[rosaclinic]

Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope.

1. The first property is exploited by transferring the cell fusion mixture to a culture medium — called HAT medium because it contains:

hypoxanthine
aminopterin
the pyrimidine thymidine

The logic:

Unfused myeloma cells cannot grow because they lack HGPRT.
Unfused normal spleen cells cannot grow indefinitely because of their limited life span. However,
Hybridoma cells (produced by successful fusions) are able to grow indefinitely because the spleen cell partner supplies HGPRT and the myeloma partner is immortal.

2. Test the supernatants from each culture to find those producing the desired antibody.

3. Because the original cultures may have been started with more than one hybridoma cell, you must now isolate single cells from each antibody-positive culture and subculture them.

4. Again, test each supernatant for the desired antibodies. Each positive subculture — having been started from a single cell — represents a clone and its antibodies are monoclonal. That is, each culture secretes a single kind of antibody molecule directed against a single determinant on a preselected antigen.

5. Scale up the size of the cultures of the successful clones.
Hybridoma cultures can be maintained indefinitely:

in vitro; that is, in culture vessels. The yield runs from 10-60 µg/ml.
in vivo; i.e., growing in mice. Here the antibody concentration in the serum and other body fluids can reach 1-10 mg/ml. However, animal welfare activists in Europe and in the U.S. are trying to limit the use of mice for the production of monoclonals.