Hybridoma technology

brief information about hybridoma technology

1. HYBRIDOMA TECHNOLOGY Done by : Lamya Alsadhan Submitted to : Dr. Elsayed Ahmed Elsayed

2. Introduction Hybridomas are cells that have been engineered to produce a desired antibody in large amounts, to produce monoclonal antibodies. Monoclonal antibodies can be produced in specialized cells through a technique now popularly known as hybridoma technology. Hybridoma technology was discovered in 1975 by two scientists, Georges Kohler and Cesar Milstein, who jointly with Niels Jerne of Denmark (now working in Germany) were awarded the 1984 noble prize for physiology and medicine . The term hybridoma was coined by Leonard Herzenberg during his sabbatical in César Milstein's laboratory in 1976/1977.

3. Monoclonal Antibodies An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. Monoclonal antibodies ( mab ) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. A monoclonal antibody can be used to detect pregnancy only 14 days after conception. Other monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal, and chlamydia infections . Monoclonal antibodies have a wide range of therapeutic applications. MAbs are used in the treatment of cancer , transplantation of bone marrow and organs, autoimmune diseases, cardiovascular diseases and infectious diseases.

4. Hybridoma technology Hybridoma technology is a method for producing large numbers of identical antibodies (also called monoclonal antibodies ). This process starts by injecting a mouse (or other mammal) with an antigen that provokes an immune response. A type of white blood cell, the B cell will be converted to Plasma cell by the presence of the antigen. The plasma cell will produce antibodies that bind to the antigen are then harvested from the mouse. These isolated plasma cells are in turn fused with immortal B cell cancer cells, a myeloma , to produce a hybrid cell line called a hybridoma .

5. The conversion of the B cell to plasma cell that secretes antibodies

6. Why do we even bother? You might wonder.. Why we even bother making the hybridoma cells when we could just isolate the plasma cells, culture them and have them produce the antibodies for us? The idea is right and possible, but the only problem is the plasma cells life span is very short, ranging from few weeks to few months maximum. In hybridoma technology, this problem is over comes by fusing the plasma cells with immortal cancerous B cells known as myeloma cells.

7. Hybridoma Technology Idea

8. PROCEDURE Mouse is immunized by giving antigen injection intravenously , meaning directly into the blood, so the antigen will be dropped into the spleen . Inside the spleen, the antigen will interact with B cells and will convert them to plasma cells , once the plasma cells are produced they will secrete a large number or monoclonal antibodies The spleen is then removed from the mouse and plasma B cells are cultured.

9. PROCEDURE After the purification of the plasma cells, it is then fused with the myeloma cells in the presence of polyethylenglycol . Since the fusion procedure is random, five different types of cells will appear :

10. How to isolate the Hybridoma between all those cells? Between all those five cells, we need the hybridoma only, to isolate it we have to use the HAT Media. The HAT stands for ( H ypoxanthine + A minopterin + T hymidine ). When ever the cell divides, it must synthesize nucleotides, the nucleotides can be synthesized in two ways : Salvage Pathway De novo Pathway The cells use the degraded parts of old nucleotides to make new nucleotides The cell completely make new nucleotides using small metabolites like sugar and amino acids Aminopterin HGPRT

11. HGPRT+ HGPRT+ HGPRT+ Short life span Short life span Immortal HAT HAT HAT only the hybridoma will remain because its immortal and have the HGPRT enzyme, in this way the hybridoma is selected and used for continues MAb production.

12. Purification of Antibodies Monoclonal antibodies may need to be purified before they are used for a variety of purposes. Antibodies can be purified by any of the following techniques : Ion-exchange chromatography. Antigen affinity chromatography.

13. Advantages of Serum Free Media in Hybridoma Cell Culture and Preparation of Monoclonal Antibodies Greatly simplified purification of antibodies due to increased Initial purity and absence of contaminating immunoglobulin. Decreased variability of culture medium. Reduced risk of infectious agents. Fewer variables for quality control/quality assurance. Increased control over bioreactor conditions. Potential for increased antibody secretion. Low or no dependence on animals. Cost effective. Overall enhanced efficiency

14. Disadvantages of Serum Free Media in Hybridoma Cell Culture and Preparation of Monoclonal Antibodies Not all serum free media are applicable to all cell lines. Cells may not grow to as high densities and may be more fragile than cells in serum Media may take longer to prepare

15. CONCLUSION Hybridoma technology is the valuable for preparing antibody in vitro condition Monoclonal antibodies, as they are known, have opened remarkable new approaches to preventing, diagnosing, and treating disease. It is helpful in various aspects .