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14. Yalow, R. S., and Berson, S. A. Assay of plasma insulin in human subjects by immunological methods. Nature (Lond.) 1959, 184, 1648.

15. Yalow, R. S., and Berson, S. A. Plasma insulin concentrations in non-diabetic and early diabetic subjects determined by a new sensitive immunoassay technique. Diabetes. In press.

16. Bauman, A., Rothschild, M. A., Yalow, R. S., and

Berson, S. A. Distribution and metabolism of Im labeled human serum albumin in congestive heart failure with and without proteinuria. J. clin. Invest. 1955, 34, 1359.

17. Pressman, D., and Eisen, H. N. The zone of locali zation of antibodies. V. An attempt to saturate antibody-binding sites in mouse kidney. J. Immunol. 1950, 64, 273.

18. Yalow, R. S., and Berson, S. A. Effect of x-rays on trace-labeled Im-insulin and its relevance to biologic studies with Im-labeled proteins. Radiology 1956, 66, 106.

19. Berson, S. A., and Yalow, R. S. Radiochemical and radiobiological alterations of Im-labeled proteins in solution. Ann. N. Y. Acad. Sci. 1957, 70, 56.

20. Ferrebee, J. W., Johnson, B. B., Mithoefer, J. C., and

Gardella, J. W. Insulin and adrenocorticotropin labeled with radio-iodine. Endocrinology 1951, 48, 277.

21. Newerly, K., and Berson, S. A. Lack of specificity of insulin-Im binding by isolated rat diaphragm. Proc. Soc. exp. Biol. (N. Y.) 1957, 94, 751.

22. Berson, S. A., Yalow, R. S., Bauman, A., Roth schild, M. A., and Newerly, K. Insulin-Im metabolism in human subjects: Demonstration of insulin binding globulin in the circulation of insulin-treated subjects. J. clin. Invest 1956, 35, 170.

23. Berson, S. A., and Yalow, R. S. Quantitative as pects of the reaction between insulin and insulin-binding antibody: Relation to problem of insulin resistance. J. clin. Invest 1959, 38, 1996.

24. Field, J. B., Tietze, F., and Stetten, D., Jr. Further characterization of an insulin antagonist in the serum of patients in diabetic acidosis. J. clin. Invest 1957, 36, 1588.

25. Fisher, A. M. Personal communication.

26. Fajans, S. S., and Conn, J. W. The early recogni tion of diabetes mellitus. Ann. N. Y. Acad. Sci. 1959, 82, 208.

27. Somogyi, M. Determination of blood sugar. J.

28. Madison, L. L., Combes, B., Unger, R. H., and Kap lan, N. The relationship between the mechanism of action of the sulfonylureas and the secretion of insulin into the portal circulation. Ann. N. Y. Acad. Sci. 1959, 74, 548.

29. Mortimore, G. E., and Tietze, F. Studies on the mechanism of capture and degradation of insulin-Im by the cyclically perfused rat liver. Ann. N. Y. Acad. Sci. 1959, 82, 329.

30. Grodsky, G., and Forsham, P. An immunochemical assay of total extractable insulin in man. J. clin. Invest 1960, 39, 000.

31. Arquilla, E. R., and Stavitsky, A. B. The produc tion and identification of antibodies to insulin and their use in assaying insulin. J. clin. Invest 1956, 35, 458.

32. Loveless, M. H. A means of estimating circulating insulin in man. Quart. Rev. Allergy 1956, 10, 374.

33. Vallance-Owen, J., and Hurlock, B. Estimation of plasma-insulin by the rat diaphragm method. Lancet 1954, 1, 68.

34. Willebrands, A. F., v. d. Geld, H., and Groen, J.

Determination of serum insulin using the isolated rat diaphragm. The effect of serum dilution. Diabetes 1958, 7, 119.

35. Randle, P. J. Assay of plasma insulin activity by the rat-diaphragm method. Brit. med. J. 1954, 1, 1237.

36. Pfeiffer, E. F., Pfeiffer, M., Ditschuneit, H., and

Ahn, C. Clinical and experimental studies of insulin secretion following tolbutamide and meta-hexamide administration. Ann. N. Y. Acad. Sci. 1959, 82, 479.

37. Yalow, R. S., Black, H., Viliazon, M., and Berson,

S. A. Comparison of plasma insulin levels following administration of tolbutamide and glucose. Diabetes. In press.

38. Anderson, E., Lindner, E., and Sutton, V. A sensi tive method for the assay of insulin in blood. Amer. J. Physiol. 1947, 149, 350.

39. Baird, C. W., and Bornstein, J. Assay of insulin like activity in the plasma of normal and diabetic human subjects. J. Endocr. 1959, 19, 74.

40. Goldner, M. G., and Clark, D. E. The insulin re quirement of man after total pancreatectomy. J. clin. Endocr. 1944, 4, 194.

41. Seltzer, H. S., and Smith, W. L. Plasma insulin activity after glucose. An index of insulogenic reserve in normal and diabetic man. Diabetes 1959, 8, 417.

42. Mirsky, I. A. The etiology of diabetes mellitus in man. Recent Progr. Hormone Res. 1952, 7, 437.

43. Cochrane, W. A., Payne, W. W., Simpkiss, M. J., and Woolf, L. I. Familial hypoglycemia precipitated by amino acids. J. clin. Invest 1956, 35, 411.

This page is intentionally left blank page 27 2. Dandliker, W. B. and Feigen, G. A. (1961)

COMMENTARY TO Quantification of the Antigen-Antibody Reaction by the Polarization of

Fluorescence. Biochemical and Biophysical Research Communications 5(4): 299-304.

Fluorescence polarization (FP) was discovered in the 1920's; was used to monitor an antigen antibody reaction for the first time in 1961; commercialized in an automated immunoassay analyzer in 1981 and within 20 years was installed in over 70 000 clinical analyzers worldwide.

FP is based on the observation that low molecular weight fluorescent molecules like rhodamine and fluorescein freely tumble and rotate in solution. When excited by plane-polarized light they emit fluorescent light with decreased polarization due to the free movement of the fluorophore during excitation. If a small molecule like fluorescein binds to a large molecule like a protein its free rotational movement is slowed down. When excited in this bound state its emitted fluorescent light remains polarized in the same plane as the excitation light. The degree of polarization of the emitted light is directly related to the amount of fluorophore that has bound. Laurence in 1952 used FP to study the binding of various fluorescent dyes to bovine serum albumin (1). Steiner measured the binding of a soybean inhibitor-fluorescein conjugate to the enzyme trypsin in 1954 (2).

Dandliker and Feigen were the first to use FP to measure antigen antibody binding. Their paper was presented at the 45th Annual Meeting of the Federation of American Societies for Experimental Biology and published as an abstract in 1961 (3). A full paper was published that same year and is presented here. The binding of fluorescein conjugated ovalbumin to rabbit anti-ovalbumin antibody was monitored. This represents the first description of a true homogeneous assay in which the antigen-antibody event is measured directly in real time. Haber and Bennett extended these observations to include the binding of insulin, ribonuclease and bovine serum albumin to their respective antibodies (4). Over the next twelve years in numerous papers Dandliker expanded on the theoretical basis of FP, coined the term fluorescence polarization immunoassay (FPIA) and extended the applications of the technology (5-7). In 1973 Spencer et al. (8) described the construction of an FPIA analyzer and applied it to assays for antitrypsin enzyme-inhibitor and insulin-insulin antibody. Researchers at Abbott Laboratories under Michael Jolley and David Kelso described the development of a fully automated FPIA analyzer with applications for the therapeutic drug monitoring of aminoglycoside antibiotics and the anticonvulstants, phenytoin and phenobarbital (9-11). The Abbott TDx™ FPIA analyzer was introduced in 1981. It went on to become one of the most successful immunoassay analyzers in clinical chemistry.

References

(1) Laurence, D.J.R. (1952) A study of the adsorption of dyes on bovine serum albumin by the method of polarization of fluorescence. Biochemical Journal. 51(2):168-177.

(2) Steiner, R.F. (1954) Reversible association processes of globular proteins VI. The combination of trypsin with soybean inhibitor. Archives of Biochemistry and Biophysics. 49(1):71-92.

(3) Dandliker, W.B. and Felgen, G.A. (1961) Detection of the antigen-antibody reaction by fluorescence polarization. Federation Proceedings. 20(1 Part 1):11, Abstracts.

(4) Haber, E. and Bennett, J.C. (1962) Polarization of fluorescence as a measure of antigen-antibody interaction. Proceedings of the National Academy of Sciences. 48(11):1935-1942.

(5) Dandliker, W.B., Schapiro, H.C., Meduski, J.W., Alonso, R., Feigen, G.A., and Hamrick, J.R. Jr. (1964) Application of fluorescence polarization to the antigen-antibody reaction. Theory and experimental method. Immunochemistry. 1(3): 165-191.

(6) Dandliker, W.B. and de Saussure, V.A. (1970) Review article: fluorescence polarization in immunochemistry. Immuno-chemistry. 7(9):799-828.

(7) Dandliker, W.B., Kelly, R.J., Dandliker, J., Farquhar, J., and Levin, J. (1973) Fluorescence polarization immunoassay. Theory and experimental method. Immunochemistry. 10(4):219-227.

(8) Spencer, R.D., Toledo, F.B., Williams, B.T., and Yoss, N.L. (1973) Design, construction, and two applications for an automated flow-cell polarization fluorometer with digital read out: enzyme-inhibitor (antitrypsin) assay and antigen-antibody (insulin-insulin antiserum) assay. Clinical Chemistry. 19(8):838-844.

(9) Jolley, M.E., Stroup, S.D., Wang, C-H.J., Panas, H.N., Keegan, C.L., Schmidt, R.L., and Schwenzer, K.S. (1981) Fluorescence polarization immunoassay I. Monitoring aminoglycoside antibiotics in serum and plasma. Clinical Chemistry. 27(7):1190-1197.

(10) Popelka, S.R., Miller, D.M., Holen, J.T., and Kelso, D.M. (1981) Fluorescence polarization immunoassay II. Analyzer for rapid, precise measurement of fluorescence polarization with use of disposable cuvettes. Clinical Chemistry. 27(7): 1198-1201.

(11) Jolley, M.E., Stroupe, S.D., Schwenzer, K.S., Wang, C.J., Lu-Steffes, M., Hill, H.D., Popelka, S.R., Holen, J.T., and Kelso, D.M. (1981) Fluorescence polarization immunoassay. III. An automated system for therapeutic drug determination. Clinical Chemistry. 27(9):1575-1579.

Biochemical and Biophysical Research Communications. 1961, 5(4): 299-304, Copyright 1961 Reprinted with permission from Elsevier.

Vol. 5, No. 4,1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

QUANTIFICATION OF THE ANTIGEN-ANTIBODY REACTION BY THE POLARIZATION OF FLUORESCENCE*

Department of Biochemistry, University of Miami, Coral Gables, Florida and Department of Physiology, Stanford University, Stanford, California.

Received June 13, 1961

The basic theory concerning the polarisation of fluorescence was developed in a series of important papers by Perrin (1926), Perrin's results have been used experimentally by Singleterry and co-workers (1951) and greatly extended, both theoretically and experimentally, by Weber (1952). Subsequent applications by Laurence (1952) and Steiner (1957) follow implicitly from the work of Weber.

The concept underlying previous work by Dandliker and Feigen (1961) and the present results is to utilize the change in rotary diffusion constant which occurs when an antigen and antibody combine in solution. An essential feature of the method is that either the antigen or antibody is made fluorescent depending upon which component is to be detected. This feature makes it possible to follow the reaction in spectral regions where adventitious fluorescence is of minor importance and also permits a choice of flu* orescence lifetime, appropriate to the range of molecular size involved; it is thus distinct from other fluorescence techniques in use, cf. Coons, et aL (1941), Boroff and Fitzgerald (1958) and Velick, et al. (I960). EXPERIMENTAL, Crystalline ovalbumin was labeled with fluorescein using fluorescein isothiocyanate (Riggs, et al. 1958); the product contained between

♦Supported by Grant A-2984 and Grant H-3693(C2), U.S. Public Health Service ♦♦Howard Hughes Medical Institute.

Vol. 5, No. 4,1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

one and two fluorescein molecules per molecule of protein assuming the molar extinction coefficient of free and bound fluorescein to be the same. Eight albino rabbits, weighing about six pounds each, were immunized to fluorescein-labeled ovalbumin (F-ovalbumin) by a series of twenty intravenous injections of 10 mg, each administered on alternate days. One week after the last injection, blood was drawn by cardiac puncture and, after clotting, the serum was separated by centrifugation. A^-globulin fraction from pooled serum was prepared by two successive precipitations in one-third saturated ammonium sulfate. For control purposes, -globulin was also prepared from normal animals. The immune globulin preparation contained 19% specifically precipitable anti-F-oval-bumin as estimated at optimal proportions by means of the quantitative precipitin method; this antibody also reacted strongly with native ovalbumin.

Measured volumes of antigen solution were added from a microburette to a constant quantity of antibody contained in a cuvette. The intensity and polarization of fluorescence were measured in a modified Brice-Phoenix apparatus using the unpolarized 4358 X mercury line for excitation.

RESULTS AND DISCUSSION. The reaction between F-ovalbumin and its antibody produces two fluorescence effects. First, there is a pronounced diminution of the fluorescence due, no doubt, to the close juxtaposition with many atomic groupings of the antibody, thus establishing favorable conditions for loss of the electronic excitation energy before fluorescence takes place; possibly a transition to the triplet state is involved. The second effect is the change in polarization of fluorescence caused by the increase in relaxation

For quantitative puposes, it is convenient to define the polarization (p) and a parameter (Q) which is proportional to fluorescence intensity divided by incident intensity. If the vertical and horizontal components in

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