PRODUCTION OF MONOCLONAL ANTIBODY USING HYBRIDOMA TECHNOLOGY Definition Monoclonal antibodies

PRODUCTION OF MONOCLONAL ANTIBODY USING HYBRIDOMA TECHNOLOGY

Definition
Monoclonal antibodies (MABs): These are the antibodies which produce from a single clone of cells. A technology which is developed by kohler and Milstein has been broadly used for the production of monoclonal antibody.

Hybridoma: Hybridoma is a form of hybrid cell produced when B cell is fused with myeloma cell (tumor cell).

Principle of Hybridoma Technology
Hybridoma technology is a method used for the production of antibodies which can be addressed as monoclonal antibodies in a large scale.
The hybrid cells are capable of producing antibody which are procured from b cells. Also it can simultaneously divide the quality derived from myeloma cells into the antibodies. Hybridoma technology combines the desired qualities of both cells therefore ensures large number of antibody production of particular specification .example: Monoclonal antibody.

STAGES INVOLVED IN HYBRIDOMA PRODUCTION

The continuous secretion of monoclonal antibody by hybridoma production contains a number of steps, specifically –

? Immunization
? Cell fusion
? Selection
? Screening
? Cloning
? Characterization and storage

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Immunization
A mixture of immunogen in microgram to milligram quantities and appropriate adjuvant is injected to the mouse intradermally or subcutaneously at several site at several times repeatedly after obtaining optimum concentration of antibody which is assayed for specificity the mouse is killed and the spleen is isolated. This spleen is dissociate to form spleenocyte by the use of enzymatic or other methods.

Cell fusion
The spleenocytes and plasmacytoma cells are mixed in a suitable medium. The medium contains high concentration of PEG (50%).Formation of hybridoma will occure if this fusion is allowed to take place over a period of time.

Selection
When the fusion of cells are done they are transferred into a medium name HAT(Hypoxantheneaminopterin thymidine) then incubation take place. Then they are moved 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 the reactivity of the antibody of interest.

Screening
The screening is done by ELISA which is most used screening method in production of hybridomas. The bottom of 96 well plates contains adsorbed antigen. The samples for assay is placed in the wells and incubated for a suitable period of time. If antibody of interest is in the sample it will bind to the antigen. Bound material remained and unbound material washed off.
The detection of this antibody is done by an immunoconjugate. It contains two component one is specific antibody for an epitope and another is an enzyme.

Cloning
The antibody of interest secreted by single cells is separated from a positive culture. It is then allowed to propagate into cell lines. Two methods for cloning is used namely limiting dilution method and soft agar method. In case of limiting dilution method cells are transferred into new well in such way that each new well possesses only single cell. Regrowth of cells occurred and this method is repeated for several times to make sure that well containing all cells are monoclonal. In soft agar method a semisolid medium containing less amount of agar is made where malignant cells are allowed to proliferate to form rounded colonies. Then the culture is distributed into single cells in the agar medium and monoclonal colonies are picked out.

Characterization
It is a method which ensures whether an antibody is monoclonal or not. The antibody can be characterized biochemically or biophysically or by other means of methods. This method helps us to know about particular antigen or a specific epitope for which the antibody of interest is monoclonal and a number of binding site by immunochemical process.

Storage
Storage condition is very important as the stability of antibody depends on this. Whether required antibody can withstand various storage condition or not like freezing storage time these factors are assayed. It must be stored in liquid nitrogen at multiple stages during cloning and cultured. It is required for preventing the destruction of cells. Some cells are stocked for further use.