Neha

1. BIONANOTECHNOLOGY
Module 1

2. MONOCLONAL
ANTIBODIES

3. Introduction
 Polyclonal antibodies
 Monoclonal antibodies
 Hybridoma
 Β lymphocytes with myeloma cells
 Hybridoma Technology

4. Principle
 Myloma cells must not be able to produce antibodies
Denova synthesis
 Require tetrahydrofolate
 Blocked by aminopterin
Salvage pathway
 Direct conversion of purine or pyrimidine into nucleotides
 Hypoxanthines guanine phosphoribosyl transferase (HGPRT)

5. Principle
Dihydrofolate
Aminopterin
Tetrahydropholate Precursors
Nucleotides De novo synthesis
HGPRT (Salvage pathway)
DNA Hypoxanthine

6. Principle
 Conversion of hypoxanthine and guanine
 Thymidine kinase (TK)
 Convert thymidine to thymidine phosphate
 Mutation in enzyme
 When cell lack in HGPRT grown in HAT medium
 Hypoxanthine, aminopterine and thymidine medium
 Only hybridoma can grow

7. Principle

8. Production
 Immunization
 Cell fusion
 Selection of Hybridoma
 Screening the products
 Cloning and propagation
 Limiting dilution method
 Soft agar method

9. Production

10. Production

11. Large scale Production
 As ascites in the peritoneal cavity of mouse
 Encapsulated hybridoma cell with alginate gel and coating
with poly-lysine
Human Monoclonal Antibodies
 Viral transformation of human β lymphocytes (Epstein bar
virus)
 SCID mouse for producing Human MAbs
 Transgenic mouse

12. Large scale Production
Genetic engineering strategies in production of Human
Monoclonal Antibodies
 Substitution of Fv region with mouse Fv
 Substitution of human Ig by mouse CDR
 Bispecific monoclonal antibodies

13. Applications
Diagnostic Applications
Mab in biochemical analysis
 Radio immuno assay and ELISA
• Hormones
• Tissue and cell products
• Tumors

14. Applications
Diagnostic Applications
Mab in biochemical analysis
 Pregnancy
• Level of gonadrotropin
 Cancer
• Plasma carcinogenic embryonic antigens

15. Applications
MAb in biochemical analysis
 Hormonal disorders
 Infectious disease
MAbs in diagnostic imaging
Imminoscintilography
 Iodine 131
 Technetium -99
 Single photon emission computed tomography

16. Applications
Cardiovascular diseases
Myocardial infection
 Meosin exposed when myocardial necrosis occurs
 Antimyosin MAbs
 Planner gamma camera
 SPECT

17. Applications
Deep vein thrombosis
 MAbs against fibrin and platelets are used
Atherosclerosis
 MAbs against fibrin and platelets are used

18. Applications
Therapeutic Applications
As direct therapeutic agents
 In destroying disease causing microorganisms
 In treatment of cancer
 Immunosuppression of organ transplantation (OK3)
 In treatment of AIDS
 In the treatment of autoimmune diseases

19. Applications

20. Applications
As targeting agent in therapy
 MAbs in use as immunotoxins
 For treatment of cancer
 Ricin –from castor oil plant
 MAbs in drug delivery

21. Applications
 MAbs in the dissolution of blood clots
 Fibrin
 Dissolved by Plasminogen in presence of fibrinogen
activators
 Drug delivery through liposomes coupled to tissue
specific MAbs
 Drug delivery through liposomes
 Use in radiotherapy

22. Applications
 Protein Purification
 Miscellaneous Applications