section 35.6

Immunoglobulin Structure

and

Function

819

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

called

avidity

to distinguish it from

affinity.

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