ELISA: Troubleshooting and Advice

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