Monoclonal Antibodies

1. Monoclonal
Antibodies

2. Introduction
Antibodies
• Immunoglobulin's
• Glycoprotein's secreted by
immune cells
• Recognize and bind antigens

3. Monoclonal Antibodies
• Antibodies generated from single clones of b cells.
• Specific antibodies.
• Have monovalent affinity.
• mAbs or moAbs
• Known as Magic bullets.

4. History
• mAbs are produced by Hybridoma technology.
• This method was introduced by Cesar Milstein &
Kohler in 1975

5. Properties and significance
• Glycoprotein in nature.
• Highly specific.
• Used in diagnostics.
• Applicable as therapeutics.

6. Formulation Steps
• Immunization
• Creation of Hybrid cells.
• Screening.
• Culturing /Propagation

7. Hybrid cells

8. Definition
• A hybrid cell used as the basis for the production of
antibodies in large amounts for diagnostic or therapeutic
use.

9. • Hybridomas are produced by injecting a specific antigen
into a mouse, collecting an antibody-producing cell from
the mouse's spleen, and fusing it with a tumor cell called
a myeloma cell.

11. Purpose
• To produce monoclonal antibodies
• To produce mass production of a single type of antibody .
• To treat foreign particles .
• Therapeutic use.

12. Hybridoma technology
• 1. Immunization
• 2. Cell fusion
• 3. Selection of hybridomas

13. Immunization
• The very first step in hybridoma technology is to
immunize an animal (usually a mouse), with appropriate
antigen.
• Activation of immune system.
• When a sufficient antibody titer is reached in serum,
immunized mice are euthanized and the spleen removed
to use as a source of cells for fusion with myeloma cells.

15. Isolation of cells
• Spleenocytes (spleen cells) are isolate.
• These cells are processed with myeloma cells through
Hybridoma technology.

17. Screening

18. Screening
• Screening is done to isolate hybrid cells.
• Use selective media for screening.
• (HAT) medium is used
The component of medium include,
• Hypoxanthine
• Aminoptrin
• Thymidine

19. • Solution is centrifuged
• Pour at HAT Medium.
• HAT medium works on principle of DNA synthesis.
• Aminoptrin inhibits folate and block Denovo DNA
synthesis of myeloma cells .
• Normal lymphocytes have short life span and die in 7days
of incubation.
• Hybrid cells retain in the medium.

21. Summary

22. Propagation

23. What is propagation?
• The term propagation refers to the proliferation of Hybrid
cells.
• The objectives is,
mass production of cells
• It is followed by culturing in nutritional media.
• Cultures are incubated at specific parameters.

24. Techniques
• The techniques include
• In vivo method
• In vitro method

25. In vivo techniques
• In vivo refers to inside of host body-
Steps include,
• Inoculation
• Incubation ( time period )
• Isolation
• Confirmatory test

26. Advantage & Limitation
• High amount of mAbs are produce
• About 5- 20µg /ml of asciti fluid.
• Drawbacks
• Require expertise
• Expensive
• Ultra-purification

27. In vitro Techniques
• Production of mAbs in bioreactors
• Automated technique
• Yield less mAbs (10 µg/ml )
• It is efficient tool and commercially used

28. Applications

29. Significance
Monoclonal Antibodies are produced and regulated by
FDA.
Reasons of production ?
• Diagnostic Applications
• Therapeutic Applications
• Clinical Applications

30. Monoclonal antibodies are used to analysis different
activities and functions,
• Serological test
• Pregnancy test
• Detection of pathogens
• Detection of cancer
• Identification of hormonal disorders
• Diagnostic imaging
• Identification of infectious agents

31. Therapeutic Applications
• Monoclonal antibodies are used as therapeutic agents.
• It is used in therapeutic tools

32. Mechanism of Action
Direct Action
• Naked mAbs
• Directly Attach to site of
action
• Induce immune reaction
• Like apoptosis
• Angiogenesis
Indirect Function
• Paul Ehrlich termed as
Magic bullets.
• Targetted delivery
• It act as delivery agent

33. • mAbs are used in inducing
• Opsonization ( RitumAb)
• Immunmodulation (ADCC)
• Used for effectors cell targetting
• Immune suppression in organ transplants
• Biospecific drugs for cancer
like TAA

34. Example
• Trastuzumab (Herceptin) is an anti-cancer antibody that
acts on HER2/neu (erbB2) receptor, which is over
expressed in breast cancer.