- Antibodies that develop in response to a single antigen are heterogeneous because they are synthesized by many different clones of plasma cells. These antibodies are named polyclonal.
- Antibodies produced by a single clone of plasma cells or by tumour plasma cells (myeloma cells) are homogeneous, being monoclonal.
- Monoclonal antibodies (mAb) can be produced by the fusion of a myeloma cell with an antibody-producing lymphocyte.
- Such hybridoma synthesizes monoclonal antibodies in vitro. Important information about the structure and function of antibodies was gained from the investigation of monoclonal antibodies.
There are several common steps in standard hybridoma technology.
- First, inbred line mice are immunized with a necessary antigen.
- After the end of immunization course mouse spleen is taken out and immune splenocytes, containing antigen-specific B cells, are derived.
- These cells are fused with non-Ig-secreting mouse myeloma cells.
- As other cancer cells, mouse myeloma cells possess immortality, but cannot produce antibodies.
- Fusion is made by polyethylene glycol (PEG) or by an electric field.
- The created hybrid cells are placed into selective media permissive only for hybridoma cells.
- Hybrid cells are diluted with cultural media to the limit of one hybridoma cell per one well of the culture plate.
- These cells are cloned. After propagation hybridoma culture is tested for specific antibody production.
- In the case of a positive test, specific hybridomas are sub-cloned.
- Mass hybrid culture is able to produce large amounts of monoclonal antibodies with the same specificity and affinity.
- Due to their homogeneity, monoclonal antibodies have become an extremely powerful tool in biology and medicine, especially for immunochemical testing.
- All cell markers (CD antigens, enzymes, signal proteins, etc.) were investigated with monoclonal Ab. They are used in any kind of immune assay (ELISA, RIA, immune histochemical testing, etc.)
- Numerous problems have primarily arisen in the use of monoclonal Abs for therapy of human diseases.
- The first generations of therapeutic mAbs were total of mouse origin; thus, they induced an immune response in humans after several injections.
- To avoid this obstacle, the humanization of mouse monoclonal Ab is performed now. In that case, variable antibody fragments of mouse origin are coupled with human constant immunoglobulin parts by methods of genetic engineering or the total sequence of primary monoclonal antibody is substituted with human one.
- These mAbs are of reduced immunogenicity, being available for therapy in humans. As they demonstrated extremely high specificity, the medical applications for therapeutic monoclonal antibodies were termed as “targeted therapy”.
- Targeted therapy is actively used now for cancer and autoimmune disease treatment, for prevention of allograft rejection, etc. The last barrier still limiting the common employment of curative mAbs is a high cost of technology.