Hybridoma Technology

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Hybridoma Technology

A hybridoma term is  stands  for being a hybrid cell obtained by fusing a B-lymphocyte with usually a tumour cell ( oma terms is basically added for cancerous cells) of the antibody forming system or of B-lymphocytes (these are called myelomas). The hybrid cells thus produced possess the ability to produce antibodies due to the B-lymphocyte genome and the capacity for indefinite growth in vitro due to the tumour (myeloma) cell involved in the fusion.

Therefore, specific hybridomas are either cultured in vitro or passage through mouse peritoneal cavity to obtain monoclonal antibodies; this is called hybridoma technology. This technology was developed by G. Kohler and C. Milstein in 1975 for which they (along with N. Jerne) were awarded the Nobel Prize for Physiology and Medicine in 1984.

Antibodies are produced by B-lymphocytes, each B-lymphocyte cell being preprogrammed to respond to a single antigenic determinant. Antigenic determinant denotes that region of an antigen molecule, which interacts with an antibody that is specific to it.

When an antigen reacts to the cell surface receptor of a B-lymphocyte, it proliferates rapidly to yield a population (clone) of B cells all of which produce antibodies of the same specificity; this is called clonal selection.

Thus a B-lymphocyte cell produces antibodies of only one specificity i.e., specific to only one antigenic determinant. In addition, an antibody producing B-lymphocyte cell, called plasma cell, is fully differentiated and does not divide when cultured in vitro; these features are critical to hybridoma technology.

B-lymphocytes are isolated from the spleen of an animal, e.g., mouse, which had been immunized with the antigen against which monoclonal antibodies are to be raised. Immunization is achieved by injecting the antigen along with a suitable adjuvant (a non-antigenic preparation known to stimulate the immune response) either subcutaneously or in peritoneal cavity, followed by booster doses of the antigen.

Immunization enhances the population of B-lymphocytes producing antibodies specific to the antigen used (clonal selection), which greatly increases the chances of obtaining the desired hybridoma clone. A large number of these B-lymphocytes are mixed with the cells of selected myeloma and induced to fuse to form hybrid cells.

The myeloma cells are selected for mainly the following two features: (1) these cells must not produce antibodies themselves, and (2) they must contain a genetic marker, e.g., HGPRT trait, which permits an easy selection of the resulting hybrid cells.

When HGPRT^– cells are fused with B- lymphocytes, the resulting cell population will consist of the following: (1) hybrid cells (hybridomes), (2) myeloma cells, and (3) B-lymphocytes (Fig. 5.5). This cell population is now cultured in HAT medium containing the drug aminopterin. The HGPRT myeloma cells will be unable to divide in the HAT medium due to aminopterin.

At the same time, the B-lymphocytes cannot grow for long periods of time in tissue culture and eventually die. In contrast, only the hybridoma cells proliferate in the HAT medium since the B-lymphocyte genome makes them HGPRT and they have the capability for indefinite growth from the myeloma cell. Thus hybridomes (myeloma + B- lymphocyte hybrid cells) are selected by using a suitable selection medium like HAT, which allows only the hybridomes to proliferate (Fig. 5.5).

 

The next step consists of identification and isolation of the hybridoma cells producing antibodies specific to the antigen used for immunization of the animals. The hybridoma cells are suspended, suitably diluted and distributed into microwells, ideally, one cell in each microwell, and allowed to grow.

The hybridoma cells grow and secrete antibodies into the medium. The supernatant from each microwell is sampled and assayed for the presence of antibodies specific to the antigen in question using one of the methods based on either precipitation or agglutination caused by the antibodies specific to the given antigen.

ELISA is the most sensitive and rapid of these assays. Wells containing the antibodies specific to the antigen are identified and the hybridoma cells from them are isolated and cloned; this ensure that a hybridoma clone produces antibodies of a single specificity.

Once the desired hybridoma clone has been obtained, it is multiplied either in vitro or in vivo to obtain monoclonal antibodies. The in vivo production system involves injection of the hybridoma cells into the peritoneal cavity of isogenic animals, e.g., Balb/C or nu/mu mice, collection of the ascitic fluid and separation of the antibodies from it. Alternatively, hybridoma cells are grown in vitro in a suitable large scale culture system and the monoclonal antibodies are purified from these cultures.

The hybridomas may be grown as a suspension culture, in a hollow fiber culture system or in an Opticell culture system. Microencapsulation in alginate beads is reported to enhance antibody production by hybridomas.

A schematic representation of the production of hybridoma clones. B-lymphocytes have a limited life in vitro, while HGPRT” myeloma cells are unable to divide due to the drug aminopterin present in the HAT medium; hence only hybridoma cells survive. Each microwell supernatant is tested for the presence of antibody specific to the given antigen, and positive testing wells are identified; cells from these wells are cloned to obtain the hybridoma clone producing the desired monoclonal antibody.

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