Hybridoma Technology

Discovered in 1975 by Georges Kohler of West Germany and Cesal Milstein of Argentina

Hybridoma technology :

A technology that involves formation of a hybrid cell(hybridoma) capable of producing/ secreting a desired product

Usually formed by fusing two types of cells:

- A secreting cell of the immune system plasma cell - A long lived cancerous immune cell myeloma(cancerous plasma cell)

The secreted product of the B cell maybe an antibody production of monoclonal antibody or a lymphokine

Uniqueness of the myeloma used in this technology - Capable of dividing indefinitely - Does not produce antibodies

Basic Methodology

For the production of monoclonal antibodies

1. (a) Immunization of a mouse with the desired antigen *(b) induction of myeloma production in a mouse: Can be done by intraperitoneal injection of mouse with mineral oil or

2. Isolation of plasma cells from the spleen Techniques used: Splenectomy

Chromatography or Rossetting or Flow cytometric cell sorting

3. Isolation, Selection and Cultivation of myeloma cells: i)Isolation done as in step 2 ii) Myeloma cells selected should -Should lack the hypoxanthine-guanine phosphoribosyltransferase (HGPRT)gene - Should not secrete antibodies

4. Fusion of myeloma and B cells - Using Polyethylene glycol for fusion - Culturing cells in selection medium Hypoxanthine aminopterin thymidine (HAT)medium is used for selection. Aminopterin is a drug which blocks one pathway for Denovo nucleotide synthesis, making the cells dependent on the salvage pathway that needs HGPRT enzyme absent in myeloma cells

5. Separation of cell lines- Done by diluting the cells in a multi-well plate Till each well contains one cell-The single cells are then cultured to produce clones or colonies

6. Screening of suitable cell lines - using colorimetric methods or - Immunocytochemical methods

8. Harvesting and purification of antibodies by: - Ion exchange chromatography - Antigen affinity chromatography - Protein A/G affinity chromatography

7. Multiplication(a)in vitro by culturing them in media(b)or in vivo: by injecting hybridomas in the intraperitoneal cavity of mice Here they produce tumors which produce antibodies contained in an antibody rich fluid called ascites fluid

Advances/Modifications of Hybridoma Technology

• Humanizing mouse monoclonal antibodies• Human antibodies• Chimeric monoclonal antibodies/hybrid

hybridomas• Abyzymes and how monoclonal antibodies are

used for therapy

Applications:

• Basic Research: - Characterizing of proteins: western blot, immuno-localisation

-In developing of new vaccines

• Diagnosis: ELISAs, Western blotting, Diagnostic histopathology as in the case of prostate cancer

• Immuno-purification• Therapy