Monoclonal Antibody Production via Hybridoma Technology

1. Monoclonal Antibody Production via Hybridoma Technology Kathryn Howard & Stephanie Pribonic

2. What are monoclonal antibodies? monoclonal antibody – an antibody that binds to one specific substance, created through identical immune cells that are clones of a unique parent cell. There are a variety of monoclonal antibodies. Because monoclonal antibodies are monospecific, each type of monoclonal antibody binds to the same epitope of an antigen (the part of an antigen that is recognized by the immune system). Currently, some of their main applications include being used in diagnostic tests and treating some types of cancer.

3. What is hybridoma technology? Hybridoma technology is the technology of forming immortalized cell lines (usually derived by fusing B-lymphocyte cells with myeloma tumor cells) that secrete a desirable protein, typically a monoclonal antibody. The cells that result are a fusion of a specific antibody-producing spleen cell with a myeloma cell. These hybrid cells have the growth characteristics of the myeloma component and the antibody-secreting characteristics of the spleen cell.

4. Hybridoma technology, cont. In order to get B cells necessary to express diverse antibodies directed against different antigens and epitopes, mice are immunized with a complex antigen mixture. The B cells are then isolated from the spleen or lymph nodes and fused with the myeloma cells. Supernatants from the resulting hybridomas . ELISA – plates are coated with the antigen of interest FACS – recognition of the native protein expressed on the surface of target cells

5. Growing the culture A selective culture medium must be used in order to grow the hybrid cells. HAT medium – contains hypoxanthine, aminopterin , and thymidine Unfused myeloma cells cannot grow because they lack HGPRT. Without HGPRT, these cells cannot replicate their DNA and therefore die. Mixture of cells is diluted, and clones are grown from single parent cells. The ability of these clones to secrete antibodies that bind to the antigen is determined through an assay. The most productive and stable clone is used for future use.

6. Growing the culture, cont. Now, we have two choices. 1. Grow the hybridomas (hybrid cells) in a suitable cell culture medium. 2. Inject mice with the hybridomas , where they will produce tumors that secrete a fluid rich in antibodies, called ascites fluid.

7. References http://www.ispe.org/glossary?term=Hybridoma http://www.cancer.gov/dictionary?cdrid=46066 http://grants.nih.gov/grants/policy/antibodies.pdf Flickinger , Michael C.  Upstream Industrial Biotechnology, 2 Volume Set.  Hoboken, New Jersey: John Wiley & Sons, Inc., 2013. Web.

8. Questions?

9. Thank you!