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1. Hybridoma Technology
Presented by
Miss Pratiksha G. Puranik
M.Sc. (Zoology) B.Ed. Ph.D pursuing
Shri Shivaji Science College Amravati

2. Contents
 Introduction
 Immunization of animals
 Isolation of stimulated spleen cells
 Myeloma cells lines used as a partner
 Fusion methods
 Detection of monoclonal antibodies
 Applications of monoclonal antibodies
 References

3. Introduction
 In 1975, Georges Köhler and Cesar
Milstein devised a method for preparing
monoclonal antibodies.
 It is a in vitro technology of forming hybrid
cell lines called as hybridomas.
 A hybridoma is a hybrid cell obtained by
fusion of B-cell usually with a tumour cells
(myelomas).
 These hybridomas possesses ability to
produce antibody due to B-cell genome &
capacity to grow indefinitely due to
myeloma cells.
 Therefore, specific hybridomas are cultured
in vitro or may passed through peritoneal
cavity of mouse to obtain monoclonal
antibodies.
 Monoclonal antibodies are derived from a
single clone & therefore are specific for
epitope of antigen.
Milstein and Kohler

4. Immunization of animals
 Mice are immunized with repeated injection of antigen which
may be whole cell, membrane fragment or complex molecules.
 This mice then produces antibody against that injected antigen
which is tested by ELISA.
 Spleen is then removed as a source of cell for cell fusion.

5. Isolation of stimulated spleen
cells
 The spleen from immunized mice i.e., stimulated spleen is
removed & spleenocytes are cultured separately.
 This cultured spleenocytes then produces antibodies & are
fused with myeloma cells.

6. Myeloma cells used as a fusion
partner
 Generally, myeloma cells are used as a fusion partner because
the spleenocytes or any normal cell has specific life-span &
they dies in culture medium when their life-span is finished.
 To produce monoclonal antibodies, continually dividing cells
are required.
 They lacks HGPRT gene.

7. Fusion methods
 PEG or Sendai virus or electrical shocks are some of the fusion
agents, causing fusion of spleenocytes with myeloma cells
which makes their cell membranes more permeable.
 Fused cells are incubated in HAT medium for 10-14 days.
 This fusion results in the formation of hybridoma.

10. Detection of monoclonal
antibodies
 Desired hybridomas are selected for screening.
 This medium is diluted into multi-well plates such that each
well will contain only one cell.
 Since antibodies in a well are produced by same spleen cell (B-
cell of spleen) they will be migrate towards the epitope of
injected antigen & thus a monoclonal antibody.

11. Applications
Monoclonal antibodies are used for –
 Detecting pregnancy, diagnosing numerous pathogenic microorganisms.
 Measuring the effect of various drugs on blood levels .
 Matching histocompatibility antigens, and detecting antigens shed by certain
tumors.
 Radiolabeled monoclonal antibodies can also be used in vivo for detecting or
locating tumor antigens, permitting earlier diagnosis of some primary or
metastatic tumors in patients.
 For classification of blood groups.

12.  Immunodiagnosis:- The diagnosis of many infectious
diseases relies on the detection of particular
antigens or antibodies in the circulation or in tissues by use
of monoclonal antibodies in immunoassays.
 Functional analysis of cell surface and secreted molecules.
 Monoclonal antibodies are also widely used to purify selected cell populations
from complex mixtures to facilitate the study of the properties and functions
of the cells.

13. References
1. Kindt, T.J, Kuby J. Goldsby A., Osborne B.A ,Immunology V Edition.
Pp:99-101
2. Abbas, A.K, Lichtman A.H., Pillai S.Cellular and Molecular Immunology
VII Edition. Pp 97-98