ANTIBODY-BASED TECHNIQUES - A Guide to Methods in the Biomedical Sciences - page 78

relied on the ability of antibodies to cross-link soluble or cell-associated
antigens, allowing for the detection of antigen-antibody interactions by
agglutination reactions, or precipitation. Both methods rely on the fact
that antibodies are multivalent (have two or more binding sites) and
therefore can bind to epitopes on more than one cell, or more than
one protein. As a result, antibodies that react with cells often cause the
cells to be clumped together, the process known as agglutination. Ag-
glutination is still the method used for ABO blood typing because it is
inexpensive and rapid, and visible to the naked eye. When proteins (or
other macromolecules) are cross-linked, they fall out of solution once
the cross-linked matrix is of sufficiently large size, a process called im-
munoprecipitation. The precipitates can easily be large enough to be
visible.
Immunoprecipitation
Immunoprecipitation is still widely used today to identify and bio-
chemically characterize molecules from cells, cell extracts, or in vitro
transcription-translation assays. Rather than relying on the antigen-
antibody matrix to become large enough to precipitate on its own, how-
ever, protein A (or protein G) coupled to agarose or Sepharose beads is
usually used to bring down the antigen-antibody complexes, which can
then be processed for further analysis.
Radioimmunoassay (RIA)
The development of the RIA was a major breakthrough in the ability to
rapidly and sensitively quantitate any biomolecule for which a antibody
existed. As the name implies, the RIA is an immunoassay (a method of
identifying and quantitating a substance using specific antibodies) that
incorporates the use of a radioactively labeled ligand. It was developed
by Yalow and Berson in 1959 and published a year later (32). The RIA
permitted, for the first time, the determination of hormone levels in the
blood (it was first developed for the measurement of insulin), and was
rapidly adapted to a number of other research and clinical applications.
The method is highly sensitive, and can detect antigens at concentra-
tions of as little as a few pg. The development of this method earned for
Rosalyn Yalow the Nobel Prize in Medicine in 1977.
The RIA is based on a competition assay in which a known amount
of antibody is mixed with a known amount of radiolabeled antigen. Un-
labeled antigen, often referred to as “cold” antigen, is then added to the
mix in increasing concentrations, which displaces increased amounts of
the labeled antigen, since the cold antigen “competes” for binding (much