A neutralizing antibody defends a cell from an antigen or infectious body by inhibiting or neutralizing any biological effect. The antibody response is crucial for preventing many viral infections and may also contribute to the resolution of an infection.
Neutralization tests are performed in vitro at cellular level including cells in the form of organs and at sub-cellular level. They assess efficacy of antibody fragments, or antibodies, to inhibit critical stages of activities which are generally deleterious to humans. These tests mostly employed to viruses and toxins. Thus, neutralization are of two types:
- Virus Neutralization test.
- Toxin Neutralization test.
Toxin neutralization tests
Toxin neutralization tests are based on the principle that biological action of toxin is neutralized on reacting with specific neutralizing antibodies called antitoxins. E.g. Adenylate cyclase toxin is an essential virulence factor of Bordetella pertussis, and antibodies to Adenylate cyclase toxin (ACT) protect against Bordetella pertussis infection in mice by neutralizing the virulent toxin.
Toxin neutralization tests can be:
- In vivo: Schick test to demonstrate immunity against diphtheria and Clostridium welchii toxin neutralization test in guinea pig or mice.
- In vitro: Antistreptolysin O test and Nagler reaction used for rapid detection of Clostridium welchii.
Other examples of toxin neutralization tests includes cellular (ricin test), sub-cellular (light chain of botulinum toxins) and at the organ level (heavy chain of botulinum toxins).
It can detect presence of toxin by the use of known antibody and identity of unknown antibody can be determined by using known antigen. ELISAs are described as an alternative method to toxin neutralization for determination of various toxins (Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon antitoxins). The assays were found to be rapid, specific, economical and showed good correlation with toxin neutralization test.
Toxin neutralization assays assess ability of antibodies within sample serum to protect cells in culture (usually macrophages, such as J7747 cell line, or other B cell lines) from toxin.
- Cells are exposed to sample sera, usually from an immunized animal, which is diluted down the plate.
- Toxin is then added, and cells are incubated.
Any live cells can be visualized using reagents such as diphenyltetrasolium bromide (MTT) which will be metabolized by live cells to give a vivid color (for MTT, resulting color is purple formazan crystals). Further towards the death cells are, less color they will develop. Completely dead cells will give no color. In presence of any antibody towards the toxin, antibody should bind the toxin which will therefore help reduce the effect of the toxin on cells, and should prevent them from dying off.
The plates in which cells have grown on can then be analyzed on a plate reader to assess the absorbance from each well and therefore concentration at which the toxin can kill half the cells can be determined.