section 35.6
Immunoglobulin Structure
F I G U R E 3 5 - 1 2
(Also see color figure.) Antigen-antibody complex formation. Optimal concentrations of antibody and antigen result in
cross-linking and precipitation of the antigen-antibody complexes. Cross-linking is important in B-cell stimulation when
antigens bind to the IgM B-cell receptors. If antibody is present in excess, then all of the epitopes on the antigen can be
occupied by single antibody molecules (subject to steric exclusion) and the antigen-antibody complexes do not
precipitate. If antigen is present in excess, then all antibody binding sites can be occupied by antigen molecules and no
cross-linking occurs.
antigen-antibody complex by precipitate formation. These
antigen-antibody complexes and the complexes at the
equivalence point are illustrated in Figure 35-12.
The strength of monoclonal antibody binding to an anti-
gen can be described by classical stoichiometric chemi-
cal equations for a bivalent acceptor (the antibody) and
a monovalent donor (the epitope of the antigen). The
strength of binding of an antigen to a monoclonal antibody
is described by a single binding constant and is therefore
called an affinity constant. Polyclonal antibodies may ap-
pear to be quantitatively more effective in forming immune
complexes, i.e., seem to have higher affinity. However,
binding strengths for polyclonal antibodies cannot be de-
scribed in this way because they are complex mixtures of
monoclonal antibodies that recognize different epitopes
and have different affinities. To distinguish these two situ-
ations, the strength of polyclonal antibody binding is now
to distinguish it from
However, lab-
oratory determinations of the effective strength of binding
are performed in the same way.
Monoclonal Antibodies: Diagnostic Tools and
Therapeutic Agents
Because each B-cell clone produces antibody to a single
epitope on an antigen, an opportunity exists for produc-
ing chemically and functionally homogeneous antibody in
substantial quantity. However, the short B-cell lifetime that
is determined by normal apoptosis initially made this im-
practical. However, the discovery by Kohler and Milstein
that B cells could be fused with “immortal” myeloma cells
to create hybridomas that can be maintained almost indef-
initely in tissue culture removed this limitation on mono-
clonal antibody production.
The now common
hybridoma method
begins with im-
munization of mice and collection of ascites (peritoneal
white blood cells) that are synthesizing antibodies to epi-
topes on the immunogen administered to the mice. Fu-
sion of the mouse cells with myeloma cells produces “im-
mortal” hybridomas. Eliminating unfused myeloma cells
from the B-cell, myeloma cell, and hybridoma cell mix-
ture was also a challenge. The use of myeloma cells that
are unable to use the purine salvage pathway for nucleotide
synthesis, e.g., mutants missing active thymidine kinase
and/or hypoxanthine-guanine phosphoribosyltransferase
(HGPRT), provided the solution to this problem. The mu-
tant myeloma cells die in a tissue culture medium that does
not support their growth. However, hybridomas having the
necessary enzymes from the B cells for the purine salvage
pathway survive and divide. Unfused B cells die a “natu-
ral, apoptotic” death. Selective immortalization provides
a way to produce monoclonal antibodies in large quantity.
The hybridoma cultures contain cells with genes that
direct synthesis of antibody to the many epitopes of the
immunogen; however, dilution of the hybridomas to form
subcultures that contain single cells permits selection of
clones. The single hybridoma cells grow, divide, and se-
crete monoclonal antibodies. Cultures from the single cells
can be chosen that produce antibody to the desired epitope.
Continued growth of the selected hybridomas in tissue cul-
ture results in production of monoclonal antibody specific
for a single defined epitope.
Immunochemical methods have provided and con-
tinue to provide the most specific and sensitive analytical
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