Large number of identical antibodies of a single specificity is called Monoclonal Antibodies (MAbs). They were first produced by Kohler and Milstein in 1975. 1
The B cells are fused with myeloma cells, called hybridoma. It is grown on an artificial media to a factory of monoclonal Antibodies. This technique is known as Hybridoma Technology. 1
Each B cell is committed to synthesize antibody of a single specificity. The stimulation of the production of plasma cells or B cell clones which propagate antibodies appointed for a number of multiple epitopes of that antigen is executed by an immunized animal with an antigen. But the propagation of MAbs is inhibited because B cells can’t be maintained in cultures. An antibody secreting plasma cell can incur malignant transformation. They can proliferate on artificial medium, called myeloma cell. 1
A powerful immunochemical technology can be achieved for the successive propagation of MAbs by the alliance of the specificity of normal plasma cells which is produced after immunization of animal with proliferative capacity of malignant plasma cells. 1
The hybridomas can survive on HAT medium (Hypoxanthine Aminopterin Thymidine medium). They can propagate MAbs continuously, if one of the cell line is B cell by using Hybridoma Technology. 1
The production of human MAbs for therapeutic use by hybridoma technology is of great interest. 1
The production of MAbs requires the following steps-
Immunization: Immunogen of microgram to milligram are combined with a suitable adjuvant. The mixture is injected intradermally or subcutaneously at multiple sites of amouse at various times in repetitive manner. After attaining optimum concentration the spleen is taken out and dissociates into single spleenocytes. 1
In an appropriate medium spleenocytes are mixed with plasmacytoma and exposed to a high concentration of PEG and allowed to take place over a period of time. 1
Selection and screening: The cells are shifted to HAT medium and incubated after fusion. The stable hybridoma are withdrawn from the medium and shifted to regular culture medium. Aliquots are distributed among the wells of 96-well containing culture plates.
ELISA is the most common test for screening assay. The antigen is adsorbed to the bottom of the plates. If desired antibody is present there, it will bind with the antigen and unbound material is washed off. Antibody is detected by an immuniconjugate. 1
The single cells which secret desire antibodies are isolated from positive cultures and propagated into cell lines. Limiting dilution and soft agar method are used for the cloning technique.
Cells are counted in cultures and diluted. The aliquot is deposited into new wells. Cells are allowed to regrow and this is done repeated times to ensure all cells are monoclonal.
In a semisolid medium containing small amounts of agar, many malignant cells will proliferated to form spherical colonies and they are most likely to be monoclonal. 1
Fig: Mabs production using hybridoma technology
Characterization ; Storage :
It is the process by which the establishment of the monoclonality of the antibody is tested. This is done on the basis of biochemical and biophysical characterization of the antibody by using electrophoretic, spectrometric and choromatographic methods.
To avoid the destruction of clones, the cell lines secreting antibodies must be frozen in liquid Nitrogen. Some are preserved for futher production. 1
1. Kulkarni, G. (2002). Chap-6,Production of Monoclonal Antibody, Biotechnology and its applications in pharmacy. New Delhi: Jaypee Bros Medical Publishers.