Neutralization Test | Its Principle types and uses


A neutralization test determines an antibody’s efficiency to prevent or lower a pathogen’s infectivity. When a person is infected with a virus, his immune system develops antibodies that recognize and bind to specific regions of the pathogen called epitopes. A subset of these antibodies has the capability to neutralize the pathogen’s infectivity through a process known as neutralization.

Neutralization might happen in vitro or in vivo. These neutralization assays rely on laboratory animals or cell cultures as “indicator systems”. These indicator systems are reacted with the toxin or virus under test, and the pathogen’s effects on the indicator system are observed. If the presence of specific antibodies neutralizes the pathogen’s effects, it shows the existence of neutralizing antibodies in the test sample.

In the situation of toxins, an antitoxin is an antiserum containing antibodies that can neutralize the toxin. Toxin neutralization assays typically evaluate the reduction in toxin effects on the indicator system if the antitoxin is present.


The neutralization test principle relies on the idea that certain antibodies or antitoxins may minimize or neutralize the biological effects of enzymes, toxins, and viruses.

Neutralization is an important part of the immune response to infections. It refers to the reduction of the negative effects generated by a specific substance, such as viral antigens, viruses, or toxins produced by pathogenic microbes, such as bacterial exotoxins. Neutralizing antibodies are very important in this procedure.

Not all antibodies produced by the body are capable of efficiently blocking antigen effects. Only certain antibodies known as neutralizing antibodies can prevent viral particles or antigens from being taken up by cells and neutralize their activity. These neutralizing antibodies can bind to the pathogen and prevent it from infecting or harming host cells.

The neutralization test is applicable to both in vitro and in vivo antigen-antibody responses. In vitro studies use isolated components in a controlled laboratory environment, whereas in vivo tests use living organisms, such as animals, to evaluate the neutralizing effects of antibodies.

Overall, the neutralization test is an important technique in immunology and virology research, providing insights into the efficacy of antibodies in preventing pathogen infectivity and understanding the immune response to diverse diseases.

Types of Neutralization tests

Virus Neutralization test

The virus neutralization test is a type of neutralization test that is used for virus identification and analyzing the neutralization of a virus’s biological activity. This test was developed specifically for detecting and neutralizing viral activity. Viruses can be grown using techniques such as animal inoculation, egg inoculation, and cell culture. Certain antibodies against a certain virus are added into the growth media during the virus neutralization test. The availability of these antibodies prevents the virus from growing and replicating. The antibodies bind the antigenic determinants on the surface of virus effectively to neutralize its effects.

Viruses have particular antigenic markers on their surface that are recognized by antibodies. When these antibodies come into contact with the virus in the test system, they begin the neutralization process. The neutralizing antibodies bind to the antigenic determinants of virus, preventing it from interacting with host cells and therefore neutralizing its effects. This prevents the virus from infecting cells or causing more damage.

The Virus Neutralization Test is a critical tool to examine the immune response to viral infections, assessing vaccination efficacy, and detecting the existence of specific viruses in diagnostic cases. Researchers can assess the efficiency of the immune response and the potential for viral neutralization by assessing the reduction or inhibition of viral activity in the presence of neutralizing antibodies.

Figure 1: this figure represents virus neutralization test (Hammerschmidt et al. 2022)

Examples of the Virus Neutralization Test

  • Neutralization of Cytopathic Effect
  • Haemagglutination Inhibition Test
Neutralization of Cytopathic Effect

The process of reducing or preventing the malfunction and death of host cells produced by viral infection is referred to as the neutralization of cytopathic effect (CPE). The term “cytopathic effect” refers to the changes in structure that take place in host cells when they are infected by a virus. These modifications might vary from changes in cell shape to cell death.

When a virus enters host cells, it replicates within them and uses the cellular machinery to do so. This mechanism frequently disrupts normal cellular processes and induces cytopathic consequences. The specific effects differ depending on the virus and the type of host cell.

When introduced into the body, microbes may release toxins that can cause damage by altering numerous biological activities. Antitoxins, on the other hand, have been developed particularly to counteract the biological effects of these toxins. The toxin neutralization test is based on the use of antitoxins to neutralize specific toxins. The negative effects of these toxins can be prevented or eliminated by adding antitoxins into the test system.

The purpose of both types of neutralization tests is to evaluate individual antibodies or the ability of antitoxins to neutralize the effects of the target pathogens. Researchers can acquire valuable knowledge into the efficiency of neutralizing agents and the overall immune response by evaluating the reduction or elimination of biological activities induced by enzymes, toxins, or viruses.

The use of particular antibodies or antiviral drugs to prevent the virus’s harmful impact on host cells is known as neutralization of cytopathic effect. These neutralizing agents can bind to virus particles, preventing them from attaching to host cells or from entering cells. In this way, the neutralizing agents disrupt the viral replication cycle and reduce the cytopathic effects.

The ability of antibodies or antiviral medicines to prevent or diminish cytopathic effects is tested in the context of neutralization testing. This can be determined by analyzing the appearance and viability of infected cells or evaluating the reduction in cell death induced by viral infection.

The neutralization of cytopathic effect is an essential aspect of researching viral diseases to develop antiviral treatments. It is feasible to minimize the level of severity of an infection, inhibit the spread of the virus, and enhance the long-term result for the infected individual by neutralizing the destructive effects of a virus on host cells.

In summary, cytopathic effect neutralization refers to the decrease of structural alterations and cell death caused by viral infection. It includes the use of particular antibodies or antiviral medicines to prevent the virus’s destructive effects on host cells, thereby minimizing the cytopathic effects and improving the infection’s outcome.

Requirements for Neutralization of Cytopathic Effect

Serum sample: The serum can be obtained from a person who has already been exposed to virus or has received the vaccination against that virus. The serum contains antibodies that may be capable of neutralizing viruses and preventing or reducing cytopathic effects.

Diluting solution: To prepare serial dilutions of the blood sample or other test compounds, a diluting solution is used. This enables for the evaluation of different concentrations of neutralizing agents. Typically, the diluting solution is a buffered medium that offers the required conditions for cell viability and virus multiplication.

Known Virus: The assay requires a known virus. This suspension contains a predetermined quantity of the pathogen/virus under investigation. It acts as a vehicle for the virus to infect host cells and cause cytopathic effects.

Cell culture system: An appropriate cell culture system is required for virus propagation as well as evaluation of cytopathic effects. Different viruses have different host cell requirements, and the correct cell line must be utilized to enable viral replication and demonstrate cytopathic effects during infection.

Procedure for Neutralization of Cytopathic Effect
  • A serum sample is taken from the patient or person being tested. A diluting solution is used to dilute the serum to various quantities. This helps in the determination of the concentrations of neutralizing antibodies in the serum.
  • A known virus suspension is combined with the diluted serum sample. A standardized amount of the target virus is present in the viral suspension. The serum and virus solution are carefully mixed in equal amount of volume.
  • The diluted serum and virus suspension mixture is then incubated for a set amount of time. The incubation settings are designed to encourage viral reproduction while also allowing interaction between neutralizing antibodies in the serum and the virus.
  • Following incubation, the mixture is poured onto an appropriate cell culture. The cell culture system utilized should be compatible to the virus under investigation. Typically, inoculation cells are cultivated in a culture dish or plate.
  • The injected cells are incubated in conditions that promote viral replication and the formation of cytopathic effects. The cells are examined on a regular basis for a set amount of time, usually several days, to determine the presence or absence of cytopathic effects. Cell morphological alterations, cell separation from the culture dish, and cell death are examples of these effects.
  • A control sample is created in addition to the test samples. A recognized positive control, which includes virus without any neutralizing agent, and a negative control, which may contain a mock or non-specific blood sample, may be used as controls.
Interpretation of results

If the test sample contains sufficient neutralizing antibodies, it will neutralize the viral components in the assay. As a result, cytopathic effects such as cell shape alterations, cell separation, or cell death will not be noticed in cell culture. This is considered a positive result, suggesting that the test sample exhibits viral neutralizing activity.

The viral components will not be effectively neutralized if the test sample lacks neutralizing antibodies or the concentration of neutralizing antibodies is insufficient. In this instance, the virus will continue to reproduce within the cells, resulting in cytopathic effects or CPEs. The presence of cytopathic effects in cell culture shows a negative result, implying that the test material lacks sufficient viral neutralizing effect.

Haemagglutination Inhibition (HI)

The Haemagglutination Inhibition (HI) test is a serological assay that determines the presence and amount of particular antibodies against a virus in a serum sample. It works by preventing the process of haemagglutination, which is the aggregation or clumping of red blood cells caused by a virus. FOR DETAIL VISIT (

Applications of virus neutralization test

  • This test is an effective method for detecting and diagnosing viral infections. It is easy to evaluate if a patient has been exposed to a specific virus, such as measles, mumps, influenza, or the novel coronavirus causing COVID-19, by examining the presence of neutralizing antibodies in their serum. This test assists in verifying the infection and determining the severity of the infection.
  • In research settings, the Virus Neutralization Test is used to examine viral infections, their behavior, and their interactions with the host immune system. It sheds light on the mechanics of viral neutralization and aids in the identification of possible targets for antiviral treatments. Furthermore, the test contributes to surveillance efforts by tracking the prevalence and transmission of viral infections in certain populations or locations.
  • This test is critical in the development and testing of vaccinations against viral infections. Researchers can measure a efficiency of vaccine in preventing viral infection by measuring the neutralizing antibodies produced in reaction to it. This test evaluates the capacity of vaccine to stimulate a strong immune response and generate neutralizing antibodies, which are essential to neutralize the virus and preventing illness.
  • Seroprevalence studies seek to find the prevalence of a certain virus in a community. The Virus Neutralization Test, which can detect the presence of neutralizing antibodies in a large number of samples, is a significant tool for such studies. These investigations aid in determining the extent of viral exposure in a community, identifying locations with higher infection rates, and directing public health policies.

Toxin Neutralization Test

This test is a laboratory assay used to assess the efficiency of antitoxins in neutralizing the adverse effects of microbial pathogen toxins. Toxins are compounds secreted by microorganisms that, when injected or exposed to them, can cause considerable damage to many biological activities within the body. Antitoxins, on the other hand, are particular antibodies that are generated to fight and diminish the biological consequences of these toxins.

Principle of Toxin Neutralization Test

This neutralization Test works on the idea of using antitoxins to neutralize the hazardous effects of the target toxin.

Figure 2: neutralization modes: Inhibiting dissociation. (1) The active toxins bind to their targets once a toxin complex is dissociated, resulting in a harmful effect. (2) Antibody binding prevents toxin complex dissociation, preventing the production of active toxins (Laustsen et al. 2018)

Requirements of Toxin Neutralization Test

Egg White Agar: it is a specific type of agar medium utilized in this test. It contains nutrients as well as egg yolk, which is a source of lecithin. Lecithin is an egg yolk component that can be degraded by bacterial toxins such as alpha-toxin generated by bacteria such as Clostridium perfringens. The presence of egg yolk in the agar permits lecithinase activity to be detected.

Test organism: The assay requires the test organism that produces the toxin of interest. This organism could be a bacterial strain or another microorganism known to produce the toxin in concern. The test organism’s inoculum is produced and introduced to the test medium, such as Egg Yolk Agar, for toxin production.

Known antitoxin: This antitoxin is a prepared antibody specific to the toxin of interest. It should be well-characterized and have a well-established neutralizing action against the toxin under test. The known antitoxin serves as a positive control and is used to evaluate the neutralizing capacity of test sample.

Incubator: Because some toxin-producing bacteria grow in oxygen-deprived situations, the Toxin Neutralization Test is often performed under anaerobic conditions. As a result, during the incubation of the test samples, an anaerobic jar or an incubator equipped with the essential parameters for sustaining anaerobic conditions, such as low oxygen and high carbon dioxide levels, may be required.

Procedure of Toxin Neutralization Test

Nagler’s reaction is a laboratory test that detects the presence of lecithinase activity, which is produced by specific bacteria, most notably Clostridium perfringens. Lecithinase is an enzyme that dissolves lecithin, which is a component of egg yolk. This test was invented by George Henry Falkiner Nuttall and Arthur Edward Boycott Nagler.

  • Agar plate with egg yolk is prepared. The egg yolk contains lecithin, which is required for the test.
  • On the agar plate, a line is drawn to divide it in half.
  • On one half of the agar plate, antitoxin, a recognized specific antibody against the lecithinase toxin, is poured. This portion is the testing area.
  • The test organism, which is known to produce lecithinase, is streaked across both the antitoxin-treated and untreated halves of the agar plate.
  • The agar plate is then placed in an anaerobic incubator for one to two days at 37 °C (98.6 °F). Clostridium perfringens and other lecithinase-producing bacteria require an anaerobic environment for growth and exist.

Results interpretation  

The presence or absence of an opaque zone on the agar plate is used to interpret the results of Nagler’s response. To evaluate the test results, follow the following guidelines:

Positive Result: The test is considered positive if an opaque zone is observed in the area of the agar plate without antitoxin treatment (antitoxin-free area) and no opaque zone is found in the area where antitoxin was administered (antitoxin infected area). This shows that the test organism produces lecithinase activity.

Negative Result: If no opaque zone is found in either the antitoxin-free or antitoxin-inoculated areas, the test is termed negative. This indicates that the test organism did not produce lecithinase activity.

It is essential to notice that the antitoxin neutralizes the activity of the alpha-toxin (lecithinase), resulting in no opaque zone in the antitoxin-inoculated area. However, the toxin activity is not neutralized in the antitoxin-free zone, allowing the lecithinase to destroy the lecithin contained in the egg yolk agar and generate an opaque zone.

The presence or absence of the opaque zone in the respective sections of the agar plate indicates the activity of lecithinase. The existence of an opaque zone in the antitoxin-free area suggests a positive result, implying the presence of bacteria that produce lecithinase. The absence of an opaque zone in any location, on the other hand, gives a negative result, suggesting the absence of lecithinase activity.

Applications of Toxin Neutralization Test

In vivo applications:
  • The Schick test is commonly used to demonstrate immunity against diphtheria infection. A little amount of diphtheria toxin is injected into the skin and the reaction is observed. There is no reaction if the person is immune to diphtheria.
  • The toxin neutralization test can be used to neutralize toxins produced by Clostridium welchii, a bacteria known to cause gas gangrene. The adverse effects of the toxins can be neutralized by adding antitoxins specific to the toxins produced by Clostridium welchii.
In vitro applications:
  • Nagler’s response, a type of toxin neutralization test, is used to detect Clostridium species quickly. The presence of lecithinase enzyme-producing Clostridium species can be determined by evaluating the presence or absence of an opaque zone in the agar plate after incubation. The above method identifies these microorganisms quickly and efficiently.
  • Anti Streptolysin O (ASO) test detects antibodies against the Streptolysin O toxin generated by Streptococcus pyogenes. It is based on toxin neutralization. The test can aid in the diagnosis of a recent or past streptococcal infection by evaluating the amount of anti-Streptolysin O antibodies in a patient’s serum.

Applications of Neutralization Tests

  • To detect the existence and activity of pathogenic components such as toxins and viruses, the neutralization tests are used. The neutralizing ability of these components can be tested by introducing antibodies or antitoxins specific to them, assisting in the identification and distinction of their pathogenicity.
  • Aside from vaccinations, the neutralization test can be used to evaluate the efficacy of drugs or therapeutic substances. Researchers can evaluate the potential of these medications to suppress infection or lessen the damaging effects of toxins by examining their capacity to neutralize the activity of pathogens or toxins.
  • The test is useful for researching the antigenic relationships between various viruses and toxins. Researchers can learn about the antigenic similarities and differences between these diseases by evaluating antibodies’ ability to neutralize distinct strains or variations.
  • These tests are essential in establishing antitoxin potency. Researchers can test the strength and efficacy of antitoxin preparations by measuring their capacity to neutralize the harmful effects of specific microbial toxins.
  • The neutralization test is used to determine vaccine efficiency in generating immunity. Researchers can measure the vaccine’s potential to give infection protection by testing the ability of vaccine-induced antibodies to neutralize the targeted pathogens.

Advantages of Neutralization Test

  • When compared to other diagnostic procedures, the neutralization test has a better sensitivity. It has the ability to detect tiny quantities of neutralizing antibodies or antigens, making it a useful tool for diagnosing infections or monitoring immune responses.
  • The test also has a better specificity, which means it may detect the presence of specific antibodies or antigens. This specificity aids in the differentiation of different infections or strains, providing precise diagnostic information.
  • The neutralization test evaluates neutralizing antibodies or antigens quantitatively. It offers information regarding the level of neutralizing activity, which can be valuable for estimating the potency of antitoxins or evaluating the intensity of immune responses.
  • The neutralization test is applicable to a wide spectrum of pathogens, including toxins-producing viruses and bacteria. Because of its versatility, it is useful in both diagnostic and research contexts, allowing for the detection, characterization, and evaluation of diverse diseases and their components.
  • The neutralizing antibodies utilized in virus neutralization assays can detect the target virus as well as its different variants. This is especially useful in cases where viruses undergo antigenic variation or evolve, guaranteeing that the test can still detect and identify the infection.

Disadvantages of Toxin Neutralization Test

  • After an infection or immunization, neutralizing antibodies take time to form in the body. This means that the test may not be suitable for early detection of acute infections because measurable quantities of neutralizing antibodies may require days or weeks to be developed.
  • To perform neutralization tests, individuals must be trained and proficient in handling and manipulating viruses, poisons, and other test components. Because of the complexity in test and the requirement for exact methodologies, it is critical to have workers with the requisite competence to assure accurate and dependable results.
  • Specific virus strains, antitoxins, and cell cultures, for example, can be difficult to develop and control for the neutralization test. The availability and quality of these materials can vary, which can affect the test results’ reliability and reproducibility.
  • High-level biosafety precautions are required: Working with live viruses and poisons in the neutralization test needs tight biosafety measures and very protective laboratory conditions. Handling infectious agents can be hazardous to laboratory personnel, necessitating proper training, containment facilities, and attention to safety protocols.
  • The neutralization test necessitates extensive preparation, including the creation of particular test materials such as viral suspensions, antitoxins, and cell cultures.  Multiple phases and meticulous execution are required for testing, which can be time-consuming and resource-intensive.


  • Hammerschmidt SI, Thurm C, Bošnjak B, Bernhardt G, Reinhold A, Willenzon S, Ritter C, Reinhold D, Schraven B, Förster RJEJoI. 2022. Robust induction of neutralizing antibodies against the SARS‐CoV‐2 Delta variant after homologous Spikevax or heterologous Vaxzevria‐Spikevax vaccination.  52(2): 356-359.
  • Laustsen AH, Gutiérrez JM, Knudsen C, Johansen KH, Bermúdez-Méndez E, Cerni FA, Jürgensen JA, Ledsgaard L, Martos-Esteban A, Øhlenschlæger MJT. 2018. Pros and cons of different therapeutic antibody formats for recombinant antivenom development.  146: 151-175.
Mubashir Iqbal
Mubashir Iqbal

Mubashir Iqbal is a highly dedicated and motivated Microbiologist with an MPhil in Microbiology from the University of Veterinary and Animal Sciences. Currently, he is researching the efficacy of commercially available SARS Cov-2 vaccines to neutralize the omicron variant in Pakistan. He holds a Bachelor's degree in Microbiology and has experience in chemical and microbiological analysis of water samples, managing SOPs and documents according to standard ISO 17025. Additionally, he has worked as an internee in BSL 3, Institute of Microbiology, UVAS, where he gained experience in RNA extraction, sample processing, and microscopy.

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