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Why Have Diverse Colors In ELISA Experiment?

Posted on October 29, 2022 by Zeng YingELISA

CatalogueEquipment For Elisa Kit Manufacturing


ELISA, also known as enzyme-linked immunosorbent assay, is a common method for the quantitative determination of the content of various target proteins in body fluids.

Its colorimetric reaction allows for qualitative or quantitative analysis, using the HRP enzyme-catalyzed TMB, the reaction produces a blue imine solution, and the addition of a termination solution transforms the blue cation into a yellow biphenylquinone.

In addition to the common blue and yellow, some unusual and diverse colors occur during ELISA experiments. Behind each color, there is a special meaning. Next, we will analyze the color secrets hidden in the "appearance" and "output" of ELISA.

ELISA appearance (component)

ELISA test shows pink color


Why do some ELISA kits contain dilutions and concentrated biotinylated antibodies that are pink in color?

Some components of the ELISA kit will have a red/pink color due to the addition of special protective agents, such as a certain series of enzyme conjugate dilutions and concentrated biotinylated antibodies. Reagents of this color perform normally and should be used with confidence according to the operating requirements within the instructions.

ELISA test shows yellow color


Why is the ELISA dilution pale yellow?

In ELISA experiments, the diluent contains preservatives and is normally a clear, transparent and odorless liquid. If the diluent becomes cloudy, smelly, and produces precipitation, the odds are that it is contaminated with bacteria and is not recommended for further use.

ELISA test appears blue or yellow-green


TMB substrate solution changes color before it is used in ELISA experiments?

In enzyme-linked immunosorbent assay experiments TMB substrate solutions not stored away from light and left open for too long will be oxidized by the HRP enzyme in the air and will appear light blue/blue. Direct use under such circumstances will lead to false positive results. Therefore, it is not recommended to continue using TMB solution that has turned blue.

Output of ELISA experiments (results)

ELISA experiment results in dark green/dark blue


After incubation with the substrate solution, the enzyme plate well solution turns dark green or dark blue

In a normal ELISA experiment, the reaction is terminated when a clear blue gradient appears in the first four wells of the marker after incubation with the substrate solution. However, when the concentration of the incubated HRP enzyme conjugate working solution is too high or the sample concentration is much higher than the detection range, the highest 1-2 gradient wells or sample wells of the standard curve may appear dark green or dark blue.

After the dark green sample terminates the reaction, the OD value read at 450 nm may be lower than the actual; the dark blue calibration curve will be too close to the gradient OD value to be fitted to get a valid calibration curve and accurately calculate the sample concentration.


1. Strictly control the incubation temperature and time in each step and prepare the HRP enzyme conjugate working solution according to the instructions.

2. During the color development phase, control the color development time by the appearance of a distinct blue gradient in the first four wells.

3. Samples with too high a concentration need to be diluted to within the detection range of the kit before determination.

ELISA experiment results in yellow color

After the ELISA reaction is terminated, the whole plate turns yellow


Possible reasons for the yellow color of ELISA experiment results

ELISA competition method is operated according to the sandwich method: the two principles are different and the operation steps are different, please pay attention to the distinction.

Improper washing: shake the plate too close to the waste bucket, resulting in liquid back splash; shake the plate not dry, resulting in liquid residue or flow into other wells; washing when each well is not injected enough washing solution, or the number of washing is not enough; too much liquid overflow contamination; immersion time is too short.

Improper storage of chromogen, or incubation without light protection, resulting in non-specific color development.

Incubation temperature is too high, or incubation time is too long.

Mixing or substitution of different kit components, resulting in matrix effect or non-specific adsorption, leading to false positive results.

ELISA test results are normal for the standard curve and yellow for the sample wells


After the readings were calculated, it was found that the standard curve fit was good, but the sample OD exceeded the maximum OD of the standard curve, indicating that the sample still needed to be diluted oh.

ELISA test results in black


After adding the termination solution, the liquid in the wells of ELISA plate turns black, or black precipitation is produced

Possible causes

HRP enzyme concentration is too high: when preparing the HRP enzyme working solution, ensure that the calculation and preparation volume is accurate, and wash the plate according to the wash volume, time and number of times in the instructions.

The concentration of the indicator in the sample is too high and the sample still needs to be diluted.

The concentration of sulfuric acid in the termination solution is too high or spoiled: the termination solution is 1M H2SO4, mixing with too high concentration of sulfuric acid may lead to charring reaction.

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