Radioimmunoassay and Related Methods Research in endocrinology owes much to the development of biological assays for many hormones in the first half of this century. Despite numer ous improvements, most of these bioassays still have deficiencies. They are usually too insensitive to permit direct, accurate detection of hormone levels in blood or other physiologic fluids. They require large groups of animals, with factors such as age, sex, weight, strain, and diet being carefully controlled. Frequently, be cause of inter-assay and intra-assay variations, even larger animal groups and statistical analyses are required to detect significant responses. Final ly, bioassays are generally not amena ble to the assay of large numbers of specimens. With the onset of subcellular frac tionation methods, numerous in vitro bioassays for hormones were devel oped which employed responses of certain metabolic pathways to these agents. However, they often had the disadvantage of nonspecific effects and the difficulty of equating the in vitro and in vivo responses. Although more sensitive than the animal bioas says, they were generally not useful for blood hormone determinations. For similar reasons instrumental methods such as gas chromatography, although capable of detecting steroid and thyroid hormones, have generally not been amenable to assaying large numbers of biological specimens and have not been applicable to protein hormones. In 1959 Rosalyn S. Yalow and the late Solomon A. Berson announced a radioimmunoassay procedure for the detection of insulin in human plasma. This pioneering work initiated the rise of a new era in endocrinology, an era which has yet to reach its zenith. One only has to glance at the endo crine journals, or papers presented at endocrine meetings, to immediately appreciate the impact of Berson and Yalow's accomplishment. Today, the
applications of the radioimmunoassay (RIA), or related techniques such as the competitive protein-binding (CPB) assay, radioenzymatic assay, radioreceptor assay, and immunoradiometric assay, are by no means re stricted to endocrinology. Any sub stance which is of sufficiently low concentration to make detection by biological, chemical, or instrumental means difficult, if not impossible, is a candidate for this type of assay. The materials being assayed by these pro cedures include protein and nonpro tein hormones, vitamins, nucleic acids, enzymes, drugs, metabolites, cancer antigens, viral antigens, anti bodies, and structural proteins. A computerized literature search re vealed over 3000 English language publications on this subject since 1964. This area has been the subject of numerous reviews, symposia, and workshops. The limited space of this report will be used to consider the basic principles and methodologies and to give examples of the numerous applications. Principle The RIA and related assays depend on the same principle. As shown in Figure 1, a compound to be measured (C) and the same compound tagged with a radioactive tracer (C*) com pete for binding to or reaction with a reactive agent. In the RIA this agent is an antibody and is depicted in Fig ure 1 as AB. In the following discus sion of the principle, only AB will be used, although it is interchangeable with the reactive agents of the other related assays. The competitive principle is quantitated by determining the amount of radioactivity (C*) in two pools: that which has bound to AB and is called Bound (B), and that which has not bound and is called Free (F). Increas ing concentrations of C result in con comitant decreases in Β and increases
878 A · ANALYTICAL CHEMISTRY, VOL. 45, NO. 1 1 , SEPTEMBER 1973
Charles D. Hawker Laboratory Procedures® Division of The Upjohn Co. Kalamazoo, Mich. 49001
in F. Therefore, the B/F ratio (alter nately, the percent of Β in the Β + F total) decreases as the concentration of C increases. Figure 2 shows the RIA principle more schematically. Test tubes are depicted with constant amounts of C* and AB, and the arrows indicate the incubation and separation of Β and F pools: the tubes on the left show the initial assay components, and the corresponding tubes on the right show the separated Β and F pools after reaction. In the top example, no stan dard or test compound (C) is added; all of the C* reacts and is found in the Β pool. In the other two cases, addition of different amounts of C re sults in binding of C and C* to AB in proportion to the C/C* ratio. Since the C/C* ratio is the same in both the Β and F pools, the C*-AB/C* ratio (or B/F) is dependent on the amount of C present. Figure 3 shows a typical RIA stan dard curve which displays this doseresponse relationship. The percent bound is plotted as a function of the quantity of the hormone testosterone (C) per assay tube. In this type of plot, the Β pool for a tube with no C is set equal to 100%. As increasing amounts of C are added, the % bound decreases. A similar curve is obtained with the B/F ratio or the B/(B + F) ratio instead of % bound on the ordi nate. This curve displays the doseresponse relationship suggested in Figure 2. By comparison of the % bound for an unknown specimen with the curve, the concentration of C in
Report
C*
+
(FREE)
AB
«=*
+
C*-AB (BOUND)
c f
C-AB Figure 1. Schematic diagram of radioimmunoassay principle
the unknown can be determined. However, it is not always easy to fit the curve to the experimental data, and reading the lower concentrations can be troublesome because they are closer together. An alternate method of plotting the standard curve which avoids these difficulties has been described. The logit of the % bound is plotted on the ordinate, and the log concentration is plotted on the abscissa. The logit function is described by the equation: logit % Β = In
——— (100 - % B) Figure 4 is a plot of logit % Β vs. log C for the same data used in Figure 3. In this example and in most cases, the standard curve obtained with this method is linear. With a standard line, it is easier to fit the line to the experimental data and to read un known concentrations with accuracy. Further, both of these steps can be computerized for automation of the RIA procedure, and numerous statis tical analyses can be incorporated to derive confidence limits or other sta tistics for both the standard line and the unknown results. Graph paper which plots these functions directly from the % bound and standard con centration is commercially available (Codex Book Co., Norwood, Mass.). 6
Basic RIA Requirements
Pure Antigen. The antigen used for radioactive labeling and as the assay standard must be pure for the assay to be specific. When used for
immunizations, pure antigen may re sult in easier assay development, but it is not an absolute requirement, be cause the assay depends on competi tion between standard (or test) anti gen and labeled antigen, and any antibody binding sites directed against other moieties do not enter into the reaction. Antiserum Preparation. Many species have been used to produce antisera; rabbits have been used most frequently, because they generally best combine the small animal ad vantages of a minimal immunogen re quirement and inexpensive procure ment and care with the large animal advantage of providing ample quan tities of serum. It is important to distinguish be tween an antigen (a substance capa ble of binding to a specific antibody) and an immunogen (a substance ca pable of provoking an immune re sponse). Proteins of greater than 5000 mol wt can usually be both antigens and immunogens. However, smaller peptides and compounds such as drugs and nonprotein hormones, al though antigenic, must be coupled to a large protein carrier such as albu min to be immunogenic. When cou pled, the small compounds are called haptens, and the carrier-hapten com plexes are called conjugates. Usually, the immunogen is mixed with an adjuvant, such as Freund's complete adjuvant, which when in jected serves to both enhance and prolong the immune response. A si multaneous injection with pertussis
vaccine also aids the response by act ing as a nonspecific stimulator. Usu ally, for booster immunizations the pertussis vaccine is omitted, and in complete adjuvant is substituted for complete adjuvant. In rabbits, in tradermal injections in the groin re gion along the lymph system are an effective means of immunization. A total of 1 ml of immunogen suspend ed in adjuvant is injected in as many as 50 sites, each receiving 20 μΐ of the suspension. The initial immunization yields "primary response" antibodies which are usually less specific. In a typical protocol, booster immunizations are given at two-month intervals fol lowing the first immunization, and serum is collected every two weeks after the first booster. This serum will contain "secondary response" an tibodies which will generally be more specific and have a higher titer (di lution at which serum can be used). Antisera must be evaluated for titer, sensitivity, and specificity; this requires the use of labeled pure anti gen. The usual working titer is that dilution which binds 50% of the total labeled antigen; antisera with titers of less than 1:100 are generally not use ful. High titers do not necessarily cor relate with sensitivity. This is evalu ated by running standard lines with unlabeled pure antigen at known con centrations. Different antisera will give parallel lines similar to the one shown in Figure 4, and the line far thest to the left will indicate the most sensitive antiserum. Sensitivity can vary among bleedings from the same animal; each bleeding should be checked. Specificity is evaluated by examining the degree of cross-reactiv ity with other compounds similar to the one to be measured. Standard lines are run for each compound, and the concentration of the test com pound at 50% bound is divided by the concentration of the standard antigen at 50% bound. The result is the frac-
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1 1 , SEPTEMBER 1973 · 879 A
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Figure 3. Standard curve for radioimmunoassay of testosterone
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(eight days) results in less frequent iodinations. Nonprotein compounds assayed by RIA or CPB methods originally used 3 H-labeled antigens which were usually commercially available. More recently, the use of iodine isotopes has been extended to these nonprotein compounds, such as digoxin, cyclic AMP, and steroid hormones by iodinating tyrosine methyl ester (TME) derivatives of the compounds. The
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specific activities of the iodinated compounds are much greater, and they can be counted with greater efficiency. Further, gamma counting is less complicated, faster, and more economical than liquid scintillation counting, although 3 H does have advantages of being a less expensive isotope and not requiring laboratory licensing for its purchase at the levels needed for an RIA. Purification of the iodinated anti-
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gen from unreacted iodide and other fractions has been achieved in many ways. For protein compounds these include gel filtration, adsorption on powdered cellulose or microfine silica particles, starch gel electrophoresis, and ion-exchange chromatography. The iodinated T M E derivatives of nonprotein compounds have usually been purified by polyacrylamide disc gel electrophoresis. Incubation Procedure. As previously described, two factors that contribute to assay sensitivity are the character of the antiserum and the specific activity of the labeled antigen. Additional sensitivity can be achieved by dilution of the assay system—the more the antiserum and labeled antigen are diluted, the lower the concentration of test antigen that can be measured. However, dilution of the assay system also results in slower reactions (longer incubation times) and increased problems such as adsorption of the antigen to the test tubes, interference with the reaction by serum proteins, and damage to the labeled antigen. The assay system should be designed to achieve an optimal balance of these factors. Steroid hormones and certain other compounds which can be extracted from serum specimens can be assayed quickly because the interfering serum proteins will not be extracted. Often the extraction or purification steps (if required) can introduce a background or blank value in the assay. However, by running appropriate controls, this background can be subtracted from the assay results. Protein hormones are usually assayed on diluted serum, and the incubation times are much longer. Most RIA incubations are conducted at 4°C to take advantage of the greater free energy of reaction and prevent microbial growth. Another variation that gives increased sensitivity is nonequilibrium incubation in which the test antigen and antiserum are first incubated in the absence of labeled antigen. The test antigen thus has a competitive advantage over the labeled antigen (which is added later) in binding to the limited number of antibody binding sites. Separation of Antibody-Bound and Free-Labeled Antigen Fractions. The original RIA methods for peptide hormones employed chromatoelectrophoresis (CME) to separate the bound and free-labeled antigen pools. This method depends on the ability of the peptides (including the Free fraction) to stick to the paper at the origin, while the Bound fraction migrates. However, CME is limited in usefulness because it is slow and laborious. It has largely been replaced by a number of faster and simpler
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methods. The double antibody (second antibody) method is now widely used. If the assay antiserum was made in a rabbit, for example, the antibodybound fraction (gamma globulins) can be specifically precipitated by reaction with an antiserum made against rabbit gamma globulins in another species such as a goat. The precipitate can then be centrifuged and counted, yielding the antibodybound labeled antigen pool. The method is applicable to antisera from any species as long as a different species is used to make the second antiserum. The antibody-bound pool can also be nonspecifically precipitated by salts such as ammonium sulfate, sodium sulfate, or trichloroacetic acid and by solvents such as dioxane, ethyl alcohol, acetone, or polyethylene glycol. A solution of ammonium acetate and ethyl alcohol in water has also been used with success. The free-labeled antigen pool can be nonspecifically adsorbed by substances such as talc, microfine silica, florisil, kaolin, cellulose powder, and anion-exchange resins. One of the most widely used adsorption methods is activated charcoal (Norit) coated with dextran. The latter is a permeable sieve which permits the smaller antigen to penetrate and bind to the charcoal but excludes the larger antibody fraction. A technique which has had some success and will probably be used more in the future is the solid-phase antibody method, in which the antibody is coupled to an inert material but is free to react in the RIA incubation. As the incubation proceeds, the separation of bound and free-labeled antigen fractions occurs simultaneously. One approach employs antiserum-coated test tubes which are merely emptied and counted after the incubation. Other approaches use antibodies coated or bound to polypropylene disks, poly (tetrafluoroethylene-g-isothiocyanatostyrene) powder, agarose and dextran gels, bromacetyl cellulose, or bentonite particles. Although solid-phase methods generally use more antiserum, their economy and simplicity are bringing them into more frequent use. Validation. After the ingredients have been obtained and the method has been established, an RIA must be validated. Primarily, assay validation consists of a demonstration that the assay is measuring what it is supposed to be measuring. This can be accomplished in several ways. Multiple dilutions of the physiological specimens should give lines parallel to the standard line. Biologically active compounds should show correla-
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tion between the concentrations de termined by bioassay and by RIA. The results of assays of drugs or other compounds that can be measured by instrumental or chemical means should also correlate with the RIA re sults. For substances such as hor mones, enzymes, and antigens which are produced in excess in pathologic states, the assay results in normal subjects and patients should correlate clinically. Finally, if the concentra tion of hormone or antigen in the specimen can be influenced by physi ologic changes or agents, the assay should be able to detect these changes. Accuracy. To a certain extent, some of the suggested methods for validating the RIA will assess its ac curacy as well. The accuracy of an RIA can only be tested if there is an alternate accepted method to mea sure the same compound. For most peptide hormones, the only other method of determination has been bioassay which often is useful as a measure of relative activity but not for accurate determinations. In addi tion, some protein hormone RIA's have used standard hormones from animal species, although the assays were used for measurement of the hormones in man. In these or similar circumstances, lack of the human standard and lack of knowledge of the degree of cross-reactivity prohibit any assessment of accuracy. For many other RIA's (drugs, steroids), there are valid standards and reliable alter nate methods of determination, so that accuracy can be easily assessed. Precision. Precision is the degree to which a group of determinations on a sample agrees with the mean of the group. It is generally expressed as the percent coefficient of variation (CV) χ
or the index of precision (λ)
where σ is the standard deviation of the determinations; χ is the mean of the determinations; and b is the slope of the response line. For RIA's using the logit method previously dis cussed, σ is the standard deviation of the logit % B, and b is the slope of the regression line. Therefore, this precision estimate incorporates the % Β value at which the variation is de termined and the slope of the stan dard line in addition to the CV. Whether λ or C Vis used, generally most RIA's have had precision better than or equal to the corresponding bioassays. Quality Control. As with any ana lytical method, RIA's require
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standardized quality control proce dures to insure consistently accurate and precise results on unknown speci mens. In addition to such checks as antibody control tubes and standard antigen tubes, each run should con tain aliquots from normal and abnor mal serum pools or other types of specimens similar to the unknowns. If these pool results are outside a range defined by the mean ± two standard deviations, then the assay results may be incorrect and require checking or repeat determinations. In this way, the assay performance can be carefully monitored and controlled. RIA Applications The original RIA methods were ap plied to protein hormones. The list of protein hormones now assayed by RIA includes insulin, glucagon, growth hormone, luteinizing hor mone, follicle-stimulating hormone, prolactin, thyroid-stimulating hor mone, adrenocorticotropin, a and β melanocyte stimulating hormones, placental lactogen (chorionic sommatomammotropin), chorionic gonado tropin, vasopressin, oxytocin, bradykinin, gastrin, secretin, pancreozymincholecystokinin, calcitonin, parathy roid hormone, and thyroglobulin. RIA has helped toward identification of other forms or prohormones for such hormones as insulin, parathyroid hor mone, growth hormone, and the go nadotropins. RIA has been extended to steroid hormones such as aldosterone, Corti sol, deoxycorticosterone, androstenedione, testosterone, dihydrotestosterone, progesterone, 17-hydroxyprogesterone, estrone, estradiol, estriol, and 2-hydroxy-estrone and to other nonpeptide compounds such as prosta glandins, thyroid hormones, and cy clic nucleotides; drugs such as digoxin, digitoxin, medroxyprogesterone, methylprednisolone, morphine, lyser gic acid diethylamide, and barbitu rates; enzymes such as carbonic anhydrase, fructose-l,6-diphosphatase, carboxypeptidases, chymotrypsin, trypsin, and elastase; large pro teins such as albumins, globulins, carcinoembryonic antigen, alpha feto protein, hepatitis-associated antigen; other tumor antigens; structural pro teins; and many other substances too numerous to list here. It is sufficient to say that any compound which can be made radioactive and which is immunogenic, or can be made im munogenic by coupling to a carrier, can be measured by RIA, provided the substance can be obtained in pure form. Related Methods In the competitive protein-binding (CPB) assay, the reactive agents are
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n a t u r a l l y occurring p r o t e i n s , u s u a l l y n o n i m m u n e globulins, which h a v e specific affinity for t h e h o r m o n e or a class of h o r m o n e s . E x a m p l e s include m a n y steroid h o r m o n e s , t h y r o x i n e , v i t a m i n D3 a n d its m e t a b o l i t e s , a n d v i t a m i n B12. T h e r a d i o e n z y m a t i c assay employs a n e n z y m e as t h e reac tive agent, a n d t h e s e p a r a t i o n of two radioactive pools involves s e p a r a t i o n of two c o m p o u n d s , one of which was formed from t h e other by t h e enzym a t i c a l l y c a t a l y z e d reaction. An ex a m p l e is t h e folic acid a s s a y b y use of folic acid r e d u c t a s e . T h e radiorecep tor assay employs a p a r t i a l l y purified tissue receptor as t h e reactive agent. E x a m p l e s include a d r e n o c o r t i c o t r o p i n ( A C T H ) , cyclic A M P , a n d cyclic G M P . T h e i m m u n o r a d i o m e t r i c assay differs from t h e RIA a n d other r e l a t e d m e t h o d s . It uses a radioactively la beled purified a n t i b o d y . U n r e a c t e d a n d r e a c t e d labeled a n t i b o d i e s are sep a r a t e d by a d s o r p t i o n to a n t i g e n which is b o u n d t o a solid s u p p o r t . E x a m p l e s include insulin, growth h o r m o n e , calcitonin, a n d p a r a t h y r o i d h o r m o n e . Generally, t h e RIA h a s n u m e r o u s a d v a n t a g e s over related m e t h o d s . C o m p a r e d t o C P B , RIA h a s greater sensitivity a n d specificity, so t h a t t h e s p e c i m e n s do n o t require c h r o m a t o g r a p h y or o t h e r purification s t e p s . T h e r a d i o r e c e p t o r assay is r e s t r i c t e d t o c o m p o u n d s for which tissue receptors can b e identified a n d isolated, al t h o u g h t h e y d o h a v e one a d v a n t a g e of m e a s u r i n g "biologic a c t i v i t y " as op posed to " i m m u n o l o g i c a c t i v i t y . " However, if t h e RIA is properly vali d a t e d , t h e r e is n o essential difference. T h e r a d i o e n z y m a t i c assay m a y be able to m e a s u r e c o m p o u n d s for which a n t i b o d i e s c a n n o t be readily m a d e b u t h a s t h e d i s a d v a n t a g e of requiring s e p a r a t i o n of c o m p o u n d s of similar size a n d s t r u c t u r e for q u a n t i t a t i o n . T h e i m m u n o r a d i o m e t r i c assay does not h a v e some of t h e " b l a n k " prob lems of t h e RIA b u t is costly in its use of a n t i s e r u m a n d a n t i g e n a n d is more difficult to set u p initially.
Bibliography The first section includes a number of reviews, symposia, and workshops on ra dioimmunoassay and related methods. The additional sections include references on specific subjects, although most of the references in the General section also cover these topics. General 1. S. A. Berson, R. S. Yalow, S. M. Glick, and J. Roth, Immunoassay of Protein and Peptide Hormones, Metabolism, 13, 1135(1964). 2. S. A. Berson and R. S. Yalow, Immu noassay of Protein Hormones, in "The Hormones," G. Pincus, K. V. Thimann, and E. B. Astwood, Eds., Vol 4, pp 557-630, Academic Press, New York, N.Y., 1964. 3. J. T. Potts, Jr., L. M. Sherwood, J. L.
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H. O'Riordan, and G. D. Aurbach, Ra dioimmunoassay of Polypeptide Hor mones, Advan. Int. Med., 13, 183 (1967). 4. S. A. Berson and R. S. Yalow, General Principles of Radioimmunoassay, Clin. Chim.Acta, 22,51 (1968). 5. E. Diczfalusy, Ed., "Immunoassay of Gonadotrophins," Acta Endocrinol., 63, Supplementum 142 (1969). 6. M. Margoulies, Ed., "Protein and Poly peptide Hormones," Part 1, Excerpta Med. Int. Congr. Series No. 161, Am sterdam, the Netherlands, 1969. 7. Β. Ε. P. Murphy, Protein Binding and the Assay of Non-Antigenic Hormones, Rec. Progr. Harm. Res., 25, 563 (1969). 8. E. Diczfalusy, Ed., "Steroid Assay by Protein Binding," Acta Endocrinol., 64, Supplementum 147 (1970). 9. G. H. Grant and W. R. Butt, Immuno chemical Methods in Clinical Chemistrv, Advan. Clin. Chem., 13,383(1970). 10. J. W. McArthur and T. Colton, Eds., "Statistics in Endocrinology," MIT Press, Cambridge, Mass., 1970. 11. F. G. Peron and B. V. Caldwell, Eds., "Immunologic Methods in Steroid De termination," Appleton-Century-Crofts, New York, N.Y., 1970. 12. W. D. Odell and W. H. Daughaday, Eds., "Principles of Competitive Pro tein-Binding Assays," Lippincott, Phila delphia, Pa., 1971. 13. K. E. Kirkham and W. M. Hunter, Eds., "Radioimmunoassay Methods," Churchill Livingstone, Edinburgh, Scot land, 1971. 14. A. R. Midgley, Jr., G. D. Niswender, V. L. Gay, and L. E. Reichert, Jr., Use of Antibodies for Characterization of Gonadotropins and Steroids, Rec. Progr. Horm. Res., 27.235(1971). 15. D. S. Skelley, L. P. Brown, and P. K. Besch, Radioimmunoassay, Clin. Chem.. 19,146(1973). Principle and Treatment of D a t a 16. G. Scatchard, The Attraction of Pro teins for Small Molecules and Ions, Ann. N.Y. Acad. Sci., 51, 660 (1949). 17. D. Rodbard, P. L. Rayford, J. A. Coo per, and G. T. Ross, Statistical Quality Control of Radioimmunoassays, J. Clin. Endocrinol. Metab., 28, 1412 (1968). 18. R. Ekins and B. Newman, Theoretical Aspects of Saturation Analysis, Acta Endocrinol., 64, Supplementum 147, 11 (1970). 19. R. S. Yalow and S. A. Berson, Ra dioimmunoassays, in "Statistics in En docrinology," J. W. McArthur and T. Colton, Eds., pp 327-44, MIT Press, Cambridge, Mass., 1970. 20. W. G. Duddleson, A. R. Midgley, Jr., and G. D. Niswender, Computer Pro gram Sequence for Analysis and Sum mary of Radioimmunoassay Data, Cornput. Biomed. Res., 5, 205 (1972). Antiserum Preparation and Evaluation 21. J. Freund, The Effect of Paraffin Oil and Mycobacteria on Antibody Forma tion and Sensitization, Amer. J. Clin. Path., 21,645(1951). 22. G. D. Niswender and A. R. Midgley, Jr., Hapten Radioimmunoassay for Ste roid Hormones, in "Immunologic Meth ods in Steroid Determination," F. G. Peron and B. V. Caldwell, Eds., pp 149-73, Appleton-Century-Crofts, New York, N.Y., 1970. 23. B. A. L. H u m and J. Landon, Antisera for Radioimmunoassay, in "Radioimmu noassay Methods," Κ. Ε. Kirkham and W. M. Hunter, Eds., pp 121-42, Chur chill Livingstone, Edinburgh, Scotland, 1971. 24. W. D. Odell, G. A. Abraham, W. R. Skowsky, M. A. Hescox, and D. A. Fish-
er, Production of Antisera for Radioim munoassays, in "Principles of Competi tive Protein-Binding Assays," W. D. Odell and W. H. Daughaday, Eds., pp 57-88, Lippincott, Philadelphia, Pa. 1971. 25. J. Vaitukaitis, J. B. Robbins, E. Nieschlag, and G. T. Ross, A Method for Producing Specific Antisera with Small Doses of Immunogen, J. Clin. Endocrinol. Metab., 33, 988 (1971). 26. G. E. Abraham and P. K. Grover, Covalent Linkage of Hormonal Haptens to Protein Carriers for Use in Radioimmu noassay, in "Principles of Competitive Protein-Binding Assays," W. D. Odell and W. H. Daughaday, Eds., pp 134-57, Lippincott. Philadelphia, Pa., 1971.
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Radiolabeled Antigen 27. R. S. Yalow and S. A. Berson, Label ing of Proteins—Problems and Prac tices, Trans. Ν. Υ. Acad. Sci., 28, 1033 (1966). 28. A. E. Freedlender, Practical and The oretical Advantages for the Use of I 125 in Radioimmunoassay, in "Protein and Polypeptide Hormones," M. Margoulies, Ed., pp 351-3, Excerpta Med. Int. Congr. Series No. 161, Amsterdam, the Netherlands, 1968. 29. G. C. Oliver, Jr., B. M. Parker, D. L. Brasfield, and C. W. Parker, The Mea surement of Digitoxin in Human Serum by Radioimmunoassay. J. Clin. Invest., 47,1035(1968). 30. A. L. Steiner, D. M. Kipnis, R. Utiger, and C. Parker, Radioimmunoassay for the Measurement of Adenosine-3'5'Cyclic Phosphate, Proc. Nat. Acad. Sci., U.S., 64,367(1969). 31. W. T. Newton, J. E. McGuigan, and Β. Μ. Jaffe, Radioimmunoassay of Pep tides Lacking Tyrosine, J. Lab. Clin. Med., 75,886(1970). 32. W. M. Hunter, The Preparation and Assessment of lodinated Antigens, in "Radioimmunoassay Methods," Κ. Ε. Kirkham and W. M. Hunter, Eds., pp 3-23, Churchill Livingstone, Edinburgh, Scotland, 1971. Incubation, Separation of Bound and Free-Labeled Antigen 33. C. N. Hales and P. J. Randle, Immu noassay of Insulin with Insulin Antibody Precipitate, Biochem. J., 88, 137 (1963)" 34. V. Herbert, K. S. Lau, C. W. Gottlieb, and S. J. Bleicher, Coated Charcoal Im munoassay of Insulin, J. Clin. Endocri nol. Metab., 25, 1375 (1965). 35. A. M. Eisentraut, N. Whissen, and R. H. linger, Incubation Damage in the Radioimmunoassay for Human Plasma Glucagon and Its Prevention with Trasylol, Amer. J. Med. Sci., 255, 137 (1968). 36. K. J. Catt and G. W. Tregear, Solid Phase Radioimmunoassay, in "Protein and Polypeptide Hormones," M. Mar goulies, Ed., pp 45 8, Excerpta Med. Int. Congr. Series No. 161, Amsterdam, the Netherlands, 1969. 37. D. Rodbard, H. J. Ruder, .]. Vaitukai tis, and H. S. Jacobs, Mathematical Analysis of Kinetics of Radioligand As says: Improved Sensitivity Obtained by Delayed Addition of Labeled Ligand, J. Clin. Endocrinol. Metab., 33, 343 (1971). 38. W. H. Daughaday and L. S. Jacobs, Methods of Separating Antibody-Bound from Free Antigen, in "Principles of Competitive Protein-Binding Assays," W. D. Odell and W. H. Daughaday, Eds., pp 303-24, Lippincott, Philadel phia, Pa., 1971. Methods Related to Radioimmunoassay 39. B. P. Murphy and C. J. Pattee, Deter
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mination of Thyroxine Utilizing the Propertv of Protein-Binding, J. Clin. Endocrinol. Metab., 24, 187 (1964). 40. Β. Ε. P. Murphy, Some Studies of the Protein-Binding of Steroids and Their Application to the Routine Micro and Ultramicro Measurement of Various Steroids in Body Fluids by Competitive Protein-Binding Radioassay.e/. Clin. Endocrinol. Metab., 27, 973 (1967). 41. S. P. Rothenburg, A Radioenzymatic Assay for Folic Acid, Nature, 206, 1154 (1965). 42. R. J. Lefkowitz, J. Roth, and I. Pastan, Radioreceptor Assay of Adrenocort icotropic Hormone: New Approach to Assay of Polypeptide Hormones in Plas ma, Science, 170,633(1970). 43. L. E. M. Miles and C. N. Hales, The Use of Labeled Antibodies in the Assay of Polypeptide Hormones, J. Nucl. Biol. Med., 13, 10(1969).
C h a r l e s D . H a w k e r is chief clinical chemist and director of research a n d development for Laboratory Proce dures®, a division of T h e Upjohn Co., K a l a m a z o o , Mich. Laboratory Proce dures is a nationwide clinical labora tory network. Dr. Hawker earned a BA degree in chemistry from Illinois Wesleyan University (Bloomington) in 1962, an M S degree in biochemis try from t h e University of Wisconsin (Madison) in 1965, a n d a P h D in bio chemistry from t h e University of Pennsylvania (Philadelphia) in 1967. H e was the recipient of an N I H post doctoral fellowship from 1967-69, and from 1967-71 engaged in research on the r a d i o i m m u n o a s s a y of p a r a t h y r o i d hormone at the University of P e n n sylvania. He has been with L a b o r a t o ry Procedures since 1971 and is di recting the d e v e l o p m e n t of radioim munoassays for m a n y c o m p o u n d s in cluding protein hormones, steroid hormones, a n d drugs. Dr. Hawker is a m e m b e r of P h i L a m b d a Upsilon, the American Chemical Society, the American Association for t h e Ad v a n c e m e n t of Science, the American Federation for Clinical Research, a n d the Endocrine Society.
