Description of results

Possible reason

Recommendations and Precautions

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

The yellowing of the whole plate may be caused by 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

Color 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.

Description of results

Possible reason

Recommendations and Precautions

After the color development step, all wells of the ELISA plate are colorless; the positive control is not obvious

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 color 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

Description of results

Possible reason

Recommendations and Precautions

All wells, including standard and samples, are lighter in color

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

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 color 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 color 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.

The standard is normal, the color of the sample is light

The sample uses NaN3 preservative, which inhibits the reaction of the enzyme

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

The visual result is normal, but the reading value of the microplate reader is low

Wrong filter used for absorbance reading

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

Description of results

Possible reason

Recommendations and Precautions

poor repeatability

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

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

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