2. HISTORY
• 1964 : A way is developed to isolate hybrid cell
from two parent cell lines using the
Hypoxanthine-aminoprotein-thymidine
(HAT) selection media
• 1975 : Georges J.F. Köhler and and César Milstein
used hybridoma to generate monoclonal
antibodies. Awarded with Nobel prize(1984)
• 1975 : Leonard Herzenberg coined the
term hybridoma.
• 1986 : First monoclonal antibody was licenced by FDA, Orthoclone OKT3
(muromonab- CD3) Which was approved for the use in Preventing kidney
transplant rejection.
• 1988 : Greg Winter gave technique to Humanize monoclonal antibodies.
3. WHAT IS HYBRIDOMA TECHNOLOGY?
• Hybridoma are formed via fusion between a splenocyte cells with tumorous
myeloma cells.
• It can divide continuously by the quality derived from myeloma cells.
• Expresses a large amount of one specefic Monoclonal antibodies.
• Can be Cryopreserved to meet never ending supply of important Mab.
4. MONOCLONAL ANTIBODIES
• Identical immunoglobulins, from
single B-Cell clone of a single parent
or a single hybridoma cell line.
• Targets a Single Epitope.
• They bind to Specefic diseased or
damaged cells.
• Treat a wide range of medical
conditions.
5. DIFFERENCE BETWEEN
MONOCLONAL AND POLYCLONAL ANTIBODIES
MONOCLONAL ANTIBODIES POLYCLONAL ANTIBODIES
Homogenous population of antibodies. Mixture of immunoglobulin molecules.
Produced by the same clones of
plasma B cells.
Produced by different clones of
plasma B cells.
Interact with a particular epitope on the
antigen.
Interact with different epitopes on the
same antigen..
Posses less cross reactivity. Posses comparatively high cross reactivity.
6. OUTLINE OF PRODUCTION OF MONOCLONAL ANTIBODIES
Purification and Characterization of
the desired antigen.
Immunization of mice with the purified
antigen.
Culture of myeloma cells which are
unable to synthesize HGPRT enzyme.
Removal of spleen cells from mice and
its fusion with the myeloma cells.
Hybridomas were grown in HAT
medium.
Hybrid cell clones are generated from
single host cells.
Tested for their ability to bind to the
antigen using ELISA.
The clone is then selected for future
use
7.
8. IMMUNIZATION SCHEDULE
• Desired protein should be in an adequate quantity (a few milligrams) and pure.
• Mice must be immunized with antigen 6–10 weeks before fusion.
• Collection of Pre-immune serum is required prior to immunization to use as a
Baseline control for antibody screening.
• Immunization schedule includes Intra-peritonial injections of 2–4 adult mice
(eg, BALB/c mice) with 20–100 µg of purified antigen in a total volume of 200 µL
(ie, 200 µL of a 1:1 emulsion of antigen in saline: adjuvant).
9. MYELOMA CELL LINE CULTURE
• Myeloma cells are cultured in presence of 8-azaguanine so they are unable to
synthesize the HGPRT enzyme.
• By using Aminopterin, de novo synthesis pathways are blocked in myeloma cells
(where salvage pathway was previously blocked) die.
• Hybridomas have a functional salvage pathway (derived from the spleen cells of
mouse) and can grow
• Elective culture medium is HAT medium hypoxanthine, aminopterin, and thymidine
10. FUSION
• The Parental myeloma cells should be Mycoplasma-free and fuse well
eg: SP2/0 and X63Ag8. 653
• B cells are mixed with HGPRT negative myeloma cells and a fusing agent, such
as Polyethylene glycol.
• Sendai virus is commercially available and still used in experiments with
hybridoma production.
11. GROWTH AND SELECTION OF MONOCLONAL ANTIBODY
• 7–14 days after fusion, Interleukin 6(IL-6) is added (Hybridoma growth factor.)
• Unfused myeloma cells die in presence of Aminopterin.
• Qualitative and Quantitative levels screened by flow cytometry or ELISA.
• Selected cultures are cloned and re-cloned to achieve a pure clone population.
12. LONG-TERM MAINTENANCE AND CRYO PRESERVATION OF MABS
• Selected hybridomas are Cryo-preserved in
Liquid Nitrogen in ampules.
• Evaluate the quality of the produced antibody
regularly.
ANTIBODY PRODUCTION AFTER FUSION
• Using Surface expanded tissue culture flasks or hollow fibre systems, such as
Technomouse.
• Growing the hybridomas in mice as an Ascitic tumor.
• Yields ascitic fluid with antibody concentrations of nearly 1–5 mg/mL.
13. APPLICATIONS OF MONOCLONAL ANTIBODIES
•DIAGNOSTIC TOOLS IN RESEARCH AND LABORATORY:
a) Western blot, immunodot blot, ELISA, radioimmuno
assay (RIA) etc.
•GENE CLONING:
a) Probe for detecting those cells that make the product
and therapy to detect the gene.
•TO IDENTIFY CELL TYPES:
a) Various types of T-cells carry cell surface antigens on
their surfaces.
b) Helpful in defining changes in T and B-cells during
development.
•PROTEIN PURIFICATION:
a) MAb-antigen complex has a Single binding affinity it is
possible to elute the required protein in a single.
16. CANCER DIAGNOSIS AND THERAPY
• Diagnosis of Solid tumors, particularly the carcinomas of the lung, breast, colon,
and rectum.
• MAbs binds complement proteins, which leads to direct cell toxicity that is
Complement Dependent Cytotoxicity (CDC).
• Block growth factors released by tumor cells by blocking Growth Factor
Receptor.
17. AUTOIMMUNE DISEASES
• Used for autoimmune diseases include infliximab and adalimumab, which are
effective in rheumatoid arthritis, Crohn’s disease, and Ulcerative Colitis.
• Due to their ability to bind to and inhibit Tumor Necrosis Factor (TNF),
TNF-α.
• The first therapeutic MAb (murine IgG 2a CD3- specific) (FDA-approved) was a
transplant rejection drug, OKT3 (or muromonab, orthoclone) in 1986.
18. ANTIBODY ENGINEERING HUMANIZATION OF MURINE ANTIBODIES
• Chimeric antibodies with human constant region and mouse variable region were
constructed.
• Transgenic mice containing human immunoglobulin germ line locus may be used
as an alternative strategy for producing Human Mabs.
• Humira is the first fully human MAb drug used for treatment of rheumatoid
arthritis and was launched in 2003.
19.
20. SIDE-EFFECTS AND LIMITATIONS OF MAbs
• Side effects include fever, headache, weakness, chills, nausea with vomiting and
diarrhea, and low blood pressure.
• Bevacizumab (used against tumor blood vessel growth) can have side effects such
as Kidney damage.
• MAb therapies are a financial burden on patients.
21. REFERENCES
• Monoclonal Antibodies: a tool in clinical research Waliza Ansar1 and
Shyamasree Ghosh- Indian Journal of Clinical Medicine
Corresponding author email: shyamasree_b@yahoo.com
• Monoclonal antibodies: Paul Nelson University of Wolverhampton, Gary
Reynolds QE Hospital & University of Birmingham
Article in Molecular Pathology · June 2000
https://www.researchgate.net/publication/264309886
