Monoclonal antibodies

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 .

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

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

12. 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.

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

14. Screening

15. Screening Screening is done to isolate hybrid cells. Use selective media for screening. (HAT) medium is used The component of medium include, H ypoxanthine A minoptrin T hymidine

16. 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.

17. Summary

18. Propagation

19. 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.

20. Techniques The techniques include In vivo method In vitro method

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

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

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

24. Applications

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

26. 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

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

28. 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

29. 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

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