ELISA: Troubleshooting and Advice


Below are a list of common problems encountered within ELISA together possible causes and subsequent actions / solutions:

  1. No signal
  2. Weak Signal
  3. High Background Signal
  4. Slow Color Development

For further help and expert advice, contact our scientist staffed technical support department here.


Problem 1: No signal

Possible causes and solutions for no signal:

a. Assay set up incorrectly or used incorrect reagents

  • Review protocol. Repeat assay using a positive control

b. Incorrect secondary antibody used

  • Retrace steps. Repeat assay using the correct secondary antibody

c. Not enough antibody used

  • Increase concentration of the primary and/or secondary antibody

d. Detection reagent too old or contaminated

  • Use fresh detection reagents

e. Antigen not coated properly

  • Try longer coating times, different coating buffers, or avidin plates with biotinylated antigen

f. Plate reader has the wrong settings

  • Check plate reader for wavelength, filters, gain etc.

g. 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


Problem 2: Weak signal

Possible causes and solutions for weak signal:

a. Insufficient amount of antigen was coated to microtiter plate

  • Use more antigen for coating or very coating buffer

b. Not enough antibody used

  • Increase concentration of the primary and/or secondary antibody. Optimize antibody concentrations for your assay

c. Detection reagent too old, contaminated, or used at the wrong pH

  • Use fresh detection reagents at the correct pH

d. Detection reagent too dilute

  • Use a higher concentration of detection reagent

e. Plate reader has the wrong settings

  • Check plate reader for wavelength, filters, gain etc

f. Incubation temperature too low

  • Optimize the incubation temperature for your assay. Reagents should be at room temperature before beginning the assay


Problem 3: High background signal

Possible causes and solutions for high background signal:

a. Too much antibody used

  • Reduce the concentration of the primary and/or secondary antibody. Optimize antibody concentrations for your assay

b. Non-specific antibody binding

  • Use a suitable blocking buffer or use an affinity-purified antibody

c. Too much detection reagent used

  • Repeat assay with a higher dilution of detection reagent

d. Too few washing cycles

  • Increase the number of washing cycles

e. Contaminated blocking agent

  • Use fresh blocking agent

f. Wrong concentration of blocking agent

  • Check the concentration of blocking agent in the recommended protocol

g. Presence of blocking buffer interferes with antibody binding

  • Wash off blocking buffer before adding antibody

h. Reaction not stopped

  • Use stop solution to prevent overdevelopment

i. Plate left too long before reading

  • Start taking measurements shortly after the addition of detection reagent

j. Wrong settings on the plate reader

  • Check settings and adjust as needed

k. Insufficient amount of Tween® in the buffers

  • Use PBS containing 0.05% Tween®

l. Incubation with substrate carried out in the light

  • Perform substrate incubation in the dark

m. Incubation temperature too high

  • Optimize the incubation temperature for your assay

n. Plates stacked during incubations leading to uneven temperature distribution

  • Avoid stacking plates

o. Pipetting errors

  • Calibrate pipettes so that they dispense the correct volumes

p. Reagents were not mixed properly

  • Before pipetting solutions into wells, make sure all reagents and samples have been throughly mixed

q. Inconsistent washing of wells

  • Take precautions to reduce variability of washes

r. Uneven evaporation of solution from wells during incubation

  • Always incubate with a lid on the plate

s. Interference caused by dirt on the bottom of the plate

  • Clean the plate carefully and reread


Problem 4: Slow color development

Possible causes and solutions for slow color development:

a. Incubation temperature is wrong

  • Ensure plates and reagents are kept at room temperature

b. Contaminated solutions

  • Make fresh solutions

c. Detection reagent too old, contaminated or used at the wrong pH

  • Use fresh detection reagents at the correct pH