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Analysis of common problems and causes of ELISA experiment

12 May 2022

When it comes to detecting and quantifying the concentration of an antigen in an unknown sample, most researchers depend on ELISA (Enzyme-Linked Immunosorbent Assay). This is completely understandable since this plate-based assay technique has a completely robust nature, exhibits exceptional sensitivity and is quick and easy to perform. 

If this is the case, why do your ELISA experiments give less than optimal results? What should you do when this happens? Here are some of the most common problems researchers run into when performing ELISA experiments along with some of the possible solutions.

High background/non-specific staining

Description of results: After termination, the whole plate results show a uniform yellow or light colour; or the standard curve is linear but the background is too high

Possible reason Recommendations and Precautions

The yellowing of the whole plate may be caused by the wrong addition of other reagents

Check the components and lot numbers of the reagents before the experiment, and confirm that all components belong to the corresponding kit. Reagents from different kits or different lot numbers cannot be mixed

ELISA plate was not washed sufficiently

Make sure that the same amount of washing solution is added to each microwell during the washing process. After washing, press the ELISA plate firmly on the absorbent paper to remove the residual buffer.

Incubation time too long

Please strictly follow the steps of the manual

Streptavidin-HRP contaminates the Pipette Tips and TMB container or positive control contaminates the Pre-coated Microplate

When absorbing different reagents, the tips should be replaced. When configuring different reagent components, different storage vessels should be used. Please use a pipette during operation.

Biotinylated Antibody or Streptavidin-HRP concentration too high

Check whether the concentration calculation is correct or use after further dilution

TMB Substrate Solution exposure or contamination prior to use

Store in the dark at all times before adding substrate

Colour development time is too long

Please strictly follow the steps of the manual

The wrong filter was used when the absorbance value was read

When TMB is used as the substrate, the absorbance should be read at 450 nm.

White plate

Description of results: After the colour development step, all wells of the ELISA plate are colourless; the positive control is not obvious

Possible reason Recommendations and Precautions
Mixed-use of component reagents Read labels clearly when preparing or using
In the process of plate washing and sample addition, the Streptavidin-HRP is contaminated and inactivated and loses its ability to catalyze the colour developing agent. Confirm that the container holding the ELISA plate does not contain HRP enzyme inhibitors (such as NaN3, etc.), and confirm that the container for preparing the wash solution has been washed.
Missing a reagent or a step Review the manual in detail and strictly follow the operating steps

Light colour

Description of results: All wells, including standard and samples, are lighter in colour

Possible reason Recommendations and Precautions
The kit has expired or been improperly stored Please use it within the expiration and store it in accordance with the storage conditions recommended in the manual to avoid contamination.
Reagents and samples are not balanced to room temperature before use All reagents and samples should be equilibrated at room temperature for about 30 minutes
The insufficient suction volume of the pipette, too fast discharge of pipetting suction, too much liquid hanging on the inner wall of the tip or the inner wall is not clean To calibrate the pipette, the tips should be matched, each time the tips should fit tightly, the pipetting should not be too fast, and the discharge should be complete. The inner wall of the tips should be clean, and it is best to use it once.
Insufficient incubation time Timer accurate timing
Insufficient colour reaction Usually 15-30min
The number of washings increases and the dilution ratio of the concentrated lotion does not meet the requirements Reduce the impact of washing, dilute the concentrated lotion and washing time according to the manual, and accurately record the washing times and dosage
Distilled water quality problem The prepared lotion must be tested to see if the pH value is neutral.
In the process of plate washing and sample addition, the Streptavidin-HRP is contaminated and inactivated and loses its ability to catalyze the colour developing agent. Confirm that the container holding the ELISA plate does not contain enzyme inhibitors (such as NaN3, etc.), confirm that the container for preparing the washing solution has been washed, and confirm that the purified water for preparing the washing solution meets the requirements and is not contaminated.


Description of results: The standard is normal, and the colour of the sample is light

Possible reason Recommendations and Precautions
The kit has expired or been improperly stored

Samples cannot use NaN3

The sample to be tested may not contain strong positive samples, so the result may be normal If in doubt, retest

Poor standard curve/poor repeatability

Description of results: Poor repeatability

Possible reason Recommendations and Precautions
Incorrect standard preparation Strictly follow the manual for standard preparation, and only use the recommended diluent to dilute the standard
Improper storage method of the kit or poor storage environment Please store under the conditions recommended in the manual, and do not keep the resuspended components at room temperature for too long
Premature dilution of working components Please configure each working component 10 minutes before use and add it to the microwell immediately
The sample was not mixed after adding For adding multiple reagent components at the same time, mix thoroughly on the mixer after adding the sample, and pay attention to hold it steady to prevent splashing.
Poor repeatability of microplate readers Calibrate the microplate reader
Inconsistency in incubation time, washing conditions, color development conditions, and operators Repeat the determination of the specimen, and keep the reaction conditions, personnel, etc. as consistent as possible with the last time
Incorrect washing Pipette accurately add 200µl/well of washing solution or fill each well, but do not overflow. There should be no blockage when washing the plate by the plate washer, and the washing should be sufficient.
Incubation temperature constant temperature effect is not good Keep the temperature constant to avoid the local temperature being too high or too low
When adding liquid, too much remains on the hole wall When adding liquid, the tip should try to add liquid along the bottom of the hole wall without touching the bottom of the hole.
Reuse of consumables The Pipette Tips should be replaced when different reagents are drawn, and different storage vessels should be used when configuring different reagent components.
The bottom of the microwell is scratched or there is dirt Be careful when operating, be careful not to touch the bottom and wipe the bottom of the microplate to remove dirt or fingerprints
Sometimes negative and sometimes positive near the threshold Make 3 duplicate wells for the same sample, with 2 (including more than 2 identical results)
Cross-contamination during sample addition Try to avoid cross-contamination when adding specimens


Description of results: The color of the Pre-coated Microplate is chaotic and irregular

Possible reason Recommendations and Precautions
Cross-contamination from manual plate washing When washing the plates by hand, the first 3 injections of the lotion should be discarded immediately, and the soaking time should be set for the next few times to reduce cross-contamination.
Cross-contamination when clapping the plate Use a suitable absorbent paper towel when clapping the plate, do not pat irrelevant substances into the hole of the plate, and try not to pat in the same position to avoid cross-contamination
The liquid filling head of the plate washer is blocked, resulting in unsatisfactory liquid addition or large residual amount of liquid suction, resulting in chaotic and irregular. Unblock the liquid addition head, so that each well is filled with washing liquid when washing the plate, and the residual amount should be small when aspirating liquid.
Incomplete centrifugation of the sample, resulting in coagulation in the reaction well or interference of sediment or residual cellular components Serum plasma should be fully centrifuged at 3000rpm for more than 6min
Samples stored too long, resulting in contamination. Samples should be kept fresh or stored at low temperature to prevent contamination
Incorrect preparation of washing solution or direct misuse of concentrated washing solution Please configure according to the manual
Should your assay not behave as expected, the solutions discussed above will provide good starting points to quickly resolve your problems. Still confused? Let us help you to make it easier. Our trained team of professionals is ready to help you through the selection process from start to finish. Reach out today to start the conversation. Call us at +603 8065 3889 today or send us a message at