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