ELISA common problems and solutions

one. Background high or non-specific staining

the reason

solution

Non-specific binding of antibodies

Replace the blocking solution (for example, use casein instead of BSA); do not wash after the end of the closure; pour off the blocking solution, blot the residual liquid on the absorbent paper and proceed directly to the next step.

Insufficient cleaning of the ELISA plate

Make sure that each microwell is filled with buffer and the cleaning instrument is working properly during the cleaning process. Wash 3 to 5 times between steps, do not increase the number of cleanings. After the cleaning is completed, the plate is pressed hard on the paper towel to remove the residual buffer. An appropriate amount of Tween-20 (recommended concentration 0.01-0.1%) should be added to the cleaning solution.

Buffer is contaminated

The buffer should be freshly prepared, used within one week, and filtered to remove bacteria.

Incubation temperature is too high

The entire experimental procedure should be at room temperature 25 °C.

Incubation time is too long

Shorten incubation time

The enzyme label contaminates the substrate or the positive control contaminates the microwell

Replace the tips with different reagents and use different reservoirs. It is best to add or remove the wash buffer with a pipette (pour/pour out may result in cross-contamination).

Detection of antibody or Avidin-HRP concentration is too high

Check if the concentration is calculated correctly or use it after further dilution.

Substrate exposure before use

Always store in the dark before adding the substrate to the microwell

Wrong filter used when reading absorbance

When ABTS is a substrate, the absorbance should be read at a wavelength of 405 nm (the absorbance should be read at a wavelength of 450 nm when TMB is a substrate), and 650 nm is used as the correction wavelength.

Color development time is too long

The absorbance is read every 5 minutes to monitor the OD reading of the blank well.

two. Color is weak or does not develop color

the reason

solution

Missing a reagent or a step

Review the protocol and follow the experimental steps.

Excessive detergent concentration in wash buffer

Remove or reduce the concentration of detergent, recommended 0.01-0.1%

The ELISA plate is not properly cleaned

Reduce the number of cleanings between steps, especially when not using a fully automatic or semi-automatic washer, 1-2 times.

Enzyme inhibitor in the sample

Sodium azide inhibits the activity of peroxidase.

Incubation time or temperature error

The incubation plate should be placed in the incubator (25 ° C) during incubation to avoid temperature fluctuations

The amount of substrate added to the microwell is incorrect.

Check pipette

Enzyme-substrate system is not suitable

Paired with Avidin-HRP and ABTS, paired with Streptavidin and TMB

Improper storage of components in the kit

Store the components as required

Wrong filter used when reading absorbance

When ABTS is a substrate, the absorbance should be read at a wavelength of 405 nm (the absorbance should be read at a wavelength of 450 nm when TMB is a substrate), and 650 nm is used as the correction wavelength.

three. Poor standard curve

the reason

solution

Standard preparation is incorrect

Dilute the standard using the recommended dilution buffer

Premature dilution

Prepare various reagents before use and use as soon as possible

Capture antibody cannot be coated

Use a plate suitable for ELISA, not a cell culture plate

Insufficient cleaning

An equal volume of cleaning solution should be added to each well to ensure that all micropores are pumped clean and filled in time.

Pipetting error

Calibrate the pipette, quickly and equally, add the reagents in the pipette along the sidewall of the microwell

Color development time is too long

The OD reading of the blank well does not exceed 0.2 or the OD reading of the highest concentration standard well does not exceed 1.2.

Improper storage of components in the kit

Store the components as required by the instructions. Do not leave the resuspended reagents at room temperature for an extended period of time.

IV. Is less accurate

the reason

solution

Insufficient reagent mixing

Mix the reagents thoroughly before pipetting

Insufficient cleaning

An equal volume of cleaning solution should be added to each well to ensure that all micropores are pumped clean and filled in time.

Pipetting error

Calibrate the pipette, quickly and equally, add the reagents in the pipette along the sidewall of the microwell

Consumables reuse

The tips should be replaced when drawing different samples; different reagents should be used for different reagents; a new seal seal should be applied during each incubation period.

Micropores are scratched by a tip or washer

Careful handling when aspirating and adding liquid

Precipitate in the sample

Centrifuge the sample tube before use

Micropores are not clean or dirt is present

Carefully inspect the micropores and remove dirt before use. Before reading the absorbance, wipe the bottom of the plate to remove dirt or fingerprints.

V. edge effects

the reason

solution

evaporation

Sealing plate seals should be used for each incubation step

Uneven temperature

During the incubation period, the plate should be placed in the incubator (25 ° C) to avoid temperature fluctuations. Do not stack the plate.