i) Polyclonal and monoclonal antibodiesii) Monoclonal antibodies (MABs)iii) Principle of hybridoma technologyiv) How HAT medium works in the selection of hybrid cells?v) Applications of MABs
i) Polyclonal and monoclonal antibodies
Antibodies produced in an animal in response to a typical antigen are heterogenous as they are formed by several different clones of plasma cells or called as polyclonal. In the last post about Antigen, we mentioned that antigens are multivalent or have many antigenic determinants or epitopes. These epitopes will induce antibody production by different plasma cells. That is why antibodies produced in response to an antigen are called as poly clonal.
Definition: MABs are antibodies that arise from a single clone of cells. They are homogenous E.g. in a plasma cell tumour (myeloma). Monoclonal antibodies are highly useful in diagnostic tests and in research. Hybridoma technology developed by Kohler and Milstein has been widely used for the production of MABs. We will discuss the technology in this post.
Let us discuss the procedure in a summarised format.
Hybridoma is a hybrid cell formed by the fusion of B cells with myeloma cells (tumor cell).
iii) Principle of Hybridoma technology
The hybrid cell has the capacity of antibody production derived from B cells at the same time it can divide continuously the quality derived from Myeloma cells. By combining the desired qualities of both cells, the technology ensures large scale antibody production of single specificity ie: monoclonal antibodies.
Step wise procedure:
Isolation of B cells:
Mice, 2-4 weeks old are immunized with the antigen against which monoclonal antibodies are to be raised by subcutaneous injection. Later B cells are isolated from the spleen of an immunized mouse
Myeloma cells are isolated from bone marrow. Myeloma cells used are HGPRT-. HGPRT- mutant cells are raised by mutations using 8-azaguanine.
Somatic cell fusion using poly ethylene glycol (PEG)
Selection of hybrid cells in HAT medium (hypoxanthine aminopterin thymidine medium).
HAT medium is used for selection of hybrid cells. Nucleotide synthesis is essential for cell survival. In HAT medium, aminopterin blocks the cellular synthesis of purines and pyrimidines from simple sugars (de novo pathway). But cells can thrive by utilizing Hypoxanthine and thymidine present in the medium by salvage pathway using the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT)
iv) How HAT medium works in the selection of hybrid cells?
Here myeloma cells are HGPRT deficient. So these cells cannot survive in HAT medium as aminopterin blocks denovo pathway.
Bcells are HGPRT+ and can survive in the HAT medium. After some division, B cells undergo normal cell death.
Hybrid cells has HGPRT enzyme from the B cells. So only hybrid cells can survive in HAT medium.
Production of Mabs
In vivo in mice but the quantity obtained is very low.
In suspended cell culture in large fermenters in controlled conditions.
v) Applications of MABs
- In Clinical diagnosis.
- Used in diagnostic kits for different cancers, malaria, Herpes virus diseases, and in pregnancy detection
- Used in the treatment of diseases including cancers of breast, lung etc and also against surface antigens of parasites.
- Used as a research tool,
- In genetic engineering, Mabs are used to screen recombinants
- In Immunogical studies, Mabs are used to identify various cell types involved in immune response.
- Used in immunologic techniques like Radio immune assay (RIA), ELISA etc.
- Used to purify proteins and enzymes especially in immunopurification