The different isotypes of the antibodies give them differences in terms of biological characteristics, structure, specificity against the antigen and distribution in the body. The isotyping antibodies is a process of great importance in such diverse applications as the evaluation of immune response after vaccination or during infection, or the development of hybridomas for production of monoclonal antibodies .
In this post we tell you more about the different isotypes of immunoglobulins, as well as the importance and methods for the isotyping of antibodies.
Immunoglobulins are categorized by isotypes based on differences in amino acid sequence of the constant region (Fc) of antibody heavy chains.
Below we detail the characteristics of the main isotypes of the antibodies:
- Functions : a key piece in the humoral immune response, activates the complement system and phagocytosis of microorganisms.
- Distribution in the organism : it is mainly found in blood, lymph, cerebrospinal fluid and peritoneal fluid.
- Functions : responsible for agglutination and cytolytic reactions. Due to its pentameric structure, it has a great appetite for antigens.
- Distribution in the organism : it is mainly found in blood and lymphatic fluid.
- Functions : it acts as a neutralizing antibody, preventing the invasion of pathogens through the epithelial surfaces.
- Distribution in the organism : it is mainly found on the surface of epithelial cells.
- Functions : it intervenes in parasite infections and in allergic and hypersensitivity reactions.
- Distribution in the organism : it is mainly found in the lungs, skin and mucous membranes.
WHY IS ANTIBODY ISOTYPING IMPORTANT?
The screening of hybridomas is one of the most important steps during their development for the production of monoclonal antibodies , in order to ensure the selection of those clones that are most productive and specific. As part of these screenings, antibody isotyping is carried out, which involves determining the class (IgG, IgM …) as well as the subclass (IgG1, IgG2 …) of the monoclonal antibody.
The isotype of the antibodies is of special importance when determining the method of purification of the antibodies, and their subsequent use in the corresponding immunoassays. Let’s take as an example that our antibody turns out to be an IgM:
- Purification method: in this case the purification cannot be carried out by the most common procedures, which are Protein A or G. (You can expand the information about the antibody purification methods in this entry ).
- Use in the immunoassay: if the immunoassay of interest were immunohistochemistry (IHC), it is very likely that this antibody must have been previously fragmented.
HOW TO PERFORM ANTIBODY ISOTYPING
Antibody isotyping requires the use of specific anti-immunoglobulin antibodies capable of detecting the different classes and subclasses of the monoclonal antibodies present in the sample.
To carry out the isotyping of antibodies, commercial antibodies can be used and the assay can be subsequently assembled in the laboratory itself, or use commercial ELISA kits , predesigned and ready for use in the isotyping of antibodies.
These ELISA kits usually include a plate coated with the specific isotyping antibody, which will capture and react with the antibody present in the sample. Each well (or series of wells) will include a specific antibody for each of the immunoglobulin classes / subclasses of the corresponding species. Subsequently, the addition of a secondary detection antibody will allow us to develop it.