The following guide serves as a checklist for possible causes and solutions with respect to some of the most commonly encountered problems in competition ELISA assays. Protocol of competition ELISA is available for you.

Note: The conditions described here may not pertain to every competition ELISA because performance requirements vary for individual assays.

Incubation time is too short: Incubate samples overnight at 4°C or strictly follow the manufacturer guidelines.

Target present below detection limits of assay: Decrease dilution factor or concentrate samples.

Assay set up incorrectly or used incorrect reagents: Review protocol and repeat assay using a positive control.

Antigen not coated properly: Try longer coating times, different coating buffers, or avidin plates with biotinylated antigen.Capture antibody not coated properly (Direct competition ELISA)

• Use ELISA plate, not tissue culture plate.
• Try longer coating time.
• Increase concentration of coating capture antibody.

Problem with the reference antigen

• Use new reference antigen.
• Check that the standard is appropriately handled.

Recognition of epitope impeded by adsorption to plate: To enhance detection of a peptide by competition ELISA, conjugate peptide to a large carrier protein before coating onto the microtiter plate.Assay buffer compatibility: Ensure assay buffer is compatible with target of interest.Not enough detection reagent

• Increase concentration of the primary and/or secondary antibody.
• Optimize antibody concentrations for your assay.
• Use fresh reagents at the correct pH.

Sample prepared incorrectly: Ensure proper sample preparation and dilution.Incubation temperature too low

• All reagents including plate should be at ambient temperature prior to use.
• Ensure the incubation steps are carried out at the correct temperature.

Incorrect wavelength: Verify the wavelength and read plate again.Plate washings too vigorous

• At least 4 times after incubation.
• Pipette wash buffer gently if washes are done manually.

Wells dried out

• Do not allow wells to become dry once the assay has started.
• Cover the plate using sealing film or tape for all incubations.

Slow color development

• Prepare substrate solution immediately before use.
• Ensure the stock solution has not expired and is not contaminated.
• Allow longer incubation.
• Ensure plates and reagents are kept at room temperature.

Antibody quality: Use a fresh aliquot of antibody that has been stored at -20°C or below.

Enzyme inhibitor present

• Avoid sodium azide in HRP reactions
• Avoid phosphate in AP reactions