Page 313 - Color Atlas of Biochemistry

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304 Tissues and organs

Monoclonal antibodies, or in vivo by producing ascites fluid in mice
immunoassay (6).

A. Monoclonal antibodies B. Immunoassay

Monoclonal antibodies (MABs) are secreted Immunoassays are semiquantitative proce-
by immune cells that derive from a single dures for assessing substances with low con-
antibody-forming cell (from a single cell centrations. In principle, immunoassays can
clone). This is why each MAB is directed be used to assess any compound against
against only one specific epitope of an immu- which antibodies are formed.
nogenic substance, known as an “antigenic The basis for this procedure is the anti-
determinant.” Large molecules contain several gen–antibody “reaction”—i. e., specific binding
epitopes, against which various antibodies are of an antibody to the molecule being assayed.
formed by various B cells. An antiserum con- Among the many different immunoassay
taining a mixture of all of these antibodies is techniques that have been developed—e. g.,
described as being polyclonal. radioimmunoassay (RIA), and chemolumines-
To obtain MABs, lymphocytes isolated from cence immunoassay (CIA)—a version of the
the spleen of immunized mice (1)are fused enzyme-linked immunoassay (EIA) is shown
with mouse tumor cells (myeloma cells, 2). here.
This is necessary because antibody-secreting The substance to be assayed—e. g., the hor-
lymphocytes in culture have a lifespan of only mone thyroxine in a serum sample—is pipet-
a few weeks. Fusion of lymphocytes with tu- ted into a microtiter plate (1), the walls of
mor cells gives rise to cell hybrids, known as which are coated with antibodies that specif-
hybridomas, which are potentially immortal. ically bind the hormone. At the same time, a
Successful fusion (2) is a rare event, but the small amount of thyroxine is added to the
frequency can be improved by adding poly- incubation to which an enzyme known as
ethylene glycol (PEG). To obtain only success- the “tracer” (1) has been chemically coupled.
fully fused cells, incubation is required for an The tracer and the hormone being assayed
extended period in a primary culture with competefor thesmall number of antibody
HAT medium (3), which contains hypoxan- binding sites available. After binding has
thine, aminopterin, and thymidine. Amino- taken place (2), all of the unbound molecules
pterin, an analogue of dihydrofolic acid, com- are rinsed out. The addition of a substrate
petitively inhibits dihydrofolate reductase and solution for the enzyme (a chromogenic solu-
thus inhibits the synthesis of dTMP (see tion) then triggers an indicator reaction (3),
p. 402).AsdTMPisessential forDNA synthe- the products of which can be assessed using
sis, myeloma cells cannot survive in the pres- photometry (4).
ence of aminopterin. Although spleen cells The larger the amount of enzyme that can
areableto circumventthe inhibitory effect bind to the antibodies on the container’s
of aminopterin by using hypoxanthine and walls, the larger the amount of dye that is
thymidine, they have a limited lifespan and produced. Conversely, the larger the amount
die. Only hybridomas survive culture in HAT of the substance being assayed that is present
medium, because they possess both the im- in the sample, the smaller the amount of
mortality of the myeloma cells and the spleen tracer that can be bound by the antibodies.
cells’ metabolic side pathway. Quantitative analysis can be carried out
Only a few fused cells actually produce through parallel measurement using stan-
antibodies. To identify these cells, the hybrid- dards with a known concentration.
omas have to be isolated and replicated by
cloning (4). After the clones have been tested
for antibody formation, positive cultures are
picked out and selected by further cloning (5).
This results in hybridomas that synthesize
monoclonal antibodies. Finally, MAB produc-
tion is carried out in vitro using a bioreactor,

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