1. www.themegallery.com KSU Microbiology section Dr. sarah I bukhari Assistant Professor of pharmaceutical microbiology

2. Monoclonal Antibodies Very specific antibodies produced against a single epitope Produced by one clone of B cells (immortal hybridoma) Definition

3. Obtained by fusion of myeloma cell (malignant plasma cells) with a B cell producing antibody against a single epitope Artificially produced in large amounts

4. Hybridomas Technique 4 B lymphocytes can mutate into tumor cells that result in a type of cancer termed myeloma. Myeloma cells become “immortal” and will grow indefinitely in culture. Fusion of a single activated B cell and a myeloma cell will create a hybridoma that can grow indefinitely in culture.

5. Monoclonal antibodies: 5 Antibodies produced from a single clone of B cells. Produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture. Monoclonal antibodies all have identical antigen- binding sites. Thus they all bind to the same epitope with the same affinity. They are all of the same antibody class (isotype).

6. 1 - An animal e.g. a mouse, is immunized with the antigen of interest. 2- Spleen cells from this animal are grown in a culture dish in the presence of mouse myeloma cells. Fusion of cells is enhanced by the addition of polyethylene glycol (PEG). Technique of synthesis Hybridoma cells are made in the following manner

7. Technique of synthesis 3 - The cells are grown in a special culture medium e.g. "HAT" medium, which is a mixture of Hypoxanthine Aminopterin and Thymidine. HAT medium supports the growth of fused cells "hybrid cells", but not the "parental" cells.

8. Technique of synthesis 4- The resulting hybridoma cells are screened for the production of antibody to the antigen, 5- This technique produces a clone of cells derived from a single progenitor, which is both immortal and produces monoclonal antibody. 6- positive clones are propagated in culture or in mice peritoneal cavity or freeze dried and stored at -70° C

9. PRODUCTION OF MONOCLONAL ANTIBODY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells HYBRIDOMA TECHNOLOGY ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification) Ab titre reached in Serum Spleen removed (source of cells)

10. PRODUCTION OF MONOCLONAL ANTIBODY Step 2: - Screening Of Mice For Antibody Production HYBRIDOMA TECHNOLOGY After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery) Titre too low BOOST (Pure antigen) Titre High BOOST (Pure antigen) 2 weeks

11. PRODUCTION OF MONOCLONAL ANTIBODY Step 3: - Preparation of Myeloma Cells HYBRIDOMA TECHNOLOGY Immortal Tumor Of Lymphocytes + 8 - azaguanine Myeloma Cells High Viability & Rapid Growth HGPRT Myeloma Cells Lacking hypoxanthine-guanine- phosphoribosyl transferase (HGPRT). -

12. PRODUCTION OF MONOCLONAL ANTIBODY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells HYBRIDOMA TECHNOLOGY FUSION PEG MYELOMA CELLS SPLEEN CELLS HYBRIDOMA CELLS ELISA PLATE Feeder Cells Growth Medium HAT Medium 1.Plating of Cells in HAT selective Medium 2.Scanning of Viable Hybridomas

13. PRODUCTION OF MONOCLONAL ANTIBODY Step 4: - Cloning of Hybridoma Cell Lines by “ Limiting Dilution” or Expansion HYBRIDOMA TECHNOLOGY A. Clone Each +ve Culture B. Test Each Supernatant for Antibodies C. Expand +ve Clones Mouse Ascites Method Tissue Culture Method

14. PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY