Neutralization Test for Virus and Toxins

When a person is infected with a pathogen, antibodies are produced against many epitopes of this pathogen. A subset of these antibodies can block infection by a process called neutralization. These antibodies block or distort the antigen sufficiently, so that pathogen fails to exert its biological activity.

Neutralization reactions can occur in vitro or in vivo. Laboratory animals or tissue culture cells are used as “indicator systems” in neutralization tests. The toxin or virus to be assayed should have known effects on the indicator system which is neutralized by using an anti-toxin.

An antiserum containing an antibody that neutralizes a toxin is called an antitoxin.

Toxin Neutralization Assay

Neutralization of a microbial toxin by a specific antibody occurs when the toxin and specific antibody combine in such a way that the active portion of the toxin is blocked. Neutralization reactions can block the effects of many bacterial exotoxins.

Examples of toxin-antitoxin neutralization tests include;

1. Schick test: It is a diphtheria toxin-antitoxin neutralization test done to assess the immune status of a person. It was used in the past to know the susceptibility of individuals to Corynebacterium diphtheriae. The test is performed by intradermal injection of 0.1 mL of a purified standardized toxin. If the patient has no antitoxin, the toxin will cause inflammation at the site 4 to 7 days later. If no inflammation occurs, anti-toxin is present and the patient is immune.
2. Nagler’s reaction: Opalescence on egg yolk agar produced by α-toxin of Clostridium perfringens is inhibited when anti-α-toxin is added to the medium, which neutralizes the α-toxin.

Applications

Antitoxin therapy is used in medicine to neutralize the toxins of Corynebacterium diphtheriae, Clostridium tetani and Clostridium botulinum to prevent the development of diphtheria, tetanus, and botulism respectively.

Virus Neutralization Assay

Virus neutralization test determines if an antibody present in patient serum or mucosal surface can neutralize the infectivity of a virus. For example, antibodies directed against the hemagglutinin and neuraminidase proteins of influenza viruses prevent the adsorption of the viruses to specific receptors on host cells, protecting them from infection or cytopathic effects. Similarly, mucosal sIgA provides intestinal immunity against poliovirus.

Neutralizing antibodies may interfere with virion binding to receptors, block uptake into cells, prevent uncoating of the genomes in endosomes, or cause aggregation of virus particles. A neutralizing antibody is directed against the surface proteins of the virus. Antibodies formed against internal components of the virus (e.g., the core antigen of hepatitis B virus) do not
neutralize the infectivity of the virus.

Non-neutralizing antibodies do not neutralize infectivity of viruses, but instead, enhance their infectivity by facilitating the entry of viruses into cells which normally do not bear specific virus receptors.

When the patient’s serum is mixed with a virus preparation and poured onto a cell line. The cell line is then monitored for cell death.

1. Presence of specific antibody in the patient’s serum: Antibody neutralizes the surface antigen of the virus, making it unable to infect the cell line. A positive neutralization test indicates that the patient has antibodies and has been exposed to the virus.
2. Absence of specific antibody: No neutralization of the virus. Thus cell lines will show cytopathic effects or cell death.

Neutralization tests have been developed for arboviruses and rabies virus as well as several others.

Applications

1. Disease diagnosis: Neutralization tests are used for the diagnosis of various infections. For example, herpes simplex virus infection.
2. Passive Immunization: Inhibition of rabies and hepatitis A and B viruses infections early in the incubation period by injecting antibodies against these viruses.
3. Active Immunization: Live attenuated Sabin poliovirus vaccine elicits a strong mucosal IgA response and provides intestinal immunity against poliovirus.
4. Serotyping of viruses: A virus neutralization assay is used for the serotyping of viruses. For example, poliovirus types 1, 2, and 3 were determined using neutralizing antibodies.

References and further readings

1. Virus neutralization by Antibodies. virology blog. Retrieved June 7, 2020.
2. Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A. (2018). Brock Biology of Microorganisms (15th Edition). Pearson.
3. Willey, Joanne M, Sherwood, Linda M, & Woolverton, Christopher J. (2016). Prescott’s Microbiology (10 edition). McGraw-Hill Education.