|High sample concentration
||Use higher sample dilutions (Determine the optimal dilutions by titration assay)
||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)
||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
||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