Neutralization Test- Definition, Principle, Types, Procedure, Results

Neutralization is a process of reducing or eliminating the biological effects of a substance by another substance that specifically binds to it. Neutralization can occur between toxins and antitoxins, or between viruses and antibodies. The substance that causes the biological effects is called the antigen, and the substance that neutralizes it is called the antibody.

The principle of neutralization is based on the concept of antigen-antibody interaction. Antigens are molecules that can elicit an immune response in the body, such as foreign proteins, polysaccharides, or nucleic acids. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. When an antibody binds to an antigen, it can block its activity or mark it for destruction by other immune cells.

Neutralization tests are laboratory methods that use antibodies to detect or measure the presence or activity of antigens. Neutralization tests can be used for various purposes, such as identifying pathogens, diagnosing infections, evaluating immunity, or testing vaccines. Neutralization tests can be classified into two main types: virus neutralization test and toxin neutralization test. These tests differ in the type of antigen and antibody used, as well as the biological effects that are measured or inhibited. In this article, we will discuss these two types of neutralization tests in detail.

Neutralization tests are based on the principle that some specific antibodies or antitoxins can reduce or neutralize the biological effects of various toxins, enzymes, and viruses. There are two main types of neutralization tests: virus neutralization test and toxin neutralization test.

Virus Neutralization Test

Virus neutralization test is a type of neutralization test used for virus detection and identification. It is based on the ability of specific antibodies to inhibit the growth and replication of viruses in different media, such as egg inoculation, animal inoculation, or cell culture. The antibodies bind to the antigenic determinants on the surface of the virus and prevent it from infecting the host cells.

There are different methods of virus neutralization test, depending on the type of virus and the medium used. Some of the common methods are:

• Neutralization of cytopathic effect: This method is used for viruses that cause visible changes in the morphology or structure of the host cells, such as poliovirus. The serum sample from the patient is diluted and mixed with a known viral suspension. The mixture is then inoculated in a cell culture and incubated. The presence or absence of cytopathic effect is observed and compared with a control. If the serum contains neutralizing antibodies, the cytopathic effect will be absent or reduced, indicating a positive result.
• Haemagglutination inhibition test: This method is used for viruses that can agglutinate or clump red blood cells (RBCs), such as influenza virus. The serum sample from the patient is diluted and mixed with a known viral suspension. The mixture is then incubated and added to a RBC suspension from a specific source. The presence or absence of haemagglutination is observed and compared with a control. If the serum contains neutralizing antibodies, the haemagglutination will be inhibited or reduced, indicating a positive result.

Toxin Neutralization Test

Toxin neutralization test is a type of neutralization test used for toxin detection and identification. It is based on the ability of specific antitoxins to counteract the biological effects of toxins produced by microbes, such as bacteria or fungi. The antitoxins bind to the toxins and prevent them from interacting with the target cells or tissues.

There are different methods of toxin neutralization test, depending on the type of toxin and the medium used. Some of the common methods are:

• In vivo toxin neutralization test: This method involves injecting a known amount of toxin into an animal, such as a mouse or a guinea pig, along with a varying amount of antitoxin. The survival or death of the animal is observed and compared with a control. If the antitoxin can neutralize the toxin, the animal will survive or show less symptoms, indicating a positive result.
• In vitro toxin neutralization test: This method involves adding a known amount of toxin to a cell culture or a tissue sample, along with a varying amount of antitoxin. The biological activity or effect of the toxin is measured and compared with a control. If the antitoxin can neutralize the toxin, the biological activity or effect will be reduced or eliminated, indicating a positive result.

One example of in vitro toxin neutralization test is Nagler`s reaction, which is used for detecting alpha-toxin or lecithinase enzyme produced by Clostridium spp., such as C. perfringens. Lecithinase can digest lecithin, which is a component of egg yolk agar. The test involves spreading antitoxin on one half of an egg yolk agar plate and streaking the test organism on the whole plate. After incubation, an opaque zone will appear on the antitoxin-free area if lecithinase is present, indicating a positive result.

Virus Neutralization Test is a type of neutralization test used for virus detection. As the name suggests, it is used for the neutralization of the biological activities of the virus. Viruses can be grown by egg inoculation, animal inoculation, or cell culture. If the antibodies specific for the respective virus are inoculated in these mediums, they can halt the growth and replication of viruses. It is the fundamental principle of the virus neutralization test. The virus contains certain antigenic determinants to which the antibodies initiate the neutralization of the effects of the virus.

There are different methods of performing virus neutralization tests, depending on the type of virus and the medium used. Some of the common methods are:

• Plaque reduction neutralization test (PRNT): This method measures the reduction in the number of plaques (clear zones) formed by the virus on a cell monolayer after incubation with serum containing antibodies. The higher the reduction, the higher the antibody titer.
• Microneutralization test (MNT): This method measures the reduction in the cytopathic effect (CPE) caused by the virus on a cell monolayer after incubation with serum containing antibodies. The CPE is usually assessed by staining or microscopy. The higher the reduction, the higher the antibody titer.
• Serum neutralization test (SNT): This method measures the reduction in the infectivity of the virus in a liquid medium after incubation with serum containing antibodies. The infectivity is usually assessed by hemagglutination assay or enzyme-linked immunosorbent assay (ELISA). The higher the reduction, the higher the antibody titer.

The procedure of virus neutralization test generally involves the following steps:

1. Prepare serial dilutions of serum sample containing antibodies in a suitable diluent.
2. Mix equal volumes of diluted serum and known virus suspension and incubate at 37°C for 1-2 hours.
3. Transfer the mixture to a suitable medium for virus growth, such as egg yolk sac, animal tissue, or cell culture.
4. Incubate at 37°C for a specific period depending on the virus type and observe for signs of infection, such as plaque formation, CPE, hemagglutination, or antigen detection.
5. Compare with a control that contains virus suspension without serum and calculate the antibody titer.

The result interpretation of virus neutralization test is based on the endpoint dilution of serum that can inhibit 50% or more of virus infection. This is called the neutralizing antibody titer (NAT) or 50% plaque reduction neutralization titer (PRNT50). The higher the NAT or PRNT50, the higher the level of immunity against the virus.

Cytopathic Effect (CPE) is the change that occurs in the structure and function of the host cells invaded by the virus. It can result in the death of the cells or the formation of syncytia (giant cells with multiple nuclei). Some examples of viruses that produce CPE are poliovirus, herpes simplex virus, and coronavirus.

The neutralization of cytopathic effect is a type of virus neutralization test that measures the ability of antibodies to prevent the virus from causing CPE in cell culture. It is based on the principle that antibodies specific for the virus can bind to its surface and block its attachment, entry, or replication in the host cells.

The procedure of neutralization of cytopathic effect is as follows:

• A serum sample from the patient is taken and diluted in a suitable diluting solution.
• A known amount of virus suspension is added to each dilution and incubated for a certain period of time. This allows the antibodies and the virus to interact and form complexes.
• The mixture of serum and virus is then inoculated into a cell culture that is susceptible to the virus. The cell culture can be in a monolayer or a suspension form.
• The inoculated cell culture is incubated and observed for CPE under a microscope. A control cell culture without serum is also prepared to compare the results.
• The presence or absence of CPE in each dilution is recorded and the neutralizing antibody titer is calculated. The titer is the highest dilution of serum that can prevent CPE in 50% of the cell culture wells.

The interpretation of results is as follows:

• If the serum sample contains neutralizing antibodies against the virus, then CPE will not be observed in the cell culture inoculated with the serum-virus mixture. This indicates a positive test and suggests that the patient has been exposed to or infected by the virus.
• If the serum sample does not contain neutralizing antibodies against the virus, then CPE will be observed in the cell culture inoculated with the serum-virus mixture. This indicates a negative test and suggests that the patient has not been exposed to or infected by the virus.

The advantages of neutralization of cytopathic effect are:

• It can detect both live and inactivated viruses.
• It can measure both humoral and cellular immunity against the virus.
• It can differentiate between different strains or serotypes of the virus.

The limitations of neutralization of cytopathic effect are:

• It requires a suitable cell culture system that supports viral growth and CPE.
• It requires a standardized virus suspension with known infectivity and concentration.
• It requires a long incubation time and careful observation for CPE.
• It may be affected by non-specific factors such as complement, cytotoxicity, or interference.

Haemagglutination is a process of aggregation or clumping of erythrocytes (red blood cells) by the virus. Some viruses have the ability to bind to the surface receptors of erythrocytes and cause them to stick together. This can be observed as a visible layer of clumped cells at the bottom of a test tube.

Haemagglutination inhibition test is a type of virus neutralization test that relies on the prevention of clump formation. It is a very convenient way of measuring the number of specific antibodies against the virus in the serum sample.

The principle of this test is that if the serum sample contains neutralizing antibodies against the virus, they will bind to the virus and block its ability to bind to erythrocytes. This will result in no haemagglutination and a clear supernatant. On the other hand, if the serum sample does not contain neutralizing antibodies, the virus will bind to erythrocytes and cause haemagglutination and a cloudy supernatant.

Requirements of Haemagglutination Inhibition Test

• Serum sample
• Known virus suspension
• RBC suspension from a specific source (E.g. For Rubella Virus, RBCs from newborn chickens can be taken. Rubella virus can agglutinate RBCs of newborn chickens)
• Diluting solution such as saline

Procedure of Haemagglutination Inhibition Test

The general procedure is as follows:

1. A serum sample is taken from the suspected patient.
2. Serial dilution of the sample is performed in saline solutions.
3. Then a typical amount of virus is added to each dilution and incubated.
4. Then RBC suspension is added to each mixture.
5. The components are allowed to mix.
6. And finally, the presence or absence of haemagglutination is observed.

Result Interpretation of Haemagglutination Inhibition Test

If the patient’s serum contains neutralizing antibodies then haemagglutination won’t be observed in any dilution and it will be considered a positive test. The neutralizing antibodies bind to different binding sites on the virus and won’t let it bind with RBC preventing the haemagglutination.

If the patient’s serum does not contain neutralizing antibodies then haemagglutination will be observed in some or all dilutions and it will be considered a negative test.

The extent of haemagglutination in different dilutions will help to determine the severity of infection or the amount of neutralizing antibodies. The higher the dilution that shows no haemagglutination, the higher the antibody titer in the serum.

Applications of Haemagglutination Inhibition Test

• It can be used to detect various viral infections such as measles, mumps, influenza, rubella, etc.
• It can be used to measure the immune response to vaccination or natural infection.
• It can be used to identify unknown viruses by comparing their haemagglutination patterns with known viruses.

Virus neutralization tests are widely used in various fields of virology, immunology and epidemiology. Some of the applications are:

• Diagnosis of viral infections: Virus neutralization tests can help to identify the presence and type of virus in a patient`s serum sample. For example, Covid-19 neutralizing antibodies test is used to detect the respective infection in an individual and its severity. It can also be used to monitor the immune response and recovery of the patient after infection or vaccination.
• Detection of viral antigens and antibodies: Virus neutralization tests can help to determine the antigenic properties of different viruses and their strains. They can also be used to measure the level and specificity of antibodies produced by the host against the virus. For example, haemagglutination inhibition test can be used to detect various viral infections such as measles, mumps, influenza, etc. It can also be used to assess the immunity status and vaccine efficacy of the population.
• Evaluation of antiviral agents: Virus neutralization tests can help to evaluate the potency and effectiveness of various antiviral drugs and vaccines. They can also be used to screen for new antiviral compounds and candidates. For example, plaque reduction neutralization test can be used to measure the ability of an antiviral agent to inhibit the replication and spread of the virus in cell culture.
• Study of viral pathogenesis and evolution: Virus neutralization tests can help to understand the mechanisms and factors involved in viral infection and disease. They can also be used to track the changes and variations in viral genomes and antigens over time and space. For example, pseudotype virus neutralization test can be used to study the role of viral envelope proteins in viral entry and tropism.

Toxin neutralization test is a type of neutralization test used for detecting and measuring the amount of toxins or antitoxins in a sample. Toxins are harmful substances produced by some bacteria, fungi, plants or animals that can cause various diseases or symptoms. Antitoxins are antibodies that can bind to and neutralize the effects of toxins.

The principle of toxin neutralization test is based on the ability of antitoxins to inhibit the biological activity of toxins in a specific assay. For example, if a toxin can cause cell death, tissue damage, enzyme inhibition or animal mortality, then the presence of antitoxins can prevent or reduce these effects.

There are two main methods of performing toxin neutralization test: in vivo and in vitro. In vivo methods involve injecting the toxin and antitoxin mixture into animals and observing the outcome. In vitro methods involve incubating the toxin and antitoxin mixture with cells, tissues or enzymes and measuring the changes.

Some examples of toxin neutralization test are:

• Schick test: It is used to determine the immunity to diphtheria toxin. A small amount of diphtheria toxin is injected into the skin of a person. If the person has antitoxin in their blood, they will not develop any reaction. If they do not have antitoxin, they will develop a red spot at the injection site.
• Nagler`s reaction: It is used to identify Clostridium perfringens, which produces alpha-toxin that can digest lecithin in egg yolk. A streak of the bacterium is inoculated on an egg yolk agar plate that has antitoxin on one half and no antitoxin on the other half. After incubation, the presence of an opaque zone around the streak on the antitoxin-free half indicates a positive result.
• Anti-streptolysin O (ASO) test: It is used to measure the level of ASO antibodies in the blood of a person who has had a streptococcal infection. Streptolysin O is a toxin produced by some strains of Streptococcus pyogenes that can lyse red blood cells. ASO antibodies can neutralize this effect. A sample of blood serum is mixed with a known amount of streptolysin O and red blood cells. The degree of hemolysis is measured by spectrophotometry. A high level of ASO antibodies indicates a recent or current infection.

Toxin neutralization test can be used for various purposes, such as:

• Diagnosis of toxin-mediated diseases or infections
• Evaluation of immune status or response to vaccination
• Identification and characterization of toxins and antitoxins
• Quality control and standardization of antitoxins and vaccines

Toxin neutralization test is a useful method for detecting and quantifying the presence of toxins and antitoxins in biological samples. It can also be used to evaluate the efficacy of vaccines and treatments against toxin-producing pathogens. Some of the applications of toxin neutralization test are:

• In vivo use: Toxin neutralization test can be performed in living animals to assess their immunity or susceptibility to certain diseases caused by toxins. For example, the Schick test is used to determine the immunity to diphtheria infection by injecting a small dose of diphtheria toxin into the skin and observing the reaction. If the skin shows no inflammation or necrosis, it indicates that the animal has sufficient antitoxins in its blood to neutralize the toxin. Another example is the test for neutralizing the toxins produced by Clostridium welchii (also known as Clostridium perfringens), which causes gas gangrene and food poisoning. In this test, a sample of tissue or blood from the infected animal is injected into a healthy animal and monitored for signs of illness. If the healthy animal does not develop any symptoms, it means that the sample contains antitoxins that can neutralize the toxins.

• In vitro use: Toxin neutralization test can also be performed in laboratory settings using artificial media and reagents to detect and measure the toxins and antitoxins in various samples. For example, Nagler`s reaction is used to identify Clostridium spp. that produce alpha-toxin or lecithinase, which can digest animal tissues. In this test, an egg yolk agar plate is divided into two halves, one with antitoxin and one without. The test organism is streaked on both halves and incubated anaerobically. If the organism produces alpha-toxin, it will cause an opaque zone on the half without antitoxin, but not on the half with antitoxin. Another example is the anti-streptolysin O (ASO) test, which is used to diagnose streptococcal infections by measuring the level of antibodies against streptolysin O, a toxin produced by Streptococcus pyogenes. In this test, a serum sample from the patient is mixed with a known amount of streptolysin O and incubated. The degree of hemolysis (red blood cell lysis) caused by the toxin is inversely proportional to the amount of antibodies in the serum.

Nagler`s reaction or lecithinase test is a biochemical test used to identify bacteria that produce a toxic enzyme called lecithinase (also known as phospholipase C) . Lecithinase is a lipolytic enzyme that hydrolyzes lecithin, a phospholipid found in animal tissues, egg yolk and some plants . Lecithinase can digest mammalian tissues and cause tissue damage and necrosis .

Nagler`s reaction is especially useful for the detection of alpha-toxin or lecithinase-producing Clostridium spp., such as Clostridium perfringens, which is a gram-positive bacillus and a common cause of gas gangrene and food poisoning . The alpha-toxin of C. perfringens can be neutralized by an antitoxin, which helps to differentiate it from other lecithinase-producing Clostridium spp. .

The principle of Nagler`s reaction is based on the observation of an opaque zone around the bacterial colonies grown on egg yolk agar medium, which indicates the breakdown of lecithin into phosphorylcholine and an insoluble diglyceride by lecithinase . The antitoxin can inhibit this reaction and prevent the formation of the opaque zone .

The procedure of Nagler`s reaction is as follows :

• Prepare an egg yolk agar plate and divide it into two halves by drawing a line.
• Inoculate one half of the plate with Clostridium perfringens type A antitoxin and spread it evenly over the agar surface. Let it absorb and dry.
• Streak the test organism on the whole agar plate.
• Incubate the plate in an anaerobic incubator at 37 °C for one or two days.
• Observe the presence or absence of an opaque zone around the bacterial growth.

The result interpretation of Nagler`s reaction is as follows :

• A positive result is indicated by an opaque zone around the bacterial growth in the antitoxin-free area and no opaque zone in the antitoxin-inoculated area. This means that the test organism produces lecithinase that can be neutralized by the antitoxin. C. perfringens usually gives a positive result.
• A negative result is indicated by no opaque zone around the bacterial growth in either area or an opaque zone in both areas. This means that the test organism does not produce lecithinase or produces lecithinase that cannot be neutralized by the antitoxin. Other Clostridium spp. may give a negative result.

Nagler`s reaction is a simple and reliable test for the identification of C. perfringens and its alpha-toxin. However, it has some limitations, such as:

• It requires anaerobic conditions and careful handling of the bacteria and the antitoxin.
• It may not differentiate between C. perfringens and Bacillus cereus, which also produces lecithinase but can be distinguished by its hemolytic activity on blood agar and motility .
• It may not detect weakly lecithinase-positive Bacillus spp., such as B. thuringiensis and B. anthracis, which can be differentiated by their nonmotility and nonhemolysis .

Neutralization test is a useful technique for detecting and quantifying the presence of specific antibodies or antigens in a sample. It can also help to evaluate the efficacy of vaccines and antitoxins against various pathogens. However, it also has some drawbacks and challenges that need to be considered. Here are some of the advantages and limitations of neutralization test:

Advantages

• It has higher sensitivity than other methods such as ELISA or PCR, as it can detect very low levels of antibodies or antigens in the sample.
• It has higher specificity than other methods, as it can distinguish between different strains or serotypes of the same pathogen based on their antigenic properties.
• It can measure the functional activity of the antibodies or antigens, not just their presence or quantity. This can provide more information about the immune response and protection level of the host.
• It can simulate the natural infection process, as it involves the interaction between the pathogen and the host cells in a suitable medium. This can provide more realistic and reliable results than other methods that use artificial substrates or indicators.

Limitations

• It requires more time than other methods, as it involves several steps such as dilution, incubation, observation, and interpretation. It can take from several hours to several days to complete a neutralization test.
• It requires more expertise than other methods, as it involves handling live pathogens and cells that need special care and precautions. It also requires proper calibration and standardization of the reagents and equipment used in the test.
• It requires more resources than other methods, as it involves using expensive and scarce materials such as cell cultures, viruses, toxins, antitoxins, and antibodies. These materials may also have limited availability or shelf life, which can affect the quality and consistency of the test results.
• It may have some variability in the results, as it depends on several factors such as the quality and quantity of the sample, the type and condition of the cells, the strain and virulence of the pathogen, the concentration and potency of the antibodies or antitoxins, and the environmental conditions during the test.

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