Immuno-Colorimetric Neutralization Test Protocol


The immuno-colorimetric neutralization test is used to measure the neutralizing activity of antibodies against specific viruses. It involves the binding of neutralizing antibodies to the virus, preventing its infection of target cells. Infected cells are then detected using an enzymatic colorimetric reaction, providing a quantitative measurement of the neutralizing activity.

Plaque assay and immune-colorimetric detection of mumps virus growing on Vero cell substrates. Fig. 1 Plaque assay and immune-colorimetric detection of mumps virus growing on Vero cell substrates. (Vaidya, 2023)


Reagents Instruments and Consumables
  • Cell culture medium: Provides a suitable environment for cell growth and virus infection.
  • Virus stock: Contains a known concentration of the virus being tested.
  • Neutralizing antibodies: Antibodies specific to the virus being tested with neutralizing activity.
  • Target cells: Cells that are susceptible to infection by the virus.
  • Wash buffer: Used to wash cells and remove unbound viruses.
  • Substrate solution: Contains a substrate for the enzymatic reaction, which produces a color change.
  • Stop solution: Terminates the enzymatic reaction.
  • Microtiter plate: Used for antibody-virus incubation.
  • 96-well plate: Used for cell culture and infection.
  • ELISA reader or spectrophotometer: Used to measure the absorbance of the colorimetric reaction.
  • Pipettes and tips: Used for precise and accurate dispensing of reagents.
  • Incubator: Used for maintaining a constant temperature during incubation steps.
  • Shaker: Used for gentle mixing of samples, if needed.


1. Preparation of target cells:
a. Culture target cells in the appropriate medium until reaching 80-90% confluency.
b. Wash the cells twice with PBS and detach them using trypsin-EDTA.
c. Count the cells and adjust the concentration to the desired level.
d. Plate the cells in a 96-well plate (10,000 cells per well) and incubate overnight at 37°C.
2. Preparation of antibodies and virus dilutions:
a. Prepare a series of serial dilutions of the neutralizing antibodies in the cell culture medium.
b. Prepare a series of virus dilutions in the cell culture medium, ranging from high to low concentrations.
3. Neutralization assay:
a. Add 50 μL of each antibody dilution to separate wells of the microtiter plate.
b. Add an equal volume (50 μL) of the virus dilution to each well containing the antibodies.
c. Incubate the plate at 37°C for 1 hour to allow antibody-virus interaction.
4. Cell infection:
a. After incubation, remove the culture medium from the target cells in the 96-well plate.
b. Add 100 μL of the antibody-virus mixture to each well of the target cells.
c. Incubate the plate at 37°C for a specific period of time (usually 1-2 hours) to allow viral entry into the cells.
5. Detection of infected cells:
a. Remove the inoculum from the target cells and wash them three times with wash buffer to remove the unbound virus.
b. Fix the cells by adding 100 μL of 4% paraformaldehyde to each well and incubate for 15 minutes at room temperature.
c. Wash the cells three times with wash buffer.
d. Add 100 μL of the substrate solution to each well and incubate for 10-15 minutes at room temperature in the dark.
e. Add 50 μL of stop solution to each well to terminate the reaction.
6. Quantification of infected cells:
a. Measure the absorbance of each well at an appropriate wavelength using an ELISA reader or spectrophotometer.
b. Calculate the percentage of neutralization by comparing the absorbance values of the antibody-virus-treated wells with the virus control wells.
c. Plot the neutralization curve and determine the neutralizing antibody titer.


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  1. Vaidya, S.R.; Immuno-colorimetric neutralization test: A surrogate for widely used plaque reduction neutralization tests in public health virology. Viruses. 2023, 15(4): 939.
For research use only, not directly for clinical use.
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