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Why do discrepancies in identifying the isotype and subclass of mouse monoclonal antibodies arise?
- Differences in isotypes occur because of ancestral gene duplication and recombinational events in the evolution of the Immunoglobulin molecule. Differences between IgG1 versus IgG2a or IgG2b are more pronounced, and therefore more easily recognisable by anti-isotyping antisera. However, the differences between IgG2a and IgG2b are much more discrete, and often not readily distinguishable.
- One reason for the complexity is that the same, say IgG2a sequences, do not occur in all murine strains i.e. there are differences in immuglobulin Heavy (H) chain allotypes as well as isotypes. For example, such differences would be manifested where anti-IgG2a isotyping antisera raised against say, the H chain of BALB/c mice, might not recognise the H chain of an antibody molecule secreted by a hybridoma made from a B6 or SJL mouse strain.
- There is no gene (IgG2a or IgG2b) that is identical between all murine strains; however, these genes do share sequences between the “prototype IgG2a or IgG2b” isotypes. Therefore, an anti-isotyping antibody can be expected to cross-react between these sub-classes, to varying extents.
- The reactivity profile of polyclonal isotyping antisera, raised in sheep, goats or rabbits will differ depending on the relative constituents of the H chains of the antigenic preparation. Monoclonal isotyping antibodies, while more specific for a single isotype, show more strain specificity, and frequently will not recognise the same isotype in all strains.