ELISA Troubleshooting

By Ryan Hamnett, PhD

ELISAs use antibodies to detect and quantify the amount of a target antigen in a liquid sample. Below is our troubleshooting guide to help solve any issues that might be encountered with an ELISA experiment.

Potential issue: Possible solution:
Blocking protein in coating solution Use coating solution containing only buffer components and the coating antigen or antibody
Capture antibody does not bind to plate Use ELISA plate, not a tissue culture plate

Try longer coating time

Increase concentration of coating antibody

Consider using a more active form of coating, such as Protein A/G or metal-coated plates
Recognition of epitope is blocked by antigen adsorption to plate Consider a more active form of antigen coating

If assaying a small peptide, try conjugating peptide to a large carrier protein before coating
Protein of interest is not present, or is present below detection limit Run positive control to ensure assay is working correctly

Decrease dilution factor, or concentrate samples

Check literature to determine if protein of interest is expected in sample type
Incompatible sample type Include positive control to ensure ELISA kits works with your sample type
Incubation time too short Increase incubation time or incubate overnight at 4°C
Incubation temperature too low Ensure all reagents including plate are at room temperature before starting assay

Carry out incubation steps according to manufacturer’s instructions
Too much washing in between steps Though washing is essential, washing too aggressively (e.g. with too much pressure) can remove detection reagents. Wash with gentle pressure
Wells scratched with pipette tips Carefully dispense and remove solutions
Wells have dried out Cover the plate for each step using sealing film
Primary and secondary antibodies not compatible Use a secondary antibody that has been raised against the host species for the primary antibody

Ensure secondary antibody can recognize the isotype of the primary antibody
Insufficient antibody Optimize antibody concentration
Enzyme inhibitors present in buffers Avoid using buffers containing sodium azide with HRP-based detection

Avoid using buffers containing phosphate with AP-based detection
Insufficient substrate Optimize substrate concentration
Plate read at incorrect detection wavelength Use the recommended wavelength and filter settings for the detection system being used
Detection system not sufficiently sensitive Consider using a more sensitive detection system such as chemiluminescence

Consider an amplification, such as the biotin-avidin system
Sample improperly stored Store samples at -80°C to avoid protein degradation, or assay immediately after collection and processing
Antibodies improperly stored Store antibodies at 4°C only for short-term usage, otherwise aliquot and store at -20°C

Use a fresh aliquot of frozen antibody

Avoid freeze-thaw cycles
Other reagents have lost activity due to improper handling or storage Follow manufacturer’s instructions for storage – many components need to be stored at 4°C

Generally avoid freeze-thawing

Handle fluorophores, fluorophore-conjugated antibodies, and enzyme substrates in the dark

Run a positive control to ensure all reagents are working appropriately
Buffers contaminated with bacteria Make up fresh buffer using sterile components
Potential issue: Possible solution:
Sample is too concentrated Determine optimal dilution by titration
Substrate changed color before use in assay Make substrate solution fresh, immediately before use
Plate read at incorrect wavelength Use the recommended wavelength and filter settings for the detection system being used
Excess time before plate reading Read plate shortly after adding substrate

Use a stop solution
Potential issue: Possible solution:
Incubation time too long Reduce incubation time

Try incubating overnight at 4°C
Buffers are contaminated with metals or enzyme Make up fresh buffers

Change pipette tips when pipetting different solutions
Insufficient blocking Increase the blocking incubation time

Try an alternative blocking agent, such as 5-10% normal serum from the same species as the enzyme-conjugated antibody
Insufficient washing Use the full volume of wash buffer for each well to ensure complete washing, as previous reagents and sample stick to sides of well

Increase number of wash steps

At the end of each wash step, flick plate over a sink and then pat on a paper towel to remove as much wash buffer as possible
Uneven evaporation of solution from wells Perform all incubations with a lid or seal on plate
Excess antibody Find optimal antibody concentration by titration
Non-specific binding by enzyme-conjugated antibody Run S0, NSB and secondary antibody controls to confirm source of background

Use a primary antibody raised in a different species to the sample

Dilute sample to reduce binding to interfering factors and anti-animal antibodies

Include Tween-20 in wash buffers to reduce non-specific interactions

Optimize incubation temperature
Contamination of reagents Change pipette tips between different solutions

Use fresh reagents
Excessive substrate used Follow manufacturer’s instructions for substrate

Dilute substrate
Precipitate in wells on addition of substrate Decrease concentration of substrate

Increase washing after enzyme-conjugate is added
Color development carried out in light Carry out substrate incubation in dark
Wells are the wrong color after stop solution addition Ensure stop solution is completely mixed with the substrate solution in the well
Overdeveloped color Use stopping solution

Read plate shortly after color development
Excessive amplification (if using) Reduce the amount of amplification, such as by conjugating less biotin to antibody
Contamination in blank control wells Change pipette tips between blanks and samples

Seal plate or put a lid to reduce spillage between wells
Potential issue: Possible solution:
Standards were not diluted correctly Confirm calculations
Standard stock solution not fully reconstituted Spin down vial before opening; inspect for undissolved material after reconstitution
Standard degraded Ensure proper handling and storage of standard stock
Pipetting error Calibrate pipettes

Use proper pipetting technique
Standard curve model does not fit data Try an alternative model, such as semi-log, 4PL, and 5PL plots
Flat portions of standard curve Some standard wells are likely saturated – reduce image acquisition time, or reduce concentrations of reagents in earlier steps
Potential issue: Possible solution:
Bubbles in wells Use proper pipetting technique

Remove bubbles with pipette
Wells not washed equally Wash all wells of the plate as recommended

Use automatic plate washers
Inconsistent pipetting Use calibrated pipettes and proper pipetting technique

Ensure all reagents are completely mixed by pipetting up and down or gently inverting
Inconsistent sample preparation and storage Store all samples under the same conditions, ideally aliquoted at -80°C
Particulates in samples Centrifuge samples to remove any particulate matter
Edge effects – outer wells of plate showing different signal to inner wells, usually due to changes in environment Check if buffer has evaporated from outer wells – if so, ensure plate cover is sealed to prevent evaporation

Ensure plate is fully equilibrated to room temperature before beginning – inner wells take longer to reach correct temperature

Do not stack plates, which may cause uneven temperature distribution across the plates
Menu Secondaire Menu Principal Nous Contacter 0Caisse
Haut