Lec 6 Monoclonal Antibody

1. Lec 6 Monoclonal Antibody

2. Most antigens possess multiple epitopes and therefore induce proliferation and differentiation of a variety of B-cell clones, each derived from B cell that recognize a particular epitope; • the resulting serum antibodies are heterogeneous composed of a mixture of antibodies each specific for one epitope, such a polyclonal antibody response facilitate phagocytosis and complement mediated lysis of antigen.

3. polyclonal antibodiesare a mixture of antibodies directed toward a variety of different epitopes of the immunizing antigen are derived from different cell lines. • For diagnostic research and therapeutic purposes monoclonal antibodies are produced. • Monoclonal antibodies (mAb)are a single type of antibody that are identical and are directed against a specific epitope, Produced by B-cell clones of a single parent (single hybridoma cell line).

5. A hybridoma cell line is formed by the fusion of a one normal B-cell lymphocyte (plasma cell) making the antibody of interest with a myeloma cell (malignant plasma cell) which have the capacity for prolonged growth in tissue culture.

6. Monoclonal Antibody Production (Hybridoma Technique): 1.Immunizing animal (mouse) with the antigen for which an antibody is to be produced.The animal responds by producing many antibodies to the epitope injected. 2. The mouse’s spleen, which contains antibody-producing plasma cells is removed and broken up to form a cell suspension.

7. 3. B-cells from mouse spleen are fused with myeloma cell line(cancerous B-cell) by the addition of polyethylene glycol (PEG)which promotes membrane fusion forming hybridoma cells, • only a small proportion of the cells fuse successfully,fusion mixture contain (spleen cells, myeloma cell, fused myeloma-spleen cells).

8. 4. Culture these cells in selective medium in which only fused cell can grow(HAT medium). HAT is a mixture of hypoxanthine, aminopterin and thymidine. 5. Separate hybridoma cell and test each one for antibody production RIA or ELISA.

9. 6. Antibody producing hybridoma cells are cloned and propagated to produce large quantities of mAbs this is done either: • - in vitroby growing in cell culture or • in vivo by reinjected into the peritoneal cavities of many mice, where the cells multiply and produce large quantities of antibody in the ascetic (peritoneal) fluid that is formed. • Asciticfluid can be removed from mice many times over the animals’lifetimes, and antibody molecules will be identical to the original clone.

10. Monoclonal Antibody Production (Hybridoma Technique)

11. Monoclonal Antibody Production (Hybridoma Technique)

12. Selection of hybridomas: Because plasma cells cannot remain viable in cell culture for very long time, they must be fused together with cells that are able to survive and multiply in tissue culture,that is, the continuously propagating, or immortal cells, of multiple myeloma (a malignant tumor of antibody-producing plasma cells).

13. The special myeloma tumor cells used for hybridoma production, however, possess a very important defect • They are deficient in the enzyme hypoxanthine guanine phosphoribosyltransferase(HGPRT). • This defect leads to their inability to survive in a medium containing hypoxanthine because it cannot utilize hypoxanthine in the culture medium as a source for nucleotides (nucleic acids precursor)and due to the presence of aminopterin in the culture is a substance that prevents myeloma cells from making their own thymidine( nucleic acids precursor).

14. Since the myeloma cells cannot use hypoxanthine and the aminopetrine they cannot make nucleic acids and will die. • Plasma cells have HGPRT enzyme and can utilize hypoxanthin and thymidine in the culture medium and survive, therefore the growth of cells in HAT medium as a selective media will select for hybrids cells (fused cells) that acquired the HGPRT gene via fusion with plasma cell.

15. Uses of monoclonal Abs: • Immune diagnosis of some infectious diseases by detection of cell membrane antigens. • Immunoassay of hormones. • Protein purification • Identifying autoantibodies and tumor antigens (e.g.leukemia & lymphoma). • Immunotherapy of tumors: conjugation of mAbswith cytotoxic drug. • Unlimited uses in research works.