HAT Medium myeloma partner has traits that make it immortal

(as it is a cancer cell). This mixture of cells is

Monoclonal antibodies (mAb or moAb) are
then diluted and clones are grown from single
monospecific antibodies that are the same
parent cells on microtitre wells. The antibodies
because they are made by identical immune cells
secreted by the different clones are then assayed
that are all clones of a unique parent cell.
for their ability to bind to the antigen (with a test
Monoclonal antibodies are typically made by
such as ELISA or Antigen Microarray Assay) or
fusing myeloma cells with the spleen cells from
immuno-dot blot. The most productive and
a mouse that has been immunized with the
stable clone is then selected for future use.
desired antigen. However, recent advances have
allowed the use of rabbit B-cells. Polyethylene
The hybridomas can be grown indefinitely in a
glycol is used to fuse adjacent plasma
suitable cell culture media, or they can be
membranes, but the success rate is low so a
injected in mice (in the peritoneal cavity, the
selective medium in which only fused cells can
gut), they produce tumors containing an
grow is used. This is because myeloma cells
antibody-rich fluid called ascites fluid. The
have lost the ability to synthesize hypoxanthine-
medium must be enriched during selection to
guanine-phosphoribosyl transferase (HGPRT),
further favour hybridoma growth. This can be
an enzyme necessary for the salvage synthesis of
achieved by the use of a layer of feeder fibrocyte
nucleic acids. The absence of HGPRT is not a
cells or supplement medium such as briclone.
problem for these cells unless the de novo purine
Culture-medium conditioned by macrophages
synthesis pathway is also disrupted. By exposing
can also be used. Production in cell culture is
cells to aminopterin (a folic acid analogue, which
usually preferred as the ascites technique is
inhibits dihydrofolate reductase, DHFR), they
painful to the animal and if replacement
are unable to use the de novo pathway and
techniques exist, this method is considered
become fully auxotrophic for nucleic acids
unethical.
requiring supplementation to survive.

The selective culture medium is called HAT

HAT Medium (Hypoxanthine Aminopterin
medium because it contains hypoxanthine,
Thymidine medium) is a selection medium for
aminopterin, and thymidine. This medium is
mammalian cell culture, which relies on the
selective for fused (hybridoma) cells. Unfused
combination of aminopterin, a drug that acts as a
myeloma cells cannot grow because they lack
folate metabolism inhibitor by inhibiting
HGPRT, and thus cannot replicate their DNA.
dihydrofolate reductase, with hypoxanthine (a
Unfused spleen cells cannot grow indefinitely
purine derivative) and thymidine (a
because of their limited life span. Only fused
deoxynucleoside) which are intermediates in
hybrid cells, referred to as hybridomas, are able
DNA synthesis.
to grow indefinitely in the media because the
Aminopterin blocks DNA de novo synthesis,
spleen cell partner supplies HGPRT and the
which is absolutely required for cell division to
proceed, but the other components Recombinant antibodies
(Hypoxanthine & thymidine) provide cells with
the raw material to evade the blockage and go The production of recombinant monoclonal

with the "salvage pathway", provided that they antibodies involves technologies, referred to as

have the right enzymes. repertoire cloning or phage display/yeast

The enzyme dihydrofolate reductase, which display. Recombinant antibody engineering

produces tetrahydrofolate (THF) by the reduction involves the use of viruses or yeast to create

of dihydrofolate, is specifically blocked by antibodies, rather than mice. These techniques

aminopterin. Without the THF required to rely on rapid cloning of immunoglobulin gene

convert dUMP (deoxyuridine monophosphate), segments to create libraries of antibodies with

there can be no TTP (thymidine triphosphate), slightly different amino acid sequences from

and DNA synthesis cannot proceed, unless TMP which antibodies with desired specificities can

(thymidine monophosphate) can be produced be selected. The phage antibody libraries are a

from another source. The alternative source is variant of the phage antigen libraries first

that thymidine present in HAT medium can be invented by George Pieczenik These techniques

absorbed by the cells and phosphorylated by can be used to enhance the specificity with

thymidine kinase (TK) into TMP. which antibodies recognize antigens, their

The synthesis of IMP, (precursor to GMP and stability in various environmental conditions,

GTP) also requires THF, and also can be their therapeutic efficacy, and their detectability

bypassed. In this case hypoxanthine-guanine in diagnostic applications. Fermentation

phosphoribosyltransferase (HGPRT) reacts chambers have been used to produce these

hypoxanthine absorbed from the medium with antibodies on a large scale.

PRPP (Phosphoribosyl pyrophosphate),

Applications
liberating pyrophosphate, to produce IMP by a
salvage pathway. Diagnostic tests
Therefore, the use of HAT medium for cell
Once monoclonal antibodies for a given
culture is a form of artificial selection for cells
substance have been produced, they can be used
containing working TK and HGPRT. Many
to detect the presence of this substance. The
useful refinements to the scheme are made
Western blot test and immuno dot blot tests
possible by poisons that kill cells, but to which
detect the protein on a membrane. They are also
they are immune if they lack one of these genes.
very useful in immunohistochemistry, which
Thus,, a cell lacking TK is resistant to
detect antigen in fixed tissue sections and
bromodeoxyuridine (BrdU) and a cell lacking
immunofluorescence test, which detect the
HGPRT is resistant to 6-thioguanine (6-TG) and
substance in a frozen tissue section or in live
8-azaguanine. Thus, selection with one of the
cells.
latter two drugs, followed by HAT medium, will
yield revertant colonies.
Therapeutic treatment
Cancer treatment

One possible treatment for cancer involves

monoclonal antibodies that bind only to cancer
cell-specific antigens and induce an
immunological response against the target cancer
cell. Such mAb could also be modified for
delivery of a toxin, radioisotope, cytokine or
other active conjugate; it is also possible to
design bispecific antibodies that can bind with
their Fab regions both to target antigen and to a
conjugate or effector cell. In fact, every intact
antibody can bind to cell receptors or other
proteins with its Fc region.