Page 471 - Veterinary Immunology, 10th Edition

containing three compounds: hypoxanthine, aminopterin, and
VetBooks.ir thymidine (known as HAT medium). Aminopterin is a drug that

prevents cells from making their own nucleotides from uridine.
Since the myeloma cells cannot use hypoxanthine or thymidine and

the aminopterin stops them from using the alternative synthetic
pathway, they cannot make nucleic acids and soon die. Hybrid cells
made from a myeloma and a normal cell are able to survive and
grow since they possess the critical enzymes. The hybridomas

divide rapidly in the HAT medium, doubling their numbers every
24 to 48 hours. On average, about 300 to 500 different hybrids can
be isolated from a mouse spleen, although not all will make
antibodies of interest.

If a mixture of cells from a fusion experiment is cultured in wells
on a plate with about 50,000 myeloma cells per well, it is usual to
obtain about one hybrid in every three wells. After culturing for 2
to 4 weeks, the growing cells can be seen, and the supernatant fluid

can be screened for the presence of antibodies. It is essential to use a
sensitive assay at this time. Radioimmunoassays or enzyme-linked
immunosorbent assays are preferred (Chapter 42). Clones that
produce the desired antibody are grown in mass culture and

recloned to eliminate non–antibody-producing hybrids.
Unfortunately, antibody-producing clones tend to lose this ability
after being cultured for several months. Thus it is usual to make
large stocks of hybridoma cells and store them frozen in small

aliquots. These can then be thawed as required and grown up in
bulk culture. Alternatively, the hybridoma cells can be injected
intraperitoneally into mice. Since they are tumor cells, the
hybridomas grow rapidly and provoke the effusion of a large

volume of fluid into the mouse peritoneal cavity. This fluid is rich
in monoclonal antibody and can be readily harvested.
The classical methods of making hybridomas produce only
mouse immunoglobulins, and these are of limited usefulness in

mammals of other species. Mouse antibodies are regarded as
foreign in these species and are removed by the recipient's antibody
response. Two strategies have been used to prevent or minimize
this. One involves the genetic manipulation of hybridomas so that
they produce antibodies of reduced antigenicity. Thus we can use

only purified Fab'2 fragments—this eliminates the immunogenic Fc

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