)
Substrate color changed before use Make substrate immediately before use
Non-specific antibody binding Try different formulations in coating solutions; Ensure wells are pre-processed to prevent non-specific binding; Use affinity-purified antibody and preferably one that is pre- adsorbed; Use serum (5-10%) from same species as secondary antibody (bovine serum is also recommended).
Incubation time too long Follow the manufacturer guidelines (If the problem persists, try incubating samples at 4°C overnight)
Excess antibody Repeat the assay with lower antibody concentrations to find the optimal one for your experiment
Contaminated buffers with metals or HRP Make and use fresh buffers
Insufficient washing Follow the manufacturer guidelines; At the end of each washing step, flick the plate over a sink and pat the plate on a paper towel.
Plate sealers not used or re-used During incubations, cover plates with plate sealers; Use a fresh sealer every time the used sealer is removed from the plate.
Plate read at incorrect detection wavelength Use recommended wavelength/filter; Ensure plate reader is set correctly for type of substrate used.
Excess time before plate reading Read your plate within 30 minutes after adding the substrate (If the reading is not performed within this time frame, add a stopping solution after sufficient color is developed in the plate)

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