Waters Symposium: Immunoassay
Development and Proliferation of Radioimmunoassay Technology Rosalyn S. Yalow † Mount Sinai School of Medicine, City University of New York, New York, NY 10029
I began my work with radioisotopes in medical research in the late 1940s, after obtaining my Ph.D. in physics, as radioisotopes became available from the reactor in Oak Ridge, Tennessee. I was essentially alone in my work until I was joined by a young clinical internist, Solomon A. Berson, when he finished his residency in 1950. From that time, until he joined the faculty at Mount Sinai, all of our work was done collaboratively. Together, we began to apply radioisotopic methods to the problems of blood volume determination, albumin and thyroid hormone metabolism, and radiation chemistry. These were important issues in their own right, but in terms of our eventual development and application of radioimmunoassay, they provided us with the intellectual approaches and technical skills that allowed us to move rapidly in new directions.
†Emeritus Senior Medical Investigator, Veterans Administration, and Solomon A. Berson Distinguished Professor-at-Large, Mount Sinai School of Medicine.
Among the disorders of the endocrine glands, diabetes affects the greatest number of people. And among peptide hormones, insulin is uniquely essential. We cannot live without insulin, nothing will suffice for its absence, only that small protein hormone will do. Other peptide hormones are crucial, such as adrenocorticotropic hormone (ACTH) or parathyroid hormone (PTH); but they are only relatively essential because, in their absence, the products of their target organs will do nicely. So insulin is a star in the firmament of peptide hormones. In 1921, the Canadians Fredrick Banting and Charles Best extracted insulin from the pancreas in a form that was capable of controlling diabetes in a dog. In a short time, bovine and porcine insulins were available to treat diabetic humans, and the previously fatal juvenile, or Type 1, diabetes could be controlled. An untold number of people, mostly young, had died from this disease, and the discovery proved nothing short of a miracle lifesaver. Banting shared the 1923 Nobel Prize for Medicine or Physiology with J. J. R. Macleod, but he shared his prize money with Best, who was only a lab assistant, and an undergraduate medical student.
The Annual James L. Waters Symposium at Pittcon The objectives of the annual James L. Waters Symposium at Pittcon are different from those of other symposia at either Pittcon or other conferences. Waters, founder of the well-known Waters Associates, Inc., and currently president of Waters Business Systems, Inc., arranged with the Society for Analytical Chemists of Pittsburgh (SACP) in 1989 to offer an annual symposium at Pittcon to explore the origins, development, and commercialization of scientific instrumentation of established and major significance. The main goals were and still are to ensure that the early history of this cooperative process be preserved, to stress the importance of contributions of workers with diverse backgrounds, objectives and perspectives, and to recognize some of the pioneers and leaders in the field. Important benefits of these symposia are creation of awareness of the way in which important new instruments and, through them, new fields are created, and promotion of interchange among inventor, development engineer, entrepreneur, and marketing organization. The topics of the first eight Waters Symposia, beginning in 1990, were gas chromatography, atomic absorption spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy, mass spectrometry, high-performance liquid chromatography, ion-selective electrodes, and lasers in chemistry. Publication of the papers presented at the Waters Symposia is a high priority of the SACP. The papers of the first symposium
were published in LC.GC Magazine and those of the next four symposia appeared in Analytical Chemistry. The next three Waters Symposia were published in this Journal: the sixth, on high-performance liquid chromatography, appeared in the January 1997 issue (pages 37–48); the seventh, on ion selective electrodes, appeared in the February 1997 issue (pages 159–182); the eighth, on lasers in chemistry, was featured in the May 1998 issue (pages 555–570). The topic of the ninth Waters Symposium, held in March 1998, was immunoassay, and is featured in this issue of the Journal. In the first paper, Nobel Laureate Rosalyn Yalow describes the insights that allowed her and Solomon Berson to invent the radioimmunoassay (RIA) method for insulin. In a complementary paper, Roger Ekins critically reviews his own work along parallel lines, and the work of others, that led to a wide variety of ligand-binding assay techniques, including the most recent ultrasensitive microarrays on a chip. The development of a variety of homogeneous immunoassays is described by Edwin Ullman in the third paper, followed by a discussion by Eugene Straus of an important application of RIA, namely, the assay of gastrointestinal hormones. Finally, Anders Weber points out the factors to be considered in technology transfer from research to the rapidly growing diagnostics industry. J. F. Coetzee University of Pittsburgh Waters Symposium Coordinator
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Waters Symposium: Immunoassay
Whereas Type 1 diabetes clearly results from a loss of the pancreas’s ability to make and secrete insulin, persons with Type 2, or adult-onset, diabetes have ample pancreatic insulin and the hormone is released into the bloodstream in response to the usual provocations, such as the ingestion of sugar. And so there was a burning question: Why do Type 2 diabetics have high blood sugar levels—why are they diabetic? The Development of Radioimmunoassay In 1952, I. Arthur Mirsky published his lectures from the Laurentian Hormone Conference, entitled The Etiology of Diabetes Mellitus in Man. Mirsky hypothesized that Type 2 diabetes might not be due to a deficiency of insulin secretion, but rather to abnormally rapid degradation of insulin by insulin-metabolizing enzyme in the liver. If this idea was correct, the rate of insulin disappearance from the bloodstream would be faster in Type 2 diabetics than in Type 1 diabetics and normal subjects. Highly purified insulin was available from the Eli Lilly Company, and radioiodinated insulin could be produced in the laboratory. We had been iodinating albumin and thyroxin and studying their metabolism in human subjects. We then knew that by testing the Mirsky hypothesis, we would be probing the center of one of the most important questions in endocrinology and medicine. We injected radioactive insulin (insulin labeled with 131I) into control subjects who had never been treated with insulin or had received insulin injections for less than one month, and into study subjects who had received insulin injections for many months or years. The control subjects included normal healthy volunteers and hospitalized patients, both diabetic (many Type 2 diabetics are not treated with insulin) and nondiabetic. What we found surprised us. The rate of disappearance of insulin from plasma was not dependent on whether the subject was diabetic, but on whether the subject had been treated with insulin for more than a few weeks. There was rapid disappearance of insulin in diabetics and nondiabetics who had not been treated with insulin. Insulin disappeared more slowly from the plasma of patients who had been given insulin, either for the treatment of diabetes or as shock therapy for schizophrenia. The Mirsky hypothesis was wrong. But we were intrigued with the question of why insulin disappearance was slower in the plasma of insulin-treated subjects. This led us to the discovery of insulin-binding antibodies, a very controversial conclusion at that time. In fact, although we had satisfied all criteria for demonstrating that our radiolabeled insulin was binding to antibody protein, the editors of The Journal of Clinical Investigation insisted that the term “insulin-binding antibody” not be used in the title of the February 1956 paper, and it was eventually entitled “Insulin-I131 Metabolism in Human Subjects: Demonstration of Insulin Binding Globulin in the Circulation of Insulin-treated Subjects” (1). The argument over “globulin” vs “antibody” was not simply a matter of semantics. We knew that we were introducing new methods for studying soluble antigen–antibody reactions, that we had extended the operational concept of antigenicity to a
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vast array of small molecules, and that the energy requirements for antigen–antibody reactions had to be reconsidered in the light of our findings. The 1956 JCI “insulin-binding globulin” paper was 20 pages long, there were 16 figures and two tables, and within this mass of data were the observations that provided the basis for the radioimmunoassay of insulin—the finding that the binding of radiolabeled insulin to a fixed concentration of antibody is a quantitative function of the amount of insulin present. In other words, incubation of insulin-binding antibody with trace amounts of radiolabeled insulin resulted in the binding of all of the labeled insulin. But addition of unlabeled insulin prevented the binding of labeled insulin in direct proportion to the number of unlabeled insulin molecules added. Our 1959 paper entitled “Assay of Plasma Insulin in Human Subjects by Immunological Methods” (2) was published in Nature and described the details of the method of insulin RIA in human blood. In 1960, we published “Immunoassay of Endogenous Plasma Insulin in Man” (3) in The Journal of Clinical Investigation, and here, for the first time, we described the pattern and quality of insulin released in response to the ingestion of sugar. This, too, was a long paper with much important data, but the most striking was the finding that Type 2 diabetics, while having lost the first rapid upstroke of insulin release in response to rising blood sugar, nevertheless release more insulin and have higher plasma insulin concentrations during the hours after ingestion of glucose. This observation, that in Type 2 diabetics high plasma glucose is accompanied by high plasma insulin, led to the realization that Type 2 diabetics are resistant to the action of insulin. Proliferation of Radioimmunoassay Technology As I have described, RIA was first applied to the measurement of insulin. We soon developed RIAs for other peptide hormones, including human growth hormone, ACTH, and parathyroid hormone. Other workers were busy developing assays for glucagon, thyroidal hormones, vitamins, steroids, prostaglandins, cyclic nucleotides, enzymes, biologic amines, and other substances. We extended our work to construct the first RIA for a virus with the assay for hepatitis B viral antigen. But the proliferation of general methodology for competitive binding assays went far beyond the immunoassay, to involve the use of specific binding substances such as intrinsic factors for vitamin B12, and membrane and other receptor molecules, and, of course, ligands were labeled with nonradioactive materials. As I look back now, I am thrilled by the bewildering scope of the work of so many talented people that came in the wake of our efforts in a small laboratory in a Veterans Administration Hospital. Literature Cited 1. Berson, S. A.; Yalow, R. S.; Bauman, A.; Rothschild, M. A.; Newerly, K. J. Clin.. Invest. 1956, 35, 170. 2. Yalow, R. S.; Berson, S. A. Nature 1959, 184, 1648. 3. Yalow, R. S.; Berson, S. A. J. Clin. Invest. 1960, 39, 1157.
Journal of Chemical Education • Vol. 76 No. 6 June 1999 • JChemEd.chem.wisc.edu
