Monoclonal antibody production and classification
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When antibodies are derived from a direct immune response they are heterogeneous. This means that B-lymphocytes derived from common stem cells differentiate into many different cell lines, each producing immunoglobulins to a particular epitope on the immunogen. The serum from an immunised animal will contain numerous epitope-specific antibody clones (hence it is polyclonal) which may comprise several Ig classes and subclasses. Conjugated polyclonal antibodies make useful secondary immunoglobulins in IHC assays.
A cell line derived from a specific B-cell clone produces monoclonal antibodies, of which there are thousands on our antibody database. However, B-cells isolated from a heterogeneous serum population cannot be cultured in useful amounts. Thus we use hybridoma technology, the established method of producing monoclonal immunoglobulins since 1975. Isolated B-lymphocytes from immunised mice are fused with murine myeloma cells. This creates immortal monoclonal hybrid cell lines specific to known epitopes. The hybridomas can be stored, and then recultured to keep producing more monoclonal cells. Culture may be in vitro, or in vivo via injection into the mouse peritoneal cavity. The cells are harvested from the ascitic fluid resulting from this.
Monoclonal antibodies are widely used as primary immunoglobulins where specificity to a single epitope is required over an extended period of time. Following culture, they are characterised to assess their titre, their class and subclass. Ab titre is the dilution factor necessary to get a signal in a given immunoassay, for example Western blot or ELISA (enzyme-linked immunosorbent assay).
Because each Ig class and subclass has specific characteristics, class determination is important to determine purification and modification procedures. For example, protein A is widely used for affinity purification of mouse IgG. However, it has a low affinity for the subclass IgG1, and so purification of this subclass should be carried out using protein G.
We at Novus Biologicals have a wide range of isotyping and purification protocols, giving us a full and comprehensive antibody database covering all the latest advances in protein biology. |
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By :
Arthor Greenwald
Submitted
2010-03-26 23:39:43 |
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