[25] Radioimmunoassay and immunotitration of human serum dopamine β-hydroxylase

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IMMUNOASSAY METHODS

[25]

Hb to 2250 ng/mg Hb. The number of reports and the range of values indicate both the degree of interest in this enzyme and the need to develop accurate assays such as the RIA. If either elevated or diminished SOD-1 levels are established as a meaningful marker for specific diseases, the scientific community must establish reference standards to ensure reproducible values from all laboratories. Until such time, however, the primary concern is to establish the importance of changes in SOD-1 levels in different diseases by comparison with normal individuals. Variations between laboratories cannot be considered significant until standardization is achieved.

[25] R a d i o i m m u n o a s s a y

and Immunotitration of Human

S e r u m D o p a m i n e f l - H y d r o x y l a s e la By JOEL DUNNETTE

and RICHARD WEINSHILBOUM

Introduction Dopamine/3-hydroxylase (dopamine fl-monooxygenase EC 1.14.17.1, DBH) is the enzyme that catalyzes the conversion of 3,4-dihydroxyphenylethylamine (dopamine) to the neurotransmitter norepinephrine, is released with catecholamines from vesicles in the adrenal medulla and sympathetic nerves, and is found circulating in blood. 1,2 It has been suggested that serum D B H levels might be a useful measure of sympathetic nervous system function. However, many procedures thought to produce alterations in sympathetic nervous system activity result in changes in serum DBH enzyme activity of only relatively small magnitude. 2 Interpretation of these changes in man is complicated by large intersubject variations in enzyme activity due mainly to the effects of inheritance. 3 Immunochemical assays of serum D B H have been performed to determine whether the magnitude or direction of change in immunoreactive DBH protein levels in response to variations in sympathetic function differ from those of enzyme activity. Immunoreactive D B H protein levels have been

la Supported in part by N I H grants NS 11014 and H L 17487. Dr. Weinshilboum is an Established Investigator of the A m e r i c a n Heart Association. R. M. Weinshilboum a n d J. Axelrod, Circ. Res. 28, 307 (1971). 2 R. M. Weinshilboum, Pharmacol. Rev. 30, 133 (1978). 3 R. M. Weinshilboum, H. G. Schrott, F. R a y m o n d , W. H. Weidman, and L. R. Elveback, Am. J. Hum. Genet. 27, 573 (1975).

METHODS IN ENZYMOLOGY, VOL. 74

Copyright © 1981by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181974-4

[25]

RIA AND IMMUNOTITRATIONOF HUMAN DBH

371

measured both with radioimmunoassay (RIA) ~-7 and immunotitration techniques. ~-1° Principles The immunotitration of D B H , like other immunotitration procedures, is based on the assumption that increasing quantities of antibody will be required to bind a given fraction of antigen as the quantity o f antigen is increased. In practice, several different quantities of anti-DBH antibody are incubated with aliquots o f the sample being analyzed. Antibodybound and free D B H may be separated by precipitation of the anti-DBH antibody with a second antibody. The D B H enzymatic activity remaining in the supernatant is measured at each antibody concentration used, and the relative quantity o f D B H protein is assumed to be related t o the quantity of antibody required to titrate a given fraction of the enzymatic activity. The radioimmunoassay of D B H , like all RIA procedures, is based on competition between radioactively labeled D B H and unlabeled enzyme for the same antibody binding sites. Both immunotitration and RIA can yield information with regard to the relative quantity of immunoreactive D B H present in an unknown sample. Both require antihuman D B H antiserum. Immunotitration, unlike RIA, does not require highly purified antigen. Although it usually requires that the unknown sample have D B H enzymatic activity, samples with no enzymatic activity can be assayed by immunotitration after mixture with a fixed amount o f enzymatically active D B H standard. An increase in the quantity of antibody required to immunotitrate the standard may indicate the presence of enzymaticaUy inactive D B H protein. Included among the disadvantages o f immunotitration are the following: only relative quantitation is possible, multiple data points are required for each sample assayed, and the method is usually not as sensitive as is an RIA. A relative disadvantage o f RIA procedures is that they require purified and radiolabeled D B H antigen. Once an RIA has been developed and validated, it has the advantages that in theory it may provide true quantitation of D B H protein and that many R. P. Ebstein, D. H. Park, L. S. Freedman, S. M. Levitz, T. Ohuchi, and M. Goldstein. 13, 769 (1973), R. A. Rush, P. E. Thomas, T. Nagatsu, and S. Udenfriend, Proc. Natl. A c , d . Sci. U.S.A. 71,872 (1974). 4;R. A. Rush, P. E. Thomas, and S. Udenfriend, Proc. Natl. Acad. Sci. U.S.A. 72, 750 (1975). 7j. Dunnette and R. Weinshilboum,J. Clin. Invest. 60, 1080 (1977). R. D. Ciaranello and G. F. Wooten, Pharmacology 12, 272 (1974). N. Kirshner, S. M. Schanberg, and H. J. Sage, Life Sci. 17, 423 (1975). "' J. Dunnette and R. Weinshilboum,Am. J. Hum. Genet. 28, 155 (1976). Life Sci.

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[25]

samples may be assayed simultaneously. The following procedures have been developed in our laboratory and used successfully in large population and biochemical genetic studies of human serum DBH. Several other procedures for the immunotitration and RIA of DBH have been described, and reference to them will be made in the subsequent discussion. Purification of Human DBH Because of a relative lack of species cross-reactivity, DBH for use in the development of antibodies and for use as an antigen for an RIA must be purified from human tissue, z Practical sources of the human enzyme are limited to the adrenal medulla, pheochromocytoma (tumors of the adrenal medulla) tissue, and, perhaps, human serum. For the specific procedures described here, DBH was purified from fresh human pheochromocytoma and from human adrenal glands obtained at autopsy. 7'~° Chromaffin granules were prepared and were lysed by freezing and thawing in a hypotonic medium. H Lysed chromaffin granules were homogenized in the presence of 0.1% Triton X-100 (Packard Instrument Co.) to solubilize DBH in the vesicle membranes. The homogenate was centrifuged at 100,000 g for 60 rain, and the supernatant was passed through a BioGel A-1.5 m (BioRad Laboratories) gel filtration column. Fractions with peak DBH enzymatic activity were pooled and concentrated. Part of this preparation was used as antigen for the development of antibodies. The preparation obtained from fresh pheochromocytoma tissue was purified further by affinity chromatography on a concanavalin A-Sepharose 4B column (Pharmacia). DBH is a glycoprotein and is apparently the only protein from chromaffin vesicle lysates that binds to the lectin concanavalin A. Elution from the concanavalin A column was accomplished with a solution of a-methyl-D-mannoside. 12 The preparation obtained after affinity chromatography migrated as a single band during disc gel electrophoresis, and was used as the antigen in the radioimmunoassay, r Protein concentrations were measured with fluorescamine (Pierce Chemical Co.), with crystallized bovine serum albumin (BSA) (Sigma) as a standard. ~a Antibody Preparation Anti-human adrenal DBH was prepared by immunization of a male New Zealand rabbit with 0.5 mg of partially purified human adrenal en~ A. D. Smithand H. Winkler,Biochem. J. 103, 480 (1967). 12R. A. Rush, P. E. Thomas, S. H. Kindler, and S. Udenfriend,Biochem. Biophys. Res. Cornmun. 57, 1301 (1974). ~3S. Udenfriend, S. Stein, P, Bbhlen, W. Dairman, W. Leimgruber, and M. Weigele, Science 178, 871 (1972).

[25]

R I A AND IMMUNOT1TRATION OF HUMAN D B H

373

zyme in an equal volume of complete Freund's adjuvant. Subcutaneous injections were made at multiple sites on the back. Blood was obtained from the ear by venipuncture before immunization and 7 weeks after immunization. Serum obtained during the second bleeding was used as an antibody. Anti-human pheochromocytoma DBH antibodies were prepared in the same fashion except that 1.0 mg of partially purified DBH was used as antigen, and a second injection was performed 1 month later with antigen plus an equal volume of incomplete Freund's adjuvant. Serum obtained 1 week after the second injection was used as antibody. 7 All rabbit blood samples were allowed to clot, serum was obtained by centrifugation, and antisera were stored at - 2 0 °. Burro antibody to rabbit "/-globulin was prepared as described in detail elsewhere. ~0 Radioiodination of DBH Purified human pheochromocytoma DBH was iodinated by the method of Bolton and Hunter.'4 The Bolton and Hunter method was used because none of the DBH enzymatic activity remained after attempts to iodinate the protein with the chloramine-T method. ''~ One millicurie of the Bolton-Hunter reagent ('2~I-labeled p-hydroxyphenylpropionic acid, N-hydroxysuccinimide ester; = 500 Ci/mmol, New England Nuclear) dissolved in benzene was purchased. The benzene was evaporated under vacuum, and, in rapid succession, 10/_tl of 0.2 M sodium borate, pH 8.5, and 10/xl (4.3/~g) of purified DBH were added to the reagent. The reaction mixture was kept on ice for 30 rain and was stirred repeatedly. The reaction was stopped by the addition of 200 /zl of 0.2 M glycine in 0.1 M borate, pH 8.5. After 5 min of mixing, the radiolabeled DBH was separated from nonprotein-bound 12~I by gel filtration chromatography on a 1.2 × 14 cm BioGel A-0.5 m (BioRad Laboratories) column. The column had been equilibrated with and was eluted with 0.2% gelatin in 50 mM potassium phosphate buffer, pH 6.5. The specific activity of the '2~Ilabeled DBH was approximately 34 mCi/mg. Sixty-five percent of the DBH enzymatic activity of the preparation was retained after iodination. Immunotitration Procedure Immunotitration of human serum DBH was performed with a twostage procedure.'° Incubation tubes for the first stage contained 50 ~l of human serum diluted 1 : 6 and 50/xl of rabbit serum diluted 1 : 250. The rabbit serum contained varying proportions of antiserum to DBH and preim,4 A. E. Bolton and W. M. Hunter, Biochern. J. 133, 529 (1973). '~ F. C. Greenwood, W. M. Hunter, and J. S. Glover, Biochem. J. 89, 114 (1963).

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mune rabbit serum to provide different points in the immunotitration. Dilutions of both human serum and rabbit serum were performed with 0.25% BSA in 0.9% NaC1 buffered to pH 7.4 with 10 mM potassium phosphate buffer. The components of the first-stage reaction were mixed, were incubated at 37° for 60 min, and were then kept at 4° for 20 hr. The second stage in the immunotitration, precipitation of the rabbit y-globulin, was initiated by the addition to the reaction mixture of 100/zl of burro antirabbit y-globulin diluted 1 : 25 with the same diluent. The reaction tubes were kept at 4° for 16 additional hours and were then centrifuged at 6000 g for 15 min to remove the precipitate. From each tube, 100 ~1 of supernatant was transferred into a reaction tube that contained 100 ~1 of a solution of 0.25% BSA, 9 ttM CuSO4, and 1 mM potassium phosphate buffer, pH 7.4. The CuSO4 was added to the reaction mixture to counteract the effects of potent endogenous inhibitors of DBH that are present in serum and in tissue homogenates, z''6 DBH enzymatic activity was assayed by a modification of the procedure of Molinoff e t al. ,.3.,7 This assay has been described in detail elsewhere.'-* Phenylethylamine at a final concentration of 1 mM served as a substrate for the reaction. The final reaction pH was 5.2. Samples were incubated at 37° for 30 min for the stage of the reaction catalyzed by DBH. An additional 30-min incubation was performed for the portion of the reaction catalyzed by noradrenalin N-methyltransferase (EC 2.1.1.28). " B l a n k s " were identical samples heated to 95° for 5 min. Heated samples that contained 100 ng of /3-phenylethanolamine HCI served as internal standards for the step in the enzymatic assay catalyzed by noradrenalin N-methyltransferase. One unit of enzyme activity represented the production of 1 nmol of/3-phenylethanolamine per hour per milliliter of serum. Discussion of Immunotitration Representative results of a series of immunotitrations of human serum DBH are shown in Fig. 1.'° Serum samples with a range of endogenous DBH enzymatic activity levels were mixed with varying quantities of anti-human adrenal DBH antibody. The enzymatic activity that remained in each sample was compared to the enzymatic activity in the same sample exposed only to preimmune rabbit serum. Exposure of human serum to preimmune rabbit serum resulted in no change in DBH enzymatic activity. Although the rabbit and burro serum used in the immunotitration did contain DBH, the amount of activity was constant and extremely t~ D. S. Duch, O. H. Viveros, and N. Kirshner, Biochem. PharmacoL 17, 255 (1968). ~7 p. B. Molinoff, R. Weinshilboum, and J. Axelrod, J. Pharmacol. Exp. Ther. 178, 425 (1971).

[25]

RIA AND tMMUNOTITRATIONOF HUMAN DBH

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FK;. 1, Immunotitration of 11 separate samples of human serum DBH graphed in a linear (A) and in a semilogarithmic fashion (B). Six samples with moderate levels of DBH enzymatic activity (453-677 units, G--O) and five samples with high activity (1377-2203 units, ~------Q) were titrated. Reprinted from Dunnette and Weinshilboum1°with the permission of the University of Chicago Press. small c o m p a r e d with that present in h u m a n serum. When the results were plotted in a linear fashion, they a p p e a r e d exponential (Fig. 1A). When they were plotted in a semilogarithmic fashion (Fig. 1B), there was a more nearly linear relationship b e t w e e n the quantity of antiserum and the titration o f D B H enzymatic activity. Linear least square lines were calculated for the semilogarithmic graphs, and the quantity of antiserum required to titrate endogenous D B H enzymatic activity by 50% (antibody dose 50% or ADs0) was used as a m e a s u r e of the relative quantity of i m m u n o r e a c t i v e D B H in the sample. The relationship of D B H enzymatic activity to i m m u n o r e a c t i v e D B H as m e a s u r e d by AD~0 values was relatively constant across the distribution of D B H e n z y m a t i c activity values in blood samples from r a n d o m l y selected subjects (Fig. 2). The coefficient o f variation o f the immunotitration p r o c e d u r e was estimated to be 10-14%. As mentioned earlier, one limitation of immunotitration p r o c e d u r e s is inability to m e a s u r e D B H protein in samples that lack enzymatic activity. For example, a p p r o x i m a t e l y 4% of individuals in a r a n d o m l y selected population sample are h o m o z y g o u s for the allele D B H ~" and have extremely low s e r u m D B H activity. 2'3'1~ T h e s e subjects have so little circulating e n z y m a t i c activity that the results of direct immunotitration of their serum would be unreliable. To o v e r c o m e this problem, im's R. M. Weinshilboum, in "Neurogenetics: Genetic Approaches to the Nervous System" (X. Breakefield, ed.), p. 257. Elsevier North-Holland, New York, 1979.

376

IMMUNOASSAY METHODS

[25]

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FiG. 2. Immunotitration of DBH in 38 samples of" human serum from randomly selected

subjects. The regression line of the ADsovalues against enzymatic DBH activity with 95 and 99% confidence limits is shown. Reprinted from Dunnette and Weinshilboum~° with the permission of the University of Chicago Press. munotitration was carried out by making 50 : 50 (v/v) mixtures of serum from these individuals and serum from subjects with high D B H enzymatic activity.l° The ratios o f e n z y m e activity to ADs0 values o f the mixtures were very similar to those of serum samples from randomly selected subjects, The values found for these mixtures fell well within the 95% confidence limits for the data shown in Fig. 2. Therefore, the blood o f individuals with genetically low serum enzymatic D B H activity does not contain a disproportionate level of immunoreactive DBH. Minor variations o f the D B H immunotitration procedure described here have been reported. In one case antibody to bovine adrenal D B H was used and D B H protein was estimated by extrapolation of the linear graphs o f titration curves to "equivalence points," the estimated points at which e n z y m e activity would have been completely titrated. ~ An excellent correlation o f estimated equivalence points with basal enzymatic activity for 50 randomly selected samples was reported (r = 0.903, p < 0.001). Unfortunately, as will be discussed subsequently, the use o f nonspecies specific antibody for RIA procedures has not resulted in values compara-

[25]

R I A AND IMMUNOTITRA'I'ION OF HUMAN D B H

377

ble to those found with anti-human D B H antibody. In another D B H immunotitration study using anti-human p h e o c h r o m o c y t o m a D B H antibodies, there was a correlation coefficient for enzymatic activity and estimated equivalence points o f 0.96 (n = 61, p < 0.001). 9 A difficulty with the use of equivalence point estimates, as can be seen in Fig. 1A, is that the nonlinear nature of the data can make such estimates difficult. An alternative approach that has been used in the immunotitration of enzyme activities is to keep the amount of antibody constant while varying the quantity of unknown sample. ~9 Although this approach has proven useful in other systems, it is not ideal for use in the measurement of immunoreactive serum D B H because of variation in the amount of endogenous D B H inhibitors as the quantity of serum is varied. Radioimmunoassay Procedure Immunoreactive D B H was measured with a double antibody technique. 7 All reagents were diluted with 1% BSA (Fraction V, Sigma) in 50 m M potassium phosphate buffer, pH 7.4. Reagents were added to disposable 12 × 75 mm polycarbonate tubes in the following order: (1) 0.5 ml D B H standard or sample to be assayed; (2) 100/zl rabbit anti-DBH diluted so that approximately 50% of the r'SI-labeled D B H would be bound in the absence of unlabeled D B H ; and (3) 100 tzl of 125Iqabeled D B H (0.25 ng). The reagents were mixed and were incubated at room temperature for 19-22 hr. At that time the following reagents were added: (4) 100 ~tl of normal rabbit serum diluted 1 : 19 (v/v) and (5) 100/zl of burro antiserum to rabbit y-globulin. The burro antiserum was added in moderate excess to result in the precipitation of all rabbit y-globulin. After an additional incubation of 4 hr at room temperature, the tubes were centrifuged at 2500 g for 10 min. The supernatant was decanted, the tubes were allowed to drain for 5 min, and the radioactivity that remained was measured in a gamma counter. A standard curve including tubes with from 0.156 to 40 ng o f D B H standard and a control sample with no unlabeled D B H were assayed each day. The amount of v'SI-labeled D B H precipitated in the presence of unknown samples was determined. The concentration of D B H in the unknown samples was calculated from a semilogarithmic transformation of the standard curve. Discussion of Radioimmunoassay A typical standard curve with superimposed data obtained by the dilution of a single human serum sample is shown in Fig. 3. 7 There was an ~" D. J. Reis, T. H. Joh, R. A. Ross, and V. M. Pickel, Brain Res. 81, 380 (1974).

378

IMMUNOASSAY METHODS

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FIG. 3. Competitive displacement of ~'~I-labeledDBH by unlabeled pheochromocytoma DBH (0) or by different dilutions of a single human serum sample (©). Reprinted from Dunnene and Weinshilboumr with the permission of the Rockefeller University Press. excellent correlation o f immunoreactive D B H measured by RIA with enzymatic serum D B H activity (Fig. 4). 7 The results shown in Figs. 3 and 4 were obtained with a single anti-human p h e o c h r o m o c y t o m a antibody. Similar results were obtained with an additional antibody against human p h e o c h r o m o c y t o m a D B H and with an antibody directed against human adrenal medullary DBH. r One example of the use o f this radioimmunoassay technique has been its application in studies of the biochemical genetic regulation o f human serum DBH. It has been shown that the genetic regulation o f immunoreactive D B H parallels the genetic regulation of D B H enzymatic activity, even in subjects with very low levels o f enzymatic activity who are homozygous for the allele D B H L . r'l° The possibility had been raised that these subjects might have " n o r m a l " levels of circulating D B H protein with decreased enzymatic activity. This has not

[25]

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Fl(;. 4. Comparison of enzymatic DBH activity with immunoreactive DBH (IDBH) measured by RIA in plasma samples from 134 randomly selected subjects, n - 134, r = 0.84, and p < 0.001. Reprinted from Dunnette and Weinshilboum 7 with the permission of the Rockefeller University Press.

proven to be the case. However, in the course of large population and family studies, one family was found in which 4 out of 8 first-degree relatives had abnormally high ratios of immunoreactive to enzymatic serum DBH activity, v This observation suggested the existence of a rare variant form of DBH with decreased enzymatic activity per molecule of enzyme protein. This observation was facilitated by the ability to assay many samples simultaneously and could not have been made without the ability to measure DBH protein. Other RIA procedures for the measurement of human serum DBH have been reported. In one study with anti-human pheochromocytoma antibodies and t2~I-labeled human pheochromocytoma antigen, a signifi-

380

IMMUNOASSAYMETHODS

[26]

cant correlation of DBH enzymatic activity with immunoreactive DBH was also found (r = 0.80, n = 60,p < 0.01). 4 The importance of speciesspecific antibody and antigen was emphasized by the experience of a group that initially reported a "complete lack of correlation" of DBH enzyme activity with immunoreactive serum DBH when antibody against bovine adrenal DBH and antigen purified from human adrenal glands was used. 5 The same group later reported an "excellent" correlation between enzymatic and immunoreactive serum DBH in 52 randomly selected serum samples (r = 0.98) with an RIA that used antibody against human adrenal DBH. 6 Conclusion There has been much interest in the assay of human serum DBH as a possible measure of the status or function of the sympathetic nervous system and the adrenal medulla. Two different approaches to the measurement of DBH protein, immunotitration and RIA, have shown a generally high degree of correlation between DBH enzyme activity and the measurement of immunoreactive protein as long as species specific DBH antibodies and protein are used in the immunological procedures. However, information beyond that obtained by enzyme activity determinations has been obtained in a few instances by measurement of immunoreactive DBH. The possibility exists that in selected pathological states or under certain physiological conditions the measurement of immunoreactive DBH protein may yield useful information beyond that provided by the measurement of enzymatic activity.

[26] A n t i b o d i e s to P r o l a c t i n R e c e p t o r s a n d G r o w t h H o r m o n e R e c e p t o r s la

By RICHARD G. DRAKE and HENRY G. FRIESEN Hormones elicit biological responses from target tissues by binding to specific, high-affinity, low-capacity cellular sites. Despite the plethora of publications in recent years describing the identification and characterization of polypeptide and steroid hormone receptor sites in numerous tissues from a multitude of species, attempts to address the problem of ~a T h i s r e s e a r c h w a s s u p p o r t e d b y g r a n t s f r o m M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a a n d United States Public Health Service HD07843-07.

METHODS IN ENZYMOLOGY, VOL. 74

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