Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogotsov 2011

Application of DNA Clones
Monoclonal Antibodies

Antibodies are synthesized by B lymphocytes, with each B lymphocyte producing a single immunoglobulin complementary to a unique specific chemical Structure (referred to as a determinant or epitope) on the antigen molecule.

When a mammal is infected with an antigen, the B lymphocytes specialized for synthesizing antibodies that recognize this antigen are stimulated to grow, proliferate, and produce antibodies.

Each B lymphocyte activated by the antigen forms a clone of Cells in the Spleen or Lymph Nodes, in which every Cell produces identical antibody molecules—namely, monoclonal antibodies.

Because Antigens possess multiple epitopes, they trigger the simultaneous growth of many different B lymphocyte clones, resulting in a polyclonal mixture of antibodies recognizing various epitopes of the given antigen. Typically, isolating monoclonal antibodies from such a polyclonal mixture using conventional biochemical Separation Methods is not feasible due to the extremely low concentration of the specific antibody.

The lifespan of normal B lymphocytes is insufficient for producing the industrial quantities of monoclonal antibodies required. Therefore, hybridomas are utilized—clones of hybrid cells obtained by fusing desired B lymphocytes from a mouse spleen with cancerous myeloma lymphocytes. This fusion is induced by special Treatment of the cells with specific viral Glycoproteins or chemically (using polyethylene glycol). As a result, the Plasma Membranes of the two cells merge, and their Cytosol and Organelles mix.

Those fused cells that retain the capacity for infinite (cancerous) division form a hybridoma. Each hybridoma synthesizes monoclonal antibodies encoded by the parent B lymphocyte.

Next, it is necessary to isolate the hybridoma cells from both un-fused parent cells and spontaneously fused cells that do not contain the hybridoma components.

The Selection scheme is illustrated in Figure 110.

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Figure 110 - Scheme for obtaining hybridomas for monoclonal antibody production

Selection is performed using "HAT" selection medium, in which mutant mouse myeloma cells (lacking HGPRT Enzymes) cannot grow and multiply, whereas normal (wild-type) mouse spleen cells can grow and multiply, with some of them capable of producing antibodies against the specific antigen X.

The HAT medium contains:

1) the purine hypoxanthine,

2) aminopterin,

3) thymidine,

which block the de novo synthesis of Glycine, purine NUCLEOTIDES, and thymidine monophosphate from tetrahydrofolate (the active form of Folic acid).

At the same time, non-mutant (wild-type) cells are able to carry out this synthesis from purine bases and thymidine using specific enzymes: thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT).

Therefore, the hybridoma cells formed as a result of fusion, having received the missing enzymes from normal cells, are able to grow and multiply in the HAT medium, whereas the un-fused myeloma cells cannot.

The "immortality" inherited from the myeloma cells sustains the longevity of the hybridoma cells, while normal mouse spleen lymphocytes die off relatively quickly.

The hybridoma obtained in this manner is subsequently cultured to yield the required amount of pure monoclonal antibody.