IgG is the most prevalent antibody in mammalian tissue, and therefore a major number of the proteins on an antibody database are of this type. The other classes that are studied are IgA, IgM, IgD and IgE. Depending on the tissue and disease being studied, polyclonal and monoclonal versions of all these antibodies are also produced against specific antigens. Cross-reactivity can occur in IHC assays using tissue-derived antibodies; therefore it is common to use fragments, rather than entire Igs as primary immunoglobulins.
Antibody classes are differentiated by their heavy chain structure. The light chains are fairly homogenous, but minor differences can occur among the H-chains of some classes. Therefore these are further divided into sub-classes. IgG has 4 such subclasses. However, they have closely related structures and so cross-reactivity is less common between subclasses than between individual classes. For example, there is little cross-reactivity between IGG1, 2, 3, or 4, but a lot of the phenomenon can occur between IgG and IgA,M,D and E.
IgA is a dimeric molecule that has a mainly protective role. It does not destroy the antigen, but prevents its entry into tissues. As it is often isolated from mammalian secretions, cross-reactivity between immunoglobulins is a problem. In 2001, 3 peptide-based ELISA systems were evaluated for detection of IgG and IgA antibodies specific to the Chlamydia bacterium. The ‘gold standard’ at the time was assay by microimmunofluorescence (MIF). However, it is a difficult process to perform and there was a problem with false positive results owing to cross-reactivity.
The results showed the ELISA assays gave much more accurate results. However, sometimes there’s no choice but to use assays like MIF, and thus antibody suppliers like us at Novus Biologicals are constantly working on specific primary antibodies that offer minimal cross-reactivity.
