PRODUCTION OF MONOCLONAL ANTIBODY USINGHYBRIDOMA TECHNOLOGYDefinitionMonoclonal antibodies (MABs): These are the antibodies which produce from a singleclone of cells. A technology which is developed by kohler and Milstein has been broadlyused for the production of monoclonal antibody.Hybridoma: Hybridoma is a form of hybrid cell produced when B cell is fused withmyeloma cell (tumor cell).Principle of Hybridoma TechnologyHybridoma technology is a method used for the production of antibodies which can beaddressed as monoclonal antibodies in a large scale.
The hybrid cells are capable of producing antibody which are procured from b cells. Also itcan simultaneously divide the quality derived from myeloma cells into the antibodies.Hybridoma technology combines the desired qualities of both cells therefore ensures largenumber of antibody production of particular specification .example: Monoclonal antibody.
STAGES INVOLVED IN HYBRIDOMA PRODUCTIONThe continuous secretion of monoclonal antibody by hybridoma production contains anumber of steps, specifically –? Immunization? Cell fusion? Selection? Screening? Cloning? Characterization and storagePage | 3SOURCEImmunizationA mixture of immunogen in microgram to milligram quantities and appropriate adjuvant isinjected to the mouse intradermally or subcutaneously at several site at several timesrepeatedly after obtaining optimum concentration of antibody which is assayed for specificitythe mouse is killed and the spleen is isolated. This spleen is dissociate to form spleenocyte bythe use of enzymatic or other methods.Cell fusionThe spleenocytes and plasmacytoma cells are mixed in a suitable medium. The mediumcontains high concentration of PEG (50%).Formation of hybridoma will occure if this fusionis allowed to take place over a period of time.SelectionWhen the fusion of cells are done they are transferred into a medium nameHAT(Hypoxanthene aminopterin thymidine) then incubation take place. Then they aremoved from this HAT medium to culture medium containing 96 well plastic culture plate.Cells are distributed among these wells and each well is assayed respectively for thereactivity of the antibody of interest.
Page | 4ScreeningThe screening is done by ELISA which is most used screening method in production ofhybridomas. The bottom of 96 well plates contains adsorbed antigen. The samples for assayis placed in the wells and incubated for a suitable period of time. If antibody of interest is inthe sample it will bind to the antigen. Bound material remained and unbound material washedoff.The detection of this antibody is done by an immunoconjugate.
It contains two componentone is specific antibody for an epitope and another is an enzyme.CloningThe antibody of interest secreted by single cells is separated from a positive culture. It is thenallowed to propagate into cell lines.
Two methods for cloning is used namely limitingdilution method and soft agar method. In case of limiting dilution method cells are transferredinto new well in such way that each new well possesses only single cell. Regrowth of cellsoccurred and this method is repeated for several times to make sure that well containing allcells are monoclonal.
In soft agar method a semisolid medium containing less amount of agaris made where malignant cells are allowed to proliferate to form rounded colonies. Then theculture is distributed into single cells in the agar medium and monoclonal colonies are pickedout.CharacterizationIt is a method which ensures whether an antibody is monoclonal or not. The antibody can becharacterized biochemically or biophysically or by other means of methods. This methodhelps us to know about particular antigen or a specific epitope for which the antibody ofinterest is monoclonal and a number of binding site by immunochemical process.StorageStorage condition is very important as the stability of antibody depends on this. Whetherrequired antibody can withstand various storage condition or not like freezing storage timethese factors are assayed.
It must be stored in liquid nitrogen at multiple stages during cloningand cultured. It is required for preventing the destruction of cells. Some cells are stocked forfurther use.