- Email: [email protected]
Radioimmunoassay
We would like to thank Dr. Shinji Morioka for valuable help in performing skin explant techniques. This work was supported by National Institutes of Health Grants AM21848, AMI9067, and CA41236-11A1. References 1. Anhait, G. J. et al. (1982). Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease. N. Engl. J. Med. 306:1189. 2. Becker, D. et al. (1981). Induction of plasminogen activator synthesis by antibodies. J. Exp. Med. 154:285. 3. Beutner, E. II. and R. E. Jordan. (1964). Demonstration of skin antibodies in sera of pemphigus vulgaris patients by indirect immunofluorescent staining. Proc. Soc. Exp. Biol. Med. 117:505. 4. Buschard, K. et al. (1981). A model for the study of autoimmune diseases applied to pemphigus: Transplants of human oral mucosa to athymic nude mice binds pemphigu s antibodies h~ vivo. J. Invest. Dermatol. 76:171. 5. Christman, J. L. et al. (1977). Plasminogen activators. In: A. J. Barrett (ed.), Proteinases in Mammalian Cells and Tissues. Elsevier, Amsterdam, p. 91.
Radioimmunoassay Rosalyn S. Yalow, Ph.D. Solomon A. Berson Research Laboratory Veterans Administration Medical Center Bronx, New York Radioimmunoassay (RIA) methodology was first applied a quarter-century ago to the measurement of insulin exogenously administered to rabbits (1). However, it was not until several years later that the assay had sufficient sensitivity to permit the determination of fasting and stimulated plasma insulin levels in man (10, 1 I). The principle is simple (Fig. 1). The concentration o f the substance in the unknown sample is made by comparing its inhibition of binding of radiolabeled antigen to antibody with the inhibition of that of known standards. A
© 1984 by Elsevier Science Publishing Co., Inc.
6. Farb, R. et al. (1978). Anti-epithelial cell-surface pemphigus antibody detaches viable epidermal cells from culture plates by activation of proteinase. Proc. Natl. Acad. Sci., USA, 75:459. 7. Hashimoto, K. et al. (1983). Anticell surface pemphigus autoantibody stimulates plasminogen activator activity of human epidermal cells: A mechanism for the loss of epidermal cohesion and blister formation. J. Exp. Med. 157:259. 8. Hashlmoto, K. et al. (1983). Molecular site of secreted and cell-associated plasminogen activators from cultured epidermal cells. Br. J. Dermatol. 109:15. 9. Isseroff, R. R. and D. B. Rifkin. (1983). Plasminogen is present in the basal layer of the epidermis. J. Invest. Dermatol. 80:297. 10. Michel, B. and C. S. Ko. (1977). An organ culture model for the study of pemphigus acantholysis. Br. J. Dermatol. 96:295. 11. O'Laughlin, S. et al. (1978), Fate of pemphigus antibody following successful therapy: Preliminary evaluation of pemphigus antibody determinations to regulate therapy. Arch. Dermatol. 114i1709. 12. Quigley, J. P. (1979). Proteolytic enzymes. In: R. O. Hynes (ed.), Surfaces of Normal and Malignant Cells.
properly validated RIA requires that the concentration determined for the unknown be independent of the dilution at which the measurement is made. There is no requirement that the standards and unknowns be identical chemically or biologically. Thus, in order to assure that measurements by RIA reflect biologic activity, it is often necessary to have additional information (vide infra). RIA is a special case of what may be termed measurement by "competitive binding" or "ligand binding" assays. The binding substance need not be an antibody, but could be a variety of other substances, including serum binding proteins, enzymes, tissue receptor sites, etc. The " l a b e l " need not be a radioisotopic label but might be, for instance, an enzyme, a fluorescent or nonfluorescent dye. Some commercial laboratories and commercial kits may employ la-
13.
14.
15.
16.
17.
18.
John Wiley and Sons, Chichester, p. 247. Reich, E. (1978). Activtion of plasminogen: A general mechanism for producing localized extracellular proteolysis. In: R. D. Berlin, et al. (eds.), Molecular Basis of Biological Degradative Processes. Academic Press, New York, p. 155. Sams, W. J. and R. E. Jordan (1971). Correlation of pemphigoid and pemphigus antibody titres with activity of disease. Br. J. Dermatol. 84:7. Schiltz, J. R. and B. Michel. (1976). Production of epidermal acantholysis in normal human skin in vitro by the IgG fraction from pemphigus serum. J. Invest, Dermatol. 67:254. Singer, K. H. et al, (1983). Stimulation of plasminogen activator following binding of antibodies to human melanoma. Fed. Proc. 42:839. Singer, K. H. et al. (1982). Antibody-induced release of cellular proteinases: Loss of adhesion of human melanoma ceils after binding of antmelanoma antibody. J. Immunol. 128:1321. Stoughton, R. B. and F. Bagateil. (1959). The nature of cantharidin acantholysis. J. Invest. Dermatol. 33:287.
bels that are not radioisotopic. Most research laboratories, however, continue to employ the more commonly used 125I, because the same or similar methodology can be used to prepare all labeled antigens, and the small size of this radioisotope offers less steric hindrance to the reaction o f antigen with antibody than the much larger enzyme or dye labels. RIA was first applied to the measurement of the peptidal hormones. Since they are generally present in plasma at concentrations of 10-13M10-1°M, the exquisite sensitivity of RIA is required for their assay. However, assay of the peptidal hormones is complicated by the observation that many of them may be found in more than one form, both in the circulation and in the tissues of origin (9). These forms may or may not have biologic activity, and may represent either pre-
23
LABELED ANTIGEN Ag"
SPECIFIC ANTIBODY +
AB
(F)
LABELED ANTIGENANTIBODY COMPLEX -
"
Ag" AB
+
(B)
UNLABELED ANTIGEN A C3" in known standard solutions or
IL
unknown samples
Ag-Ab UNLABELED ANTIGENANTIBODY COMPLEX Figure I. Compethlg reactions that form the basis of radiohnmunoassay (RIA).
cursor(s) or metabolic product(s) of the well known, well characterized, biologically active hormone. For some hormones, such as insulin, the intact biologically active peptide predominates in plasma although, under certain circumstances (e.g., patients with insulin-secreting tumors or those with a rare genetic abnormality that prevents cleavage of the C-peptide), a biologi-
cally inactive precursor, such as proinsulin, may be prominent. In the case of other peptides, including secretin or pancreatic polypeptide, neither a precursor nor a metabolic fragment has yet been identified. However, the predominant form of gastrin in plasma is usually, but not always, a biologically active 34 amino acid precursor (G34), rather than the 17 amino acid peptide
Figure 2. Partial listing of peptidal and nonpeptidal hormones and other substances measured by radioimmunoassay. (Reproduced from Yalow, R. S. (1978). Radioimmunoassay: A Probe for the fine structure of biologic systems. Sciences 200:1236--1245.) BLOOD GLUCOSE
PLASMA INSULIN
200.
2OO-
/
Iol'srt'
I
j
1 •
i
m
:
',ool
,oo
d
i
fi
i
g
g
TIME IN HOURS
24
,
z
3
4
5
6
(GI7), which had been purified from the antrum. For a given plasma concentration, G17 is 3 - 5 times more potent in promoting acid secretion than is G34. Thus, it is necessary to know the hormonal form in order to interpret the significance of any given plasma level of the hormone. Differential diagnosis between early Zollinger-Ellison syndrome and non-tumor-associated hypergastrinemia often requires appropriate stimulatory tests. The application of the parathyroid hormone (PTH) assay is even more complicated. Most laboratories employ an antiserum directed towards the C-terminus of the PTH molecule. Such an antiserum also reacts with a biologically inactive C-terminal fragment of PTH that generally is the major immunoreactive form of PTH in plasma. The use of such an antiserum enhances the sensitivity of the assay and makes it quite useful for the diagnosis of primary hyperparathyroidism. However, since the C-terminal fragment is removed almost solely by the kidney, elevated levels of this peptide are found in patients with renal disease with or without secondary hyperparathyroidism. Since the standards and the immunoreactive PTH in plasma may not be immunochemically identical, the assay usually cannot be properly validated. It is not surprising, therefore, that discrepancies are found among the same samples assayed in different laboratories (4). Even when not complicated by a multiplicity of hormonal forms, the range of normal values for many of the peptide hormones in the basal and stimulated state often is quite large (Fig. 2). For differential diagnosis between normal states and those associated with hypo- or hyperendocrine function, appropriate stimulatory or suppression tests generally are employed. In spite of these complications in interpretation, virtually all that is known about the regulation of the secretion of peptide hormones and the interrelationships among the hormones and their substrates has been gained through the use of RIA. Of particular interest over the past
ClinicalImmunologyNewsletter
few years has been the finding that there are numerous peptides common both to the brain and to the gut. Included among these are substance P, neurotensin, somatostatin, vasoactive intestinal polypeptide, and the cholecystokinin family (CCK). The finding of these peptides widely distributed in brain and peripheral tissues raises the question of the commonality of their precursors. It is now apparent that the enkephalins, once thought to be derived solely from a 13-endorphin precursor in the pituitary (3), are now known to be derived also from an unrelated precursor in the adrenal medulla (6).
Nonpeptidal Hormones The direct chemical assay of thyroidal and steroidal hormones had been
possible before the RIA era because these hormones possess distinctive chemical groupings and solubilities, and because their molar concentrations in plasma are 103-105 higher than those of the peptide hormones in the basal state. Soon after the development of RIA, it was appreciated that natural binding proteins in plasma could serve as the ligand in place of antibody for the assay of thyroxine (T4) and cortisol. For almost a decade, the competitive protein binding assay (CPBA) was used extensively for the determination of these nonpeptidal hormones. Although CPBAs are still employed, the increased sensitivity and specificity of RIA has resuited in its more common application. The specificity of RIA, indeed, is remarkable. Antisera that can distinguish among the thyronines containing
I - 4 iodine atoms or between cortisol or corticosterone, which differ only in a single OH group, are easily produced. The sensitivity of RIA permits its use for the screening of neonatal hypothyroidism (2). This disease occurs in 1 in 4000 births in the United States. If it is not recognized and treated within a few weeks after birth, before recognition of clinical symptoms is feasible, irreversible mental retardation ensues. For screening, a few drops of blood taken shortly after birth are placed on filter paper and sent to central laboratories. Usually, T 4 is determined. However, if the T4 is low, confirmation should be obtained by measurement of thyrotropin (TSH) in the blood spots. Using RIA, the diagnosis can be made within 1 week. The availability of RIAs for the thy-
Figure 3. Blood glucose and plasma insulin concentrations in 39 nonobese nondiabetic sllbjects. Note the very narrow range of blood glucose and the very wide range of plasma hzsulin concentrations. (Reproduced from Yaiow, R. S. and W. A. Bauman. (1983). Plasma insulin in health and Disease. In: M. Ellenberg and H. Rifkin (eds.), Diabetes Mellitus, Theory and Practice, Third ed. Medical
Examination Publishing, New York, pp. 119-150.) SUBSTANCESMEASUREDBY RADIOIMMUNOASSAY PEPTIDAL HORMONES PITUITARY HORMONES Growth hormone Adrenocorticotropic hormone (ACTH) Melanocyte stimulating hormone (MSH) a-MSH B-MSH Glycoprotelns Thyroid stimulating hormone {TSH) Follicle stimulating hormone (FSH) Lutelnlzlng hormone (LH) Prolactln tlpotropln gasopressln Oxytocln CHORIONIC HORMONES Human chorionic gonadotropin (HCG) Human chorionic somatemammotropln {HCS) PANCREATICHORMONES Insulin Glucagon Pancreatic Polypeptlde CALCITROPIC HORMONES Parathyroid hormone (PTH) Calcltonln (CT) GASTROINTESTINALHORMONES Gastrin Secretin Cholecystoklnln (CCK) Vasoactlve intestinal polypeptlde {VIPI Gastric inhibitory polypeptlde (GIP) VASOACIIVE TISSUE HORMONES Anglotenslns Bradykinins RELEASING AND RELEASE INHIBITING FACTORS Thyrotropin releasing factor (TRF) LHRF Sematostatin OTHERPEPTIDES Substance P Endorphins Enkephalins
© 1984 by Elsevier Science Publishing Co., Inc.
ffON-PEPTIDAL HORMONES
NON-HORMONALSUBSTArlCES
THYRDIDAL HORMONES Thyroxine (T4) Triiodothyronlne iT31 Reverse T3 STEROIDS Aldosterone Dortlcosterolds Estrogens Androgens Progesterones PROSTAGLAMDINS BIOLOGICAMINES Serotonln Melatonln
DRUGS & VITAMINS Cardlac glycosides Drugs of Abuse Psychoactive Drugs Antibiotics CNS Depressants Vitamin A, Folic acid CYCLIC NUCLEOTIDES ENZYMES CI esterase Fructose I , 6 diphosphatase Plasminogen, Plasmtn Chymutrypsln, Trypsin Carbonic anhydrase Isoenzymes A1dose reductase Carboxypeptldase B Pancreatic elastase VIRUSES Hepatitis associated antigen Murlne Leukemia viruses {Gross, Rauscher, Moloney) Mason-Pflzer monkey virus TUMORAMTIGENS Carclnoembryonlc antigen a-Fetoproteln SERUM PROTEINS Thyroxine binding globulin IgG, IgE, IgA, IgM Properdln Fibrlnogen ApolIpoproteln B Myoglobln Myelin Basic Protein OTHER Intrinsic factor Rheumatoid factor Hageman factor Neurophysins Staphylococcal B-Enterotoxin
25
i
roid related hormones has made it possible to detect and treat neonates with thyroid disease in time for the simple and inexpensive therapy to be effective. Mass screening programs are now in place throughout the United States. Infectious Diseases
The application of RIA in the field of infectious disease began with the development of an assay for hepatitis B antigen, then known as Australia antigen (8). Within 2 years after this report, kit assays were developed commercially that were suitable for routine detection of hepatitis B antigen with much greater sensitivity than had been obtained with classic methodology. Contamination of blood with this virus had been responsible for almost all cases of posttransfusion hepatitis. Now all blood used in the United States is tested for both hepatitis B and hepatitis A antigen. Nearly all of the current remaining incidence of transfusion hepatitis is due to another virus (or viruses) called non-A, non-B (NANB). This report (8) served as the model for the application of RIA in virology. Since that time, there has been a veritable explosion of applications of RIA in this field. More recently, an RIA for a tuberculoprotein has been described (7), offering the potential to substantially shorten the time and make possible the diagnosis of active tuberculosis in a less expensive, simpler, and far safer fashion than with the use of classic microbiologic methodology. Even if this technology were applicable only to the diagnosis of tuberculosis, it would remain a major breakthrough. However, it should serve as a model for the development of assays for proteins associated with other slow growing organisms, the diagnosis of which may be difficult with microbiologic methodology. Pharmacology and Toxicology RIA has already added a completely new dimension to the identification
26
and measurement of pharmacologically active substances in plasma and tissue (5). The list of compounds for which such assays are available is growing rapidly (Fig. 3). In general, achieving adequate sensitivity is not difficult, since the molar concentrations of drugs at pharmacologic levels are high when compared, for instance, with the concentration of peptide hormones in body fluids. However, the requirement for the specificity of RIA of drugs merits some consideration. Structurally related compounds or metabolites may have significant immunoreactivity with some antisera, but not with others, and maY or may not constitute a problem, depending on the purpose of the assay. For instance, if the clinical problem relates to the toxicity of a particular drug, then the question as to whether or not the assay measures only the biologically active form is relevant. If the question relates simply to whether or not a drug had been taken surreptitiously, however, then the reactivity of metabolites or variation of the immunoreactivity with the exact form of the drug may be irrevelant. Conclusion
In the 25 years since its first description, RIA has had a major impact on clinical diagnosis and in most areas of investigative medicine. Although an increasing number of peptidal hormones have been identified since RIA methodology was first described, their number is infinitesimal compared with the number of nonhormonal substances that are now or potentially will be measurable by RIA. It can be concluded that if there is a need to measure an organic substance of biologic interest and if there is no other sensitive, specific, and inexpensive method for doing so, some perspicacious investigator will develop an RIA for the purpose. Nevertheless, although RIA permits the determination of a large number of clinical parameters of health and disease simply and expeditiously its use in a causal manner, without insight into its pitfalls, can be destruc-
ii
i
i
tive of its very important role in clinical medicine.
References 1. Berson, S. A. and R. S. Yalow. (1958). Isotopic tracers in the study of diabetes. In: J. H. Lawrence and C. A. Tobias (eds.), Advances in Biological and Medical Physics, Vol. VI. Academic Press, New York, pp. 349430. 2. Fisher, D. A. and G. N. Burrow (eds.) (1975). Perinatal Thyroid Physiology and Disease. Raven Press, New York, pp. 197-269. 3. Hughes, J. el al. (1975). Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 28:577-580. 4. Raisz L. G. et al. (1979). Comparison of commercially available parathyroid hormone immunoassays in the differential diagnosis of hypercalzemia due to primary hyperparathyroidism or malignancy. Ann. Intern. Med. 91:739-740. 5. Spector, S. (1973). Radioimmunoassay of drugs. In: Radioimmunoassay and Related Procedures in Medichw, Vol. II. IAEA-SM-177/212, Proceedings of a Symposium, Istanbul, September 10-14, pp. 233-249. 6. Stern, A. S. et al. (1981). Two adrenal opioid polypeptides: Proposed intermediates in the processing of proenkephalin. Proc. Natl. Acad. Sci., USA 78:1962-1966. 7. Strans, E. et al. (1981). Clinical applications of the radioimmunoassay of secretory tuberculoprotein. Proc. Natl. Acad. Sci., USA 78:3214-3217. 8. Walsh, J. H., R. S. Yalow, and S. A. Berson. (1970). Detection of Australia antigen and antibody by means of radioimmunoassay techniques. J. Infect. Dis. 121:550-554. 9. Yalow, R. S. (1974). Heterogeneity of peptide hormones. Recent. Prog. Horm. Res. 30:597-633. 10. Yaiow, R. S. and S. A. Berson. (1960). Immunoassay of endogenous plasma insulin in man. J. Clin. Invest. 39:1157-1175. 11. Yalow, R. S. and S. A. Berson. (1959). Assay of plasma insulin in human subjects by immunological methods. Nature 184:1648-1649. 12. Yang, R.-K. et ai. (1983). Secretin responses to feeding and acid load. J. Lab. Clin. Med. 102:17-13.
Clinical ImmunologyNewsletter
Radioimmunoassay Rosalyn S. Yalow, Ph.D. Solomon A. Berson Research Laboratory Veterans Administration Medical Center Bronx, New York Radioimmunoassay (RIA) methodology was first applied a quarter-century ago to the measurement of insulin exogenously administered to rabbits (1). However, it was not until several years later that the assay had sufficient sensitivity to permit the determination of fasting and stimulated plasma insulin levels in man (10, 1 I). The principle is simple (Fig. 1). The concentration o f the substance in the unknown sample is made by comparing its inhibition of binding of radiolabeled antigen to antibody with the inhibition of that of known standards. A
© 1984 by Elsevier Science Publishing Co., Inc.
6. Farb, R. et al. (1978). Anti-epithelial cell-surface pemphigus antibody detaches viable epidermal cells from culture plates by activation of proteinase. Proc. Natl. Acad. Sci., USA, 75:459. 7. Hashimoto, K. et al. (1983). Anticell surface pemphigus autoantibody stimulates plasminogen activator activity of human epidermal cells: A mechanism for the loss of epidermal cohesion and blister formation. J. Exp. Med. 157:259. 8. Hashlmoto, K. et al. (1983). Molecular site of secreted and cell-associated plasminogen activators from cultured epidermal cells. Br. J. Dermatol. 109:15. 9. Isseroff, R. R. and D. B. Rifkin. (1983). Plasminogen is present in the basal layer of the epidermis. J. Invest. Dermatol. 80:297. 10. Michel, B. and C. S. Ko. (1977). An organ culture model for the study of pemphigus acantholysis. Br. J. Dermatol. 96:295. 11. O'Laughlin, S. et al. (1978), Fate of pemphigus antibody following successful therapy: Preliminary evaluation of pemphigus antibody determinations to regulate therapy. Arch. Dermatol. 114i1709. 12. Quigley, J. P. (1979). Proteolytic enzymes. In: R. O. Hynes (ed.), Surfaces of Normal and Malignant Cells.
properly validated RIA requires that the concentration determined for the unknown be independent of the dilution at which the measurement is made. There is no requirement that the standards and unknowns be identical chemically or biologically. Thus, in order to assure that measurements by RIA reflect biologic activity, it is often necessary to have additional information (vide infra). RIA is a special case of what may be termed measurement by "competitive binding" or "ligand binding" assays. The binding substance need not be an antibody, but could be a variety of other substances, including serum binding proteins, enzymes, tissue receptor sites, etc. The " l a b e l " need not be a radioisotopic label but might be, for instance, an enzyme, a fluorescent or nonfluorescent dye. Some commercial laboratories and commercial kits may employ la-
13.
14.
15.
16.
17.
18.
John Wiley and Sons, Chichester, p. 247. Reich, E. (1978). Activtion of plasminogen: A general mechanism for producing localized extracellular proteolysis. In: R. D. Berlin, et al. (eds.), Molecular Basis of Biological Degradative Processes. Academic Press, New York, p. 155. Sams, W. J. and R. E. Jordan (1971). Correlation of pemphigoid and pemphigus antibody titres with activity of disease. Br. J. Dermatol. 84:7. Schiltz, J. R. and B. Michel. (1976). Production of epidermal acantholysis in normal human skin in vitro by the IgG fraction from pemphigus serum. J. Invest, Dermatol. 67:254. Singer, K. H. et al, (1983). Stimulation of plasminogen activator following binding of antibodies to human melanoma. Fed. Proc. 42:839. Singer, K. H. et al. (1982). Antibody-induced release of cellular proteinases: Loss of adhesion of human melanoma ceils after binding of antmelanoma antibody. J. Immunol. 128:1321. Stoughton, R. B. and F. Bagateil. (1959). The nature of cantharidin acantholysis. J. Invest. Dermatol. 33:287.
bels that are not radioisotopic. Most research laboratories, however, continue to employ the more commonly used 125I, because the same or similar methodology can be used to prepare all labeled antigens, and the small size of this radioisotope offers less steric hindrance to the reaction o f antigen with antibody than the much larger enzyme or dye labels. RIA was first applied to the measurement of the peptidal hormones. Since they are generally present in plasma at concentrations of 10-13M10-1°M, the exquisite sensitivity of RIA is required for their assay. However, assay of the peptidal hormones is complicated by the observation that many of them may be found in more than one form, both in the circulation and in the tissues of origin (9). These forms may or may not have biologic activity, and may represent either pre-
23
LABELED ANTIGEN Ag"
SPECIFIC ANTIBODY +
AB
(F)
LABELED ANTIGENANTIBODY COMPLEX -
"
Ag" AB
+
(B)
UNLABELED ANTIGEN A C3" in known standard solutions or
IL
unknown samples
Ag-Ab UNLABELED ANTIGENANTIBODY COMPLEX Figure I. Compethlg reactions that form the basis of radiohnmunoassay (RIA).
cursor(s) or metabolic product(s) of the well known, well characterized, biologically active hormone. For some hormones, such as insulin, the intact biologically active peptide predominates in plasma although, under certain circumstances (e.g., patients with insulin-secreting tumors or those with a rare genetic abnormality that prevents cleavage of the C-peptide), a biologi-
cally inactive precursor, such as proinsulin, may be prominent. In the case of other peptides, including secretin or pancreatic polypeptide, neither a precursor nor a metabolic fragment has yet been identified. However, the predominant form of gastrin in plasma is usually, but not always, a biologically active 34 amino acid precursor (G34), rather than the 17 amino acid peptide
Figure 2. Partial listing of peptidal and nonpeptidal hormones and other substances measured by radioimmunoassay. (Reproduced from Yalow, R. S. (1978). Radioimmunoassay: A Probe for the fine structure of biologic systems. Sciences 200:1236--1245.) BLOOD GLUCOSE
PLASMA INSULIN
200.
2OO-
/
Iol'srt'
I
j
1 •
i
m
:
',ool
,oo
d
i
fi
i
g
g
TIME IN HOURS
24
,
z
3
4
5
6
(GI7), which had been purified from the antrum. For a given plasma concentration, G17 is 3 - 5 times more potent in promoting acid secretion than is G34. Thus, it is necessary to know the hormonal form in order to interpret the significance of any given plasma level of the hormone. Differential diagnosis between early Zollinger-Ellison syndrome and non-tumor-associated hypergastrinemia often requires appropriate stimulatory tests. The application of the parathyroid hormone (PTH) assay is even more complicated. Most laboratories employ an antiserum directed towards the C-terminus of the PTH molecule. Such an antiserum also reacts with a biologically inactive C-terminal fragment of PTH that generally is the major immunoreactive form of PTH in plasma. The use of such an antiserum enhances the sensitivity of the assay and makes it quite useful for the diagnosis of primary hyperparathyroidism. However, since the C-terminal fragment is removed almost solely by the kidney, elevated levels of this peptide are found in patients with renal disease with or without secondary hyperparathyroidism. Since the standards and the immunoreactive PTH in plasma may not be immunochemically identical, the assay usually cannot be properly validated. It is not surprising, therefore, that discrepancies are found among the same samples assayed in different laboratories (4). Even when not complicated by a multiplicity of hormonal forms, the range of normal values for many of the peptide hormones in the basal and stimulated state often is quite large (Fig. 2). For differential diagnosis between normal states and those associated with hypo- or hyperendocrine function, appropriate stimulatory or suppression tests generally are employed. In spite of these complications in interpretation, virtually all that is known about the regulation of the secretion of peptide hormones and the interrelationships among the hormones and their substrates has been gained through the use of RIA. Of particular interest over the past
ClinicalImmunologyNewsletter
few years has been the finding that there are numerous peptides common both to the brain and to the gut. Included among these are substance P, neurotensin, somatostatin, vasoactive intestinal polypeptide, and the cholecystokinin family (CCK). The finding of these peptides widely distributed in brain and peripheral tissues raises the question of the commonality of their precursors. It is now apparent that the enkephalins, once thought to be derived solely from a 13-endorphin precursor in the pituitary (3), are now known to be derived also from an unrelated precursor in the adrenal medulla (6).
Nonpeptidal Hormones The direct chemical assay of thyroidal and steroidal hormones had been
possible before the RIA era because these hormones possess distinctive chemical groupings and solubilities, and because their molar concentrations in plasma are 103-105 higher than those of the peptide hormones in the basal state. Soon after the development of RIA, it was appreciated that natural binding proteins in plasma could serve as the ligand in place of antibody for the assay of thyroxine (T4) and cortisol. For almost a decade, the competitive protein binding assay (CPBA) was used extensively for the determination of these nonpeptidal hormones. Although CPBAs are still employed, the increased sensitivity and specificity of RIA has resuited in its more common application. The specificity of RIA, indeed, is remarkable. Antisera that can distinguish among the thyronines containing
I - 4 iodine atoms or between cortisol or corticosterone, which differ only in a single OH group, are easily produced. The sensitivity of RIA permits its use for the screening of neonatal hypothyroidism (2). This disease occurs in 1 in 4000 births in the United States. If it is not recognized and treated within a few weeks after birth, before recognition of clinical symptoms is feasible, irreversible mental retardation ensues. For screening, a few drops of blood taken shortly after birth are placed on filter paper and sent to central laboratories. Usually, T 4 is determined. However, if the T4 is low, confirmation should be obtained by measurement of thyrotropin (TSH) in the blood spots. Using RIA, the diagnosis can be made within 1 week. The availability of RIAs for the thy-
Figure 3. Blood glucose and plasma insulin concentrations in 39 nonobese nondiabetic sllbjects. Note the very narrow range of blood glucose and the very wide range of plasma hzsulin concentrations. (Reproduced from Yaiow, R. S. and W. A. Bauman. (1983). Plasma insulin in health and Disease. In: M. Ellenberg and H. Rifkin (eds.), Diabetes Mellitus, Theory and Practice, Third ed. Medical
Examination Publishing, New York, pp. 119-150.) SUBSTANCESMEASUREDBY RADIOIMMUNOASSAY PEPTIDAL HORMONES PITUITARY HORMONES Growth hormone Adrenocorticotropic hormone (ACTH) Melanocyte stimulating hormone (MSH) a-MSH B-MSH Glycoprotelns Thyroid stimulating hormone {TSH) Follicle stimulating hormone (FSH) Lutelnlzlng hormone (LH) Prolactln tlpotropln gasopressln Oxytocln CHORIONIC HORMONES Human chorionic gonadotropin (HCG) Human chorionic somatemammotropln {HCS) PANCREATICHORMONES Insulin Glucagon Pancreatic Polypeptlde CALCITROPIC HORMONES Parathyroid hormone (PTH) Calcltonln (CT) GASTROINTESTINALHORMONES Gastrin Secretin Cholecystoklnln (CCK) Vasoactlve intestinal polypeptlde {VIPI Gastric inhibitory polypeptlde (GIP) VASOACIIVE TISSUE HORMONES Anglotenslns Bradykinins RELEASING AND RELEASE INHIBITING FACTORS Thyrotropin releasing factor (TRF) LHRF Sematostatin OTHERPEPTIDES Substance P Endorphins Enkephalins
© 1984 by Elsevier Science Publishing Co., Inc.
ffON-PEPTIDAL HORMONES
NON-HORMONALSUBSTArlCES
THYRDIDAL HORMONES Thyroxine (T4) Triiodothyronlne iT31 Reverse T3 STEROIDS Aldosterone Dortlcosterolds Estrogens Androgens Progesterones PROSTAGLAMDINS BIOLOGICAMINES Serotonln Melatonln
DRUGS & VITAMINS Cardlac glycosides Drugs of Abuse Psychoactive Drugs Antibiotics CNS Depressants Vitamin A, Folic acid CYCLIC NUCLEOTIDES ENZYMES CI esterase Fructose I , 6 diphosphatase Plasminogen, Plasmtn Chymutrypsln, Trypsin Carbonic anhydrase Isoenzymes A1dose reductase Carboxypeptldase B Pancreatic elastase VIRUSES Hepatitis associated antigen Murlne Leukemia viruses {Gross, Rauscher, Moloney) Mason-Pflzer monkey virus TUMORAMTIGENS Carclnoembryonlc antigen a-Fetoproteln SERUM PROTEINS Thyroxine binding globulin IgG, IgE, IgA, IgM Properdln Fibrlnogen ApolIpoproteln B Myoglobln Myelin Basic Protein OTHER Intrinsic factor Rheumatoid factor Hageman factor Neurophysins Staphylococcal B-Enterotoxin
25
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roid related hormones has made it possible to detect and treat neonates with thyroid disease in time for the simple and inexpensive therapy to be effective. Mass screening programs are now in place throughout the United States. Infectious Diseases
The application of RIA in the field of infectious disease began with the development of an assay for hepatitis B antigen, then known as Australia antigen (8). Within 2 years after this report, kit assays were developed commercially that were suitable for routine detection of hepatitis B antigen with much greater sensitivity than had been obtained with classic methodology. Contamination of blood with this virus had been responsible for almost all cases of posttransfusion hepatitis. Now all blood used in the United States is tested for both hepatitis B and hepatitis A antigen. Nearly all of the current remaining incidence of transfusion hepatitis is due to another virus (or viruses) called non-A, non-B (NANB). This report (8) served as the model for the application of RIA in virology. Since that time, there has been a veritable explosion of applications of RIA in this field. More recently, an RIA for a tuberculoprotein has been described (7), offering the potential to substantially shorten the time and make possible the diagnosis of active tuberculosis in a less expensive, simpler, and far safer fashion than with the use of classic microbiologic methodology. Even if this technology were applicable only to the diagnosis of tuberculosis, it would remain a major breakthrough. However, it should serve as a model for the development of assays for proteins associated with other slow growing organisms, the diagnosis of which may be difficult with microbiologic methodology. Pharmacology and Toxicology RIA has already added a completely new dimension to the identification
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and measurement of pharmacologically active substances in plasma and tissue (5). The list of compounds for which such assays are available is growing rapidly (Fig. 3). In general, achieving adequate sensitivity is not difficult, since the molar concentrations of drugs at pharmacologic levels are high when compared, for instance, with the concentration of peptide hormones in body fluids. However, the requirement for the specificity of RIA of drugs merits some consideration. Structurally related compounds or metabolites may have significant immunoreactivity with some antisera, but not with others, and maY or may not constitute a problem, depending on the purpose of the assay. For instance, if the clinical problem relates to the toxicity of a particular drug, then the question as to whether or not the assay measures only the biologically active form is relevant. If the question relates simply to whether or not a drug had been taken surreptitiously, however, then the reactivity of metabolites or variation of the immunoreactivity with the exact form of the drug may be irrevelant. Conclusion
In the 25 years since its first description, RIA has had a major impact on clinical diagnosis and in most areas of investigative medicine. Although an increasing number of peptidal hormones have been identified since RIA methodology was first described, their number is infinitesimal compared with the number of nonhormonal substances that are now or potentially will be measurable by RIA. It can be concluded that if there is a need to measure an organic substance of biologic interest and if there is no other sensitive, specific, and inexpensive method for doing so, some perspicacious investigator will develop an RIA for the purpose. Nevertheless, although RIA permits the determination of a large number of clinical parameters of health and disease simply and expeditiously its use in a causal manner, without insight into its pitfalls, can be destruc-
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tive of its very important role in clinical medicine.
References 1. Berson, S. A. and R. S. Yalow. (1958). Isotopic tracers in the study of diabetes. In: J. H. Lawrence and C. A. Tobias (eds.), Advances in Biological and Medical Physics, Vol. VI. Academic Press, New York, pp. 349430. 2. Fisher, D. A. and G. N. Burrow (eds.) (1975). Perinatal Thyroid Physiology and Disease. Raven Press, New York, pp. 197-269. 3. Hughes, J. el al. (1975). Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 28:577-580. 4. Raisz L. G. et al. (1979). Comparison of commercially available parathyroid hormone immunoassays in the differential diagnosis of hypercalzemia due to primary hyperparathyroidism or malignancy. Ann. Intern. Med. 91:739-740. 5. Spector, S. (1973). Radioimmunoassay of drugs. In: Radioimmunoassay and Related Procedures in Medichw, Vol. II. IAEA-SM-177/212, Proceedings of a Symposium, Istanbul, September 10-14, pp. 233-249. 6. Stern, A. S. et al. (1981). Two adrenal opioid polypeptides: Proposed intermediates in the processing of proenkephalin. Proc. Natl. Acad. Sci., USA 78:1962-1966. 7. Strans, E. et al. (1981). Clinical applications of the radioimmunoassay of secretory tuberculoprotein. Proc. Natl. Acad. Sci., USA 78:3214-3217. 8. Walsh, J. H., R. S. Yalow, and S. A. Berson. (1970). Detection of Australia antigen and antibody by means of radioimmunoassay techniques. J. Infect. Dis. 121:550-554. 9. Yalow, R. S. (1974). Heterogeneity of peptide hormones. Recent. Prog. Horm. Res. 30:597-633. 10. Yaiow, R. S. and S. A. Berson. (1960). Immunoassay of endogenous plasma insulin in man. J. Clin. Invest. 39:1157-1175. 11. Yalow, R. S. and S. A. Berson. (1959). Assay of plasma insulin in human subjects by immunological methods. Nature 184:1648-1649. 12. Yang, R.-K. et ai. (1983). Secretin responses to feeding and acid load. J. Lab. Clin. Med. 102:17-13.
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