Making antibodies work
MILESTONES
M I L E S TO N E 4
Making antibodies
work
Crossed immunoelectrophoresis—just one analytical
application of antibodies—
can simultaneously identify
dozens of serum proteins.
Courtesy T.C.Bøg-Hansen.
Because antibodies are able to
specifically bind target molecules,
the possibility of their having an
analytical application was recognized
early on. Robin Coombs, Arthur
Mourant and Robert Race, working
for the UK’s Medical Research
Council emergency blood-transfusion service, were keen to determine
whether specific antibodies, such as
those binding to red blood cells in
autoimmune hemolytic anemia, were
present in the human body. However,
ambiguous results were often
obtained with the existing methodology. In 1945 they described how
to use anti-human-immunoglobulin
serum to agglutinate red blood cells
that had antibody bound to their cell
surface. This enabled them to better
distinguish whether antibodies to Rh
factor were present in the blood. The
ability to detect such antibodies had a
practical application, as the presence
of antibodies could be indicative of
the susceptibly of newborn babies to
hemolytic disease. The assay developed was a forerunner to the Coombs
tests that are used to this day.
Another major advance in the
technical application of antibodies
came in 1960. Rosalyn Yalow and
Solomon Berson developed a system
using antibodies to quantify insulin
present in blood plasma. The principle behind this was the competition
S8 | DECEMBER 2016
between the binding of antibodies
to endogenous insulin versus their
binding to radioactive insulin. This
radioimmunoassay was used to
measure insulin present in the blood
and provided greater sensitivity than
that of previous approaches. It was
subsequently applied to a multitude
of serum antigens. The importance
of the radioimmunoassay was recognized by a Nobel Prize awarded to
Rosalyn Yalow in 1977; unfortunately,
Solomon Berson had already died and
so was unable to be a co-recipient.
In 1971 researchers described
replacing the radioactive label with
components detectable in alternative
ways. Bauke van Weeman and Anton
Schuurs chose to conjugate their
molecule of interest (human chorionic
gonadotrophin) with the enzyme
horseradish peroxidase. The antibody
thus bound a molecule that would
change the color of a substrate when
oxidized by horseradish peroxidase.
They used this system to assay
the amount of human chorionic
gonadotrophin in the urine of women,
thereby identifying those who were
pregnant. The general principle
behind this assay remains the basis of
many pregnancy-testing kits today.
Concurrently, Eva Engvall, a graduate
student in Stockholm, and Peter
Perlmann, her advisor, performed
a similar assay but used antibody
monoclonal
antibodies…
have had
a truly
transformative
effect on
laboratory
sciences.
conjugated to the enzyme alkaline
phosphatase. They named their assay
the ‘enzyme-linked immunosorbent
assay’, which resulted in the catchy
acronym ‘ELISA’. In addition to its
application in the detection and
quantification of serum components,
ELISAs are routinely used to detect
viral infections, such as infection with
human immunodeficiency virus, and
the technique remains a mainstay of
laboratories around the world.
In addition to detecting the
tagging of antibodies to molecules or
cells of interest, it was clearly desirable to be able to separate the tagged
components. This was achieved in
1979 by David Parks, Virginia Bryan,
Vernon Oi and Leonard Herzenberg,
who used the newly invented
fluorescence-activated cell sorter. The
light-scattering and fluorescent properties of the cells enabled cells bound
with antigen-coupled microspheres
to be distinguished and directed
into alternative collection pots, thus
facilitating phenotypic separation,
description and categorization.
Since the pioneering work of
César Milstein and Georges J. F.
Köhler (MILESTONE 9) there have
been innumerable applications
and refinements to the application of monoclonal antibodies.
Immunohistochemistry, immunofluorescence, immunoprecipitation,
immunoblot analysis, Ouchterlony
double immunodiffusion, radial
immunodiffusion and rocket immunoelectrophoresis are additional
techniques dependent on the use
of antibodies that have been implemented widely across the biological
sciences. Thanks to their specific
binding and the possibility for investigators to select the target of choice,
monoclonal antibodies have proven
to be an indispensable research and
analytical tool that have had a truly
transformative effect on clinical and
laboratory sciences.
Katharine Barnes
Managing Editor, Nature Protocols
ORIGINAL RESEARCH PAPERS Coombs, R. R. A. et al. A new test for the detection of
weak and “incomplete” Rh agglutinins. Br. J. Exp. Pathol. 26, 255–266 (1945) | Yalow, R. S.
& Berson, S. A. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest. 39,
1157–1175 (1960) | Van Weemen, B. K. & Schuurs, A. H. W. M. Immunoassay using
antigen-enzyme conjugates. FEBS Lett. 15, 232–236 (1971) | Engvall, E. & Perlmann, P.
Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G.
Immunochemistry 8, 871–874 (1971) | Parks, D. R. et al. Antigen-specific identification
and cloning of hybridomas with a fluorescence-activated cell sorter. Proc. Natl. Acad.
Sci. 76, 1962–1966 (1979)
www.nature.com/milestones/antibodies
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