Antibody

antibody its stucture and function

1. WELCOME SUBMITTED TO: Dr. NARAYAN MOGER DEPARTMENT OF BIOTECHNOLOGY SUBMITTED BY: Balaji Rathod ID NO. PGS16AGR7165

2. Principles of Antibody Production 1900 *Paul Erlich , Antibody formation theory

3. The blood contains two types of white blood cell or leukocyte Phagocytes ingest bacteria by endocytosis Lymphocytes produce antibodies

4. Antibodies Antibodies are proteins secreted by B cells that specifically bind to a foreign substance- Antigen Antigens are foreign substances that stimulate the production of antibodies Many of the molecules on the surface of viruses and bacteria are antigens

5. Antibodies are specific – they usually bind to only one specific antigen . Antigen Antibodies Microbe

6. Production of Antibodies by Lymphocytes A lymphocyte can produce only one type of antibody so a huge number of different types are needed Each lymphocyte has some of its antibody on its surface…

7. The antigens of a pathogen bind to the antibodies in the surface membrane of a lymphocyte… …This activates the lymphocyte.

8. The active lymphocyte divides by mitosis to produce a clone of many identical cells MITOSIS The clone of cells starts to produce large quantities of the same antibody… … the same antibody needed to defend against the pathogen!

9. Most microbes have more than one antigen on their surface, so… …they stimulate more than one type of lymphocyte… …resulting in the production of many different antibodies. These are called polyclonal antibodies.

10. Structure of an antibody

11. What is an antibody? Protein secreted by B-cells that specifically bind a foreign substance (antigen) Immunoglobulin domains Complementarity-determining Regions (CDRs ) Fab= Fragment antigen binding Hinge Fc = Fragment crystalline F( ab )’2= Protease digestion still useful to bind antigen

12. Stages in Antibody Production

13. Antibody Production: The Primary Response Macrophages take in antigen by endocytosis Antigen Macrophage The macrophage processes the antigen and attaches it to a membrane protein called a MHC protein The MHC protein is moved to the cell surface membrane by exocytosis so that the antigen is displayed on its surface. MHC protein Step 1: Antigen Presentation

14. Helper T-cell binds to macrophage presenting the antigen Step 2: Activation of Helper T-cell Helper T-cells have receptors on their cell surface membranes which can bind to antigens presented by macrophages. receptor Helper T-cell Macrophage sends a signal to activate the helper T-cell

15. Step 3: Activation of B-lymphocytes B-cells have antibodies in their cell surface membranes Inactive B-cell Antibody Antigens bind to the antibodies in the surface membranes of B-cells Antigen

16. An activated helper T-cell with receptors for the same antigen binds to the B-cell SIGNAL The helper T-cell sends a signal to the B-cell, activating the B-cell.

17. Step 4: Proliferation Plasma cells are activated B-cells with a very extensive network of rough endoplasmic reticulum. Plasma cells synthesis large amounts of antibody, which they excrete by exocytosis. The activated B-cell starts to divide by mitosis to form a clone of plasma cells.

18. The Secondary Response: Memory Cells If an antigen invades your body a second time, a much faster response occurs which produces much larger quantity of the required antibody. When activated B-cells are dividing during the primary response, some cells stop dividing and secreting antibody and become memory cells. Large numbers of memory cells remain in the body for a long time… …they are capable of producing large amounts of antibody very quickly when stimulated.

19. B-cell Macrophage Antigen Activate Antigen Helper T-cell Activate Clone Memory Cell Plasma Cell Antibodies Antibody Production: Summary

20. Principles of antibody production Clonal selection > 10 15 antibodies B-cells clone themselves Challenge and response Immunity developed only when disease challenges the immune system

21. Antigen dependent maturation of B cells

22. Classes of antibodies

23. Production of monoclonal antibodies Antigens injected to an animal B-cells extracted from the animal Tumour cells obtained B-cells fused with tumou r cells Hybridoma cells- produc e antibody Antibodies are extracted and purified http://highered.mcgraw-hill.com/olc/dl/120110/micro43.swf

24. Monoclonal Antibody Production Monoclonal Antibody Production technology was developed in 1975 . Since its development it has been very important in the modern medical science with the diagnosis, therapy, research and even basic science today. It is still largely dependent upon animal testing however. Because it requires immunization of mice in order for them to create the antibodies to be grown. Monoclonal Antibody Production or mAb is produced by cell lines or clones obtained from the immunized animals with the substance to be studied . Cell lines are produced by fusing B cells from the immunized animal with myeloma cells . To produce the desired mAb , the cells must be grown in either of two ways: by injection into the peritoneal cavity of a suitably prepared mouse (the in vivo, or mouse ascites , method) or by in vitro tissue culture. The vitro tissue culture is the method used when the cells are places in culture outside the mouse's body in a flask.

25. Why this method is used!! This method is used because antibodies must be formed from the immunization of the substance being studied. So antibodies must be produced. Once the antibodies are produced the animal aspect of the study can be eliminated and tissue culture can then be used. When using live mice researchers have found that it is the better option because in vitro doesn’t always produce adequate cell lines that are adaptive to tissue culture. Protein denaturation can occur from purification techniques and antibody activity is decreased with normal activity not represented. Also cell lines could possibly become contaminated when using in vitro technique.

26. Polyclonal antibodies: If an animal is immunized with a protein, a wide array of B cells will be stimulated to produce anti-protein antibodies. Antibodies may be made to a number of different epitopes of the protein. Even antibodies that bind to the same epitope may have different antigen-binding sites and bind the epitope with different affinity. The mixture of antibodies produced in response to an antigen are referred to as polyclonal antibodies (they are produced by many different clones of B cells).

27. Producing polyclonal antibodies

28. MONOCLONAL VS POLYCLONAL ANTIBODIES MONOCLONAL ANTIBODIES POLYCLONAL ANTIBODIES Expensive to produce Inexpensive to produce Training is required for the technology used Skills required are low Time scale is long for hybridization. Time scale is short Can produce large amount of specific antibodies. Produces large amount of non specific antibodies. Recognizes only 1 epitope on an antigen Recognizes multiple epitopes on anyone antigen Once hybridoma is made it is a constant and renewable source. No or low batch to batch variability. Can have batch to batch variability.

29. Active and passive immunity Active immunity: antibodies by the organism itself Passive immunity: antibodies received from another organism During pregnancy antibodies passed to the fetus

30. Antigenic determinants An antibody will recognize Epitope: defined segment of an antigen Immunoreactivity of epitopes may depend on primary, secondary, tertiary or quaternary structure of an antigen Define the possible applications Variability of epitopes depends on the species Antibodies are antigen themselves

31. Commercial production of antibodies: polyclonal vs monoclonal Slected clones from a polyclonal each recognizing a single epitope can be fused to a tumor cell ( hybridoma ) to proliferate indefinitely Host animals ca be used to raise antibodies against a given antigen

32. Laboratory use of antibodies Quantitation of an antigen RIA, Elisa Identification and characterization of protein antigens Immunoprecipitation Western blotting Cell surface labelling and separation Localisation of antigens within tissues or cells Expression librairies Phage display

33. Elisa: Enzyme-linked immunosorbent assay

34. Western blotting

35. Phage display

36. Clinical use of antibodies Diagnostic Detection of peptides and other molecules in various diseases Endocrine diseases: hyperinsulinemia , diabetes, hyperparatyroidism Tumor antigens (p53 tumor suppressor, PSA, a - foetoprotein ) Antibodies against viral proteins (AIDS, hepatitis) Therapeutic Neutralizing antibodies Anti-ErbB2 for breast and ovarian cancer Anti-CD20 for B-cell non-Hodgkin's lymphoma Antisera and antidotes (viruses and venoms ) Drug discovery Identification of therapeutic targets (phage display )

37. Physiological roles of antibodies Protect against Viral infections Bacterial infections Foreign bodies Antigens Deleterious in Autoimmune diseases Reumathoid arthritis Lupus Type 1 diabetes Croh’n disease Graft rejection and hypersensitivity responses

38. Lacking an antibody for your protein or antigen of interest is limiting the progression of your research! THANK YOU