ELISA Troubleshooting

Problem Possible Causes Action/solution
No signal Assay set up incorrectly or used incorrect reagents   Review protocol. Repeat assay using a positive control  
  Incorrect secondary antibody used Retrace steps. Repeat assay using the correct secondary antibody  
  Not enough antibody used Increase concentration of the primary and/or secondary antibody
  Detection reagent too old or contaminated   Use fresh detection reagents
  Antigen not coated properly Try longer coating times, different coating buffers, or avidin plates with biotinylated antigen
  Plate reader has the wrong settings Check plate reader for wavelength, filters, gain etc.  
  Antibody stored at 4°C for several weeks or subjected to repeated freeze/thaw cycles Use a fresh aliquot of antibody that has been stored at -20°C or below
Weak signal Insufficient amount of antigen was coated to microtiter plate Use more antigen for coating or very coating buffer
  Not enough antibody used Increase concentration of the primary and/or secondary antibody. Optimize antibody concentrations for your assay
  Detection reagent too old, contaminated, or used at the wrong pH Use fresh detection reagents at the correct pH
  Detection reagent too dilute Use a higher concentration of detection reagent
  Plate reader has the wrong settings Check plate reader for wavelength, filters, gain etc
  Incubation temperature too low Optimize the incubation temperature for your assay. Reagents should be at room temperature before beginning the assay
High background signal Too much antibody used Reduce the concentration of the primary and/or secondary antibody. Optimize antibody concentrations for your assay
  Non-specific antibody binding Use a suitable blocking buffer or use an affinity-purified antibody
  Too much detection reagent used Repeat assay with a higher dilution of detection reagent
  Too few washing cycles Increase the number of washing cycles  
  Contaminated blocking agent   Use fresh blocking agent  
  Wrong concentration of blocking agent   Check the concentration of blocking agent in the recommended protocol
  Presence of blocking buffer interferes with antibody binding   Wash off blocking buffer before adding antibody  
  Reaction not stopped Use stop solution to prevent overdevelopment
  Plate left too long before reading Start taking measurements shortly after the addition of detection reagent
  Wrong settings on the plate reader Check settings and adjust as needed
  Insufficient amount of Tween® in the buffers Use PBS containing 0.05% Tween®
  Incubation with substrate carried out in the light Perform substrate incubation in the dark
  Incubation temperature too high Optimize the incubation temperature for your assay
  Plates stacked during incubations leading to uneven temperature distribution Avoid stacking plates
  Pipetting errors Calibrate pipettes so that they dispense the correct volumes
  Reagents were not mixed properly Before pipetting solutions into wells, make sure all reagents and samples have been throughly mixed
  Inconsistent washing of wells Take precautions to reduce variability of washes
  Uneven evaporation of solution from wells during incubation Always incubate with a lid on the plate
  Interference caused by dirt on the bottom of the plate Clean the plate carefully and reread
Slow color development Incubation temperature is wrong Ensure plates and reagents are kept at room temperature
  Contaminated solutions Make fresh solutions
  Detection reagent too old, contaminated or used at the wrong pH Use fresh detection reagents at the correct pH

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