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2) WHAT IS HYBRIDOMA TECHNOLOGY? HOW THE FUSION TECHNIQUE IS FOLLOWED TO DO HYBRIDOMA?

A) Hybridoma
Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by fusing a specific antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis. The antibodies produced by the hybridoma are all of a single specificity and are therefore monoclonal antibodies (in contrast to polyclonal antibodies). The production of monoclonal antibodies was invented by Cesar Milstein and Georges J. F. Khler in 1975. They shared the Nobel Prize of 1984 for Medicine and Physiology with Niels Kaj Jerne, who made other contributions to immunology. The term hybridoma was coined by Leonard Herzenberg during his sabbatical in Cesar Milstein's laboratory in 1976/1977.

EXPERIMENT TECHNIQUE:
To produce the monoclonal antibody first of all the mouse immunized with specific antigen then spleen cells of mouse are removed out. Monoclonal antibodies are made by fusing myeloma cells and spleen cells of mouse. Polyethylene glycol (PEG) is used to increase the somatic cell division, only fused cells can grow. This is because myeloma cells are not able to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) enzyme. This enzyme necessary for the salvage pathway of nucleic acids. The cells are not affected in the absence of HGPRT unless the de novo synthesis pathway is also disrupted. In the presence of aminopterin the cells are unable to use the de novo pathway and thus these cells become auxotrophic for nucleic acids as supplement to (Hypoxanthine Aminopterin Thymidine medium). In this medium only fused cells will grow. Unfused myeloma cell does not have ability to grow in this HAT medium because they lack HGPRT, and thus the cell are not able to produce the DNA.

Non fused spleen cells cannot grow because of short life span 460 fused hybrid cells are called hdybridomas. Hybrid cells have capicity to grow in the HAT medium because the spleen cell partner produce HGPRT. This hybrid cell clones are generatate from single host cells. The antibodies secreted by the different clones are then tested for to check the ability to bind to the antigen (this test known as ELISA).

THE BASIC TECHNIQUE

STEPS

ARE

INVOLVED

IN

HYBRIDOMA

The basic steps involved are: 1. Immunization. 2. Generation of cell hybridomas by fusing prime cells and myeloma cells. 3. Selection and the screening of resulting clones. 4. Cloning by propagating the desire hybridomas

FUSION TECHNIQUE:
These are the agents which induse the somatic cell fusion. There are following type of agents: PHYSICALTECHNIQUE: A single-beam gradient force optical trap is combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. CHEMICAL TECHNIQUE: Poly ethylene glycol (PEG) is used to induced cell fusions and a high number of cells can be fused in the presence of PEG in a short time. ELECTROCHEMICAL TECHNIQUE: In this technique electric potential is applied in the fusion medium to induce cell fusion. This is known as electro fusion. These factors included specific resistance and osmotic strength the ionic composition, of the fusion medium and field strength and proteolytic pre-treatment of the cells effect the electrofusion.

FORMATION OF HYBRIDOMA CELL

Procedure of fusion of cells by hybridoma technology

In order for us to isolate a B lymphocyte producing a certain antibody, we first have to induce the production of such a B cell in an organism. For example, if we need an antibody for avian SERCA2 protein, we would inject the protein into a mouse. This is typically done in two doses, an initial "priming" dose and a second "booster" dose 10 days later (Campbell MA, pers. comm.). Since the protein is of foreign origin, the mouse immune system recognizes it as such and soon some of the B cells in the mouse would begin production of the antibody to avian SERCA2. A sample of B cells is extracted from the spleen of the mouse and added to a culture of myeloma cells (cancer cells). The intended result is the formation of hybridomas, cells formed by the fusion of a B cell and a myeloma cell. The fusion is done by using polyethylene glycol, a virus or by electroporation (Campbell MA, pers. comm.) The next step is to selct for the hybridomas. The myeloma cells are HGPRTand the B cells are HGPRT+. HGPRT is hypoxanthine-guanine phosphoribosyl transferase, an enzyme involved in the synthesis of nucleotides from hypoxanthine, an amino acid. The culture is grown in HAT (hypoxanthine-aminopterin-thymine) medium, which can sustain only HGPRT+ cells .The myeloma cells that fuse with another myeloma cell or do not fuse at all die in the HAT medium since they are HGPRT-. The B cells that fuse with another B cell or do not fuse at all die because they do not have the capacity to divide indefinitely.

Only hybridomas between B cells and myeloma cells survive, being both HGPRT+ and cancerous.The initial collection of B cells used is heterogenous, i.e. they do not all produce the same antibody. Therefore the hybridoma population too does not produce a single antibody. There is also another complication. A hybridoma cell is initially tetraploid, having been formed by the fusion of two diploid cells. However the extra chromosomes are somehow lost in subsequent divisions in a random manner (Campbell MA, pers. comm.). This means that we cannot be certain that the hybridomas will all produce the desired antibody or even any antibody at all. Screening is required to decide which hybridoma cells are actually producing the desired antibody. Each hybridoma is cultured and screened after doing SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis) and Western blots. The probe used is the epitope of the antibody that is desired, which may be labeled by radioactivity or immunofluorescence. Once we are sure that a certain hybridoma is producing the right antibody, we can culture that hybridoma indefinitely and harvest monoclonal antibodies from it.

PROCEDURE FOR HYBRIDOMA TECHNIQUE

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