Purified antibodies can be labelled with a variety of detection reagents for use in immunoassays. These include:
- Biotin for streptavidin/avidin linked detection
- Fluorescent dyes such as fluorescein
- Enzymes such as alkaline phosphatase and horse radish peroxidase
Immunoglobulins (IgG, IgM) can be selectively cleaved by proteases to give the antibody binding fragments (Fab, F(ab’)2 or Fab’) and the class defining fragment (Fc). These can be separated by affinity chromatography using Protein A which binds the Fc fragment and intact IgG but not the Fab fragments. At least 10mg purified IgG or IgM is required for this procedure.
Antibody fragments offer a number of potential advantages over the intact molecule including reduced non-specific binding, higher sensitivity and better tissue penetration for ICC applications. The purified antibody fragments can be labelled in the same way as the intact antibody.