50 Years Ago in Immunofluorescence

In a Citation Classic, a virologist recalls developing a green dye that is now a staple in immunofluorescence

John L. Riggs

Editor's note: Citation Classics Commentaries were written by the authors of studies that were some of the most highly cited papers between 1961 and 1975. The essays were originally published between 1977 and 1993 in Current Contents, a publication of the Institute for Scientific Information (ISI), now Thomson Scientific. (ISI was founded by Eugene Garfield, also the founder of The Scientist.) In this essay, published in 1977, a virologist recalls his 1958 paper describing a modified technique for immunofluorescence.

View other Citation Classics

<figcaption>Reference >> J.L. Riggs et al., " />
Reference >> J.L. Riggs et al., "Isothio cyanate compounds as fluorescent labeling agents for immune serum," Am J Pathol, 34:1081-92, 1958. Credit: Reprinted from Am J Pathol, 34(6), 1958 cover image with permission from the American Society for Investigative Pathology

Immunoflourescence originally used isocyanate as a dye, an unstable substance that required the poisonous gas phosgene in its preparation. As a graduate...

"John, why don't you try to substitute a sulfur atom for the oxygen in the isocyanate portion of the molecule? Perhaps that will stabilize the compound." --Joseph Burckhalter

One morning Dr. Burckhalter came into the laboratory and said, "John, why don't you try to substitute a sulfur atom for the oxygen in the isocyanate portion of the molecule? Perhaps that will stabilize the compound." Many more hours were spent in the laboratory by Seiwald and myself attempting to synthesize the isothiocyanate by different procedures suggested by Dr. Ray Brewster, then Chairman of the Department of Chemistry at the University of Kansas. We could not at that time find a commercial source of thiophosgene, and although many commercial laboratories would custom synthesize it for us, the cost was prohibitive. We were on the verge of attempting to synthesize it ourselves when an advertisement of Rapter Laboratories of Chicago was noted which stated that they had thiophosgene available. We immediately obtained some and carried out the experiments to produce the isothiocyanates. We were successful in producing the isothiocyanates (they were indeed more stable compounds) and, with Dr. Downs in the bacteriology laboratories, were also successful in coupling them to antibodies which were subsequently used in staining and specifically identifying different bacterial, rackettsial and viral antigens. "Fluorescein and rhodamine isothiocyanate soon became available commercially and the fluorescent antibody procedure then became available as a tool in almost any laboratory that wished to use the technic," quoting Dr. Coons.1 Now it was no longer true, as Pressman had told me, that immunofluorescence was impossible because those who could do the chemistry couldn't do the histology, and vice versa.

Dr. John L. Riggs, Viral and Rickettsial Disease Laboratory, California Department of Health, Berkeley, California, February 10, 1977

1. A.H. Coons, "The development of immunohistochemistry," Ann NY Acad Sci, 177:5-9, 1971.

Interested in reading more?

Magaizne Cover

Become a Member of

Receive full access to digital editions of The Scientist, as well as TS Digest, feature stories, more than 35 years of archives, and much more!
Already a member?