Journal of Clinical InvestigationVol. 41, No. 2, 1962

STUDIES ONHUMANGROWTHHORMONE. I. A RADIO-IMMUNOASSAYFORHUMANGROWTHHORMONE*

By ROBERTD. UTIGER, MARYL. PARKERAND WILLIAM H. DAUGHADAY

(From the Metabolism Division, Department of Medicine, Washington University School ofMedicine, St. Louis, Mo.)

(Submitted for publication June 29, 1961; accepted October 5, 1961)

Since highly purified human pituitary growthhormone has become available, much informationconcerning its metabolic effects has been accumu-lated. However, simple and specific methods ofmeasuring endogenous levels of human growthhormone have been difficult to achieve. Attemptsto assay human growth hormone by injectingwhole serum into hypophysectomized rats andmeasuring the width of the tibial plate have gener-ally failed to show any activity in normal serum,although some acromegalic sera have been foundto contain stimulating activity (1-3). More re-cently, Gemzell (4) assayed extracts of plasmausing the tibial-line assay and found up to 12.3,ug growth hormone activity per ml of plasma inacromegalic subjects. Growth hormone-like ac-tivity was also found in plasma extracts fromcertain patients with diabetes, liver disease, andseveral other conditions; however, no activity wasdetected in extracts of normal plasma (5). Thevariability, insensitivity, and questionable speci-ficity of the tibial assay system have raised doubtsas to the significance of these results. In thislaboratory, measurement of the serum sulfationfactor has proved a useful indicator of growthhormone activity, but it is, nevertheless, not adirect measurement of growth hormone (6).

Immunologic methods for assaying humangrowth hormone have recently attracted much at-tention. Li, Moudgal and Papkoff (7) have beenable to detect as little as 2.5 ug of growth hor-

* Supported by Research Grant A-1526 from the Na-tional Institute of Arthritis and Metabolic Diseases, Na-tional Institutes of Health, and by an institutional grantto Washington University from the American CancerSociety. Clinical studies were carried out on theWashington University-Barnard Hospital Clinical Re-search Center, under support of Grant OG-12, Division ofGeneral Medical Sciences, National Institutes of Health.Presented in part before the Fifty-third Annual Meetingof the American Society for Clinical Investigation, At-lantic City, N. J., May, 1961.

mone by direct precipitation with specific anti-serum -and reported finding approximately 200m/Ag of growth hormone per ml in acromegalicserum. Greenspan, Cofell, Lew and Peng (8)have increased the sensitivity of the precipitin re-action by the use of radioiodinated antiserum tohuman growth hormone, so that as little as 1 jug ofgrowth hormone could be detected.

Read and Bryan (9) have utilized the principleof hemagglutination inhibition for the measure-ment of minute quantities of growth hormone. Intheir technique, as little as 2 to 20 mug of hor-mone can be dectected. Normal human serumhas been found to contain 100 to 600 mug per mlby this method, with appropriately higher andlower levels in acromegalic and hypopituitary sera,respectively. Hartog and Fraser (10), using thesame technique, have reported somewhat lowerlevels in normal individuals. The hemagglutina-tion inhibition technique has certain disadvantagesin that the manipulations are tedious and notadapted for assaying many samples simultane-ously. A number of extraneous factors preventsday-to-day reproducibility, and the evaluation ofthe complete hemagglutination pattern introducesa subjective judgment.

Isotopic techniques have been applied previouslyto the study of the reaction of protein hormoneswith specific antibodies. Frequently, at the con-centrations of reactants employed, immunopre-cipitation does not occur and special techniquesare necessary to separate the hormone-antibodycomplex from unreacted hormone. Yalow andBerson (11) have achieved this separation bychromatoelectrophoresis in their insulin assay.Another procedure has been immunologic precipi-tation of the hormone-antibody complex. Intheir study of sera of diabetic patients containingantibodies that react with heterologous insulin,Skom and Talmage (12) have precipitated in-sulin-I131 bound to human y-globulin by the ad-

254

HUMANGROWTHHORMONERADIO-IMMUNOASSAY

dition of antihuman y-globulin serum. Recently,both chromatoelectrophoresis and immunoprecipi-tation have proved successful in separating solu-ble glucagon-I'31-antibody complexes from un-reacted glucagon-I131 in a sensitive radio-immuno-assay (13).

In this paper, the radioiodination of humangrowth hormone and utilization of the labeledhormone for a highly sensitive radio-immunoassayare described. In this assay the soluble growthhormone-I131-antibody complex has been precipi-tated immunologically by the technique of Skomand Talmage.

MATERIALS AND METHODS

Human growth hormone (HGH). HGH was pre-pared from acetone-dried pituitaries by the Rabenmethod (14). The final product was lyophilized and keptat 50 C until use. Monkey growth hormone was sup-plied by Dr. A. E. Wilhelmi. The porcine and bovinehormones were obtained from Dr. Joseph Fisher, Ar-mour Laboratories, Chicago, Ill.

Stock solutions of HGHwere prepared in a concen-tration of 0.2 mg per ml in 0.01 M phosphate - 0.15 MNaCl buffer, pH 7.2, and kept at - 140 C until use.

Preparation of HGH-I'. HGHhas been iodinated ina number of ways. At first, iodination was carried outby a modification of the procedure used by Yalow andBerson (11) for iodinating insulin. In this method,which has been described in detail (15), iodide is oxi-dized to molecular iodine in acid solution with nitrousacid. The iodine is then extracted into chloroform andfrom the chloroform into alkaline solution containingHGH. Although useful preparations were frequentlyobtained, other preparations proved to be extensively de-natured, perhaps due to trace impurities in the chloroformor the initial oxidation mixture.

More recently, HGHhas been iodinated by a modifica-tion of the distillation procedure used by Staub, Springsand Elrick in the preparation of glucagon-I.3. (16).Their apparatus was modified by addition of a Y-jointwith Teflon stopcocks to allow refluxing and additionof reagents without disconnecting the reaction vessel(Figure 1). To carry out the iodination 10 to 20 mccarrier-free NaI.3. (Union Carbide Nuclear Corp., OakRidge, Tenn.), 1.5 ml 2 N H2SO4, and 2.5 ml H20 wereadded to the distilling flask, refluxed gently for 30 min-utes, and allowed to cool. Then 3.0 ml H20 and 0.5 to1.0 ml of a solution containing 0.48 mmole KI and 0.09mmole KIO3 per L were added (in recent iodinations,we have omitted the KI). The reaction mixture wasnext boiled gently until 4.0 ml distillate had accumulatedin the collecting tube. Under these conditions, 30 to 60per cent of the starting radioactivity was recovered inthe distillate. After the stopcocks were opened the distil-late was added dropwise to a: 10 X 40 mmtube containing

FIG. 1. DISTILLATION APPARATUS USED FOR HGHIODINATION.

0.2 to 1.0 mg HGHin 1.0 ml 0.2 M borate buffer, pH8.0, in an ice-bath. During the addition of I.3. and for 3minutes thereafter, the growth hormone was mixed witha small Teflon-covered magnet and a magnetic stirrer.The reaction mixture was next transferred to a cellophanebag, one end of which was tied to a rubber serum-bottlecap, and dialyzed for 2 hours against a continuous flowof 0.15 MNaCi at a rate of 14 L per hour. Bovine se-rum albumin (Nutritional Biochemicals, Cleveland, Ohio),sufficient to provide a final concentration of 5 per cent,was injected into the bag after 1 hour. After the sec-ond hour of continuous flow dialysis, the HGH-111 wasdialyzed against 2.5-L volumes of 0.15 M sodium chlo-ride for 16 to 20 hours. The prolonged dialysis wasnecessary to remove trichloroacetic acid (TCA)-solublefragments of HGH. The bovine serum albumin (BSA)was added to minimize radiation damage to the hor-mone (17) and adsorption of the labeled hormone toglassware (18).

Both iodination procedures have yielded HGH-I1'with calculated specific activities of up to 5 mc per mg.These preparations were over 97 per cent precipitable in7.5 per cent TCA and 75 to 85 per cent precipitable im-munologically. This discrepancy between TCA and im-mune precipitability has been attributed to the presence ofnondialyzable TCA-precipitable labeled protein fragmentswhich do not react with anti-HGH serum. Much of thisprobably represents partially degraded HGH, but thepresence of labeled protein contaminants of the RabenHGHhas not been completely excluded. After 2 to 3weeks' storage at 50 C, the concentration of TCA-solu-ble P"31 increased to 5 to 10 per cent. The calculatedI: GH molecular ratios have ranged from 0.25 to 2.0.

255

R. D. UTIGER, M. L. PARKERAND W. H. DAUGHADAY

P II

a I

a.._

0

a.Mo

60 -

120-

80

40_

1800 400 200 100 50

Reciprocal of Rabbit Anti-HGH Serum Dilution

FIG. 2. PRECIPITATION CURVE OBTAINED WITH CON-

STANT AMOUNTSOF GOAT ANTI-RGG SERUMAND VARY-ING CONCENTRATIONSOF RABBIT ANTI-HGH SERUM. Eachtube contained 300 1l anti-RGG diluted 1: 6, 200 Ald anti-HGHas shown, and 500 Al 1 per cent BSA in phos-phate-saline buffer. At dilutions of anti-HGH serum

greater than 1: 200, there is anti-RGG excess and maxi-mal precipitation of the anti-HGH serum.

Preparations made with higher I: GH ratios have beenfound less reactive with antibody.

The iodinated growth hormone was added to normalhuman serum and subjected to starch block electrophore-sis in a barbital buffer of pH 8.6, ionic strength 0.1, for18 hours at 250 v. After electrophoresis, imprints were

made and stained. Segments of 1 cm width were cut,protein was eluted with water, and aliquots of the eluatewere counted in a well-type scintillation counter. In thissystem nearly all radioactivity migrated as a single com-

ponent with the mobility of the a-globulins.Preparation of anti-HGH serum. The antigen was

prepared by emulsifying 2.5 mg HGH in 2.5 ml salineand 2.5 ml Ramon's (19) or Freund's (20) adjuvant.This mixture was given subcutaneously to New Zealandwhite rabbits at weekly intervals for 3 weeks. Twoweeks after the final injection the rabbits were bled bycardiac puncture. Because of the possibility of tracecontamination with other pituitary hormones or with se-

rum proteins which could serve as effective antigens, itis probably undesirable to give booster injections to thesame rabbit.

The double diffusion gel technique of Preer (21) was

used to test the specificity of our antihuman growthhormone immune sera. Antigens tested were our prepa-

ration of HGH, HGH-I1 3, human serum albumin(Squibb), human y-globulin (Squibb), and serum froma hypophysectomized patient. Using undiluted anti-HGHantiserum, a single precipitin zone was observed withHGH (10 jg per ml), HGH-1.31 (10 ptg per ml), andwith hypophysectomized human serum (diluted 1: 10).No precipitin zone was noted with albumin or y-globulin.

Preparation of antirabbit y-globulin serum (anti-RGGserum). Two injections of 40 mg of rabbit y-globulin(Pentex, Inc., Kankakee, Ill.) in 8 ml of adjuvant were

given subcutaneously to adult female goats 6 weeks apart,and 2 months later the animals were bled by jugularpuncture.

The goat anti-RGG serum was fractionated by pre-

cipitation at 1.4 M (NH4)2SO4, suspension of the pre-

cipitin in water, and dialysis against buffered saline, un-

til the dialysant was free of sulfate. All antisera were

stored at - 140 C until use and then dilutions of theantisera were prepared with 0.01 M phosphate-0.15 MNaCl buffer, pH 7.2.

Titration of goat anti-RGG. A constant volume ofgoat anti-RGG serum and varying amounts of the rab-bit anti-HGH serum were allowed to interreact for 20 to24 hours at 40 C. After this period, the precipitates were

separated by centrifugation, washed twice with phos-phate-saline buffer and then dissolved in 0.25 M aceticacid. The protein concentration was determined bymeasuring the absorbance in the Beckman spectrophotom-eter at 278 my (22). On the basis of such a titration(Figure 2), the amount of anti-HGH serum maximallyprecipitated by a given amount of anti-RGG was foundand the zone of antibody (i.e., anti-RGG) excess was

defined.To confirm the results of the precipitin titration, a

second titration was performed employing constantamounts of HGH-131, rabbit anti-HGH serum, and varyingamounts of goat anti-RGG serum. After incubationand centrifugation, aliquots of the supernatant solutionwere counted. A parallel titration was run for com-

parison, using 1 per cent human -y-globulin as the dil-uent to evaluate the effect of any cross reaction of thegoat antisera and human -y-globulin.

Titration of HGH-I"1' preparations and anti-HGHsera. The antigenic reactivity of different HGH-I.3.preparations and the potency of various rabbit sera were

determined by the following procedure. A series of tubeswas prepared in duplicate with constant amounts of anti-HGH serum and variable amounts of HGH-P"31. Alltubes contained at least 0.5 per cent BSA (final concen-

tration) in order to minimize adsorption of radioactivityin glassware. After 2 hours, the anti-RGG serum inexcess was added. Eighteen hours later, the per cent of

v

w 1004-

a. 75

_Q20

80 500

-6O 25

%I-0

IF

5 10 15 20 25

HGH-I131 meg. per Tube (Calculated )

FIG. 3. TITRATION OF AN ANTI-HGH IMMUNE SE-

RUMDILUTED 1: 1,000 WITH INCREASING CONCENTRATIONS

OF HGH-1I31.

256

onnZI

HUMANGROWTHHORMONERADIO-IMMUNOASSAY

-A.

l .i ... , , 4- C

a> 4000

.> 3000

.0

cr

B.

2004

10 40 70 100 r

Unlobelled HGH - nryg / Tube200 10 100

Unlabelled HGH- ming / Tube

FIG. 4. STANDARDASSAY CURVES SHOWINGTHE RELATIONSHIP BETWEEN

UNLABELEDHGHAND THE TOTAL SUPERNATANTRADIOACTIVITY WITH HGHCONCENTRATIONPLOTTED LINEARLY (A) AND LOGARITHMICALLY (B).

total radioactivity precipitated was determined (Figure3) by counting the entire tube and an aliquot of thesupernatant solution. At the concentrations of rabbitserum and anti-RGG serum employed, 48 hours' incuba-tion failed to increase the amount of radioactivity pre-

cipitated over that obtained at 18 hours. At dilutions ofantibody used in the assay, 65 to 70 per cent of the HGH-I131 preparations was precipitated. Omission of anti-HGHor anti-RGG serum from the complete system, or sub-stitution of nonimmune rabbit serum for anti-HGH re-

sulted in precipitation of less than 5 per cent of the totalradioactivity.

Standard curves and assay of plasma or extracts. Theimmunoassay depends on ability of unlabeled HGH toinhibit competitively the binding of HGH-1.3. by anti-HGHserum. The concentration of HGHin unknownsolutions is determined from a standard curve obtainedwith known concentrations of HGH.

The following volumes and conditions of reaction haveproved most satisfactory for HGH assay. Duplicatetubes were prepared containing: 1) 100 jd of HGH-13'(1 to 5 mtg, having a radioactivity of about 5,000 cpm)in 1 per cent BSA in phosphate-saline buffer. 2) 400 1.lof a solution of 1 per cent BSA in phosphate-saline buf-fer containing known amounts of HGHor the sample tobe assayed. In each run a blank tube without HGHand6 to 9 tubes containing from 3 to 400 m/ig HGHwere

used. 3) 200pul of anti-HGH serum diluted in phosphate-saline buffer on the basis of prior titration with theHGH-I131 preparation in use. The dilution of anti-HGHserum used in the assay ranged from 1 to 600 to 1 to3,200. When dilutions greater than 1 to 1,200 were used,normal rabbit serum, 1 to 1,200, was used as the diluent.After 2 hours at 4° C: 4) 300 ,gl of anti-RGG serum di-luted in phosphate-saline buffer on basis of prior titrationwas added.

The tubes were shaken and allowed to stand at 50 Cfor 18 hours. The precipitates were then separated bycentrifugation and 400-jul aliquots of the supernatewere transferred to plastic tubes for determination of

radioactivity in a well-type scintillation detector.Extraction of HGHfrom plasma. Heparinized plasma

(serum was equally satisfactory) was extracted by themethod of Gemzell (4). Two to 4 ml of plasma was

precipitated with an equal volume of 10 per cent TCA.The precipitate was extracted with 3 vol absolute ethanoland the alcohol extract adjusted to pH 6.1 with 0.1 MNaOH. The resulting precipitate was collected by cen-

trifugation, suspended in water, dialyzed for 18 hours,then lyophilized. All operations were done at 5° C.After lyophilization, the extracts were kept at 5° C un-

til assayed, at which time they were dissolved in phos-phate-saline buffer to the original volume. With eachseries of extractions, the recovery of 25 to 500 mgg HGHfrom plasma was determined so that correction for lossescould be made. Recovery of the added HGHhas rangedfrom 40 to 98 per cent and has generally been about75 per cent. Results given below have all been correctedto 100 per cent recovery. Reproducibility of recovery

of HGHadded to a series of plasma samples simultane-ously extracted and assayed has varied 12 per cent.Some of the acromegalic plasma used has been kept at- 140 C for up to 3 years and still contained measurableactivity.

RESULTS

Effects of HGHon HGH-I131 immunoprecipi-tation. The addition of increasing amounts ofHGHto the assay system led to a progressiverise in the concentration of HGH-I'131 which hadnot been immunologically precipitated (super-natant HGH-I131) (Figure 4A). In most assays

as little as 3 to 6 mug of HGHcaused a sig-nificant rise in supernatant radioactivity. Al-though several methods of plotting the assay re-

sults have been examined, the most satisfactory was

a simple plot of the supernatant radioactivity as a

function of the logarithm of the added HGHcon-

centration (Figure 4B). With this type of anal-ysis a straight line function was calculated by the

4000

3200

2400

1600

1000

257

cTo

F

:3ac

R. D. UTIGER, M. L. PARKERANDW. H. DAUGHADAY

method of least squares over the workable portionof the assay curve. If the values at the lower endof this curve fell significantly above this line, anexcess of anti-HGH in the system was suspected.The standard curve was used to calculate theHGHconcentration of unknown solutions.

Studies with heterologous growth hormones.The ability of pituitary growth hormones fromnonhuman sources to inhibit the reaction ofHGH-I31 with anti-HGH has been examined.Purified porcine and bovine growth hormone, evenwhen added in large amounts, produced virtuallyno inhibition (Figure 5). In contrast, monkeygrowth hormone did inhibit the precipitation ofHGH-I131. The slope of the lines relating thelogarithms of growth hormone concentration tosupernatant radioactivity for both primate growthhormones is not significantly different (p > 0.5).The monkey growth hormone preparation testedwas found 40 per cent as effective by weight asthe HGHstandard preparation in competing withHGH-I131 for binding with anti-HGH serum.

Observations on unfractionated plasma. In thepresent assay system, unfractionated plasma inter-fered with the precipitation of HGH-I131 by its anti-body in a nonspecific manner. Evidence for thisview is provided by a series of observations onhuman plasma samples in which there was no

E

ua 3000

>I-

CL)4 20000

a

4

I-z

1000z

(I)

STANDARDCURVE

)F Bovine Growth oAbmoneAll A A

Porcine Growth Hormone

10 100 1000UNLABELLEDGROWTHHORMONE-rug/Tube

FIG. 5. A COMPARISONOF THE EFFECTS OF HUMAN,

MONKEY,BOVINE, ANDPORCINEGROWTHHORMONESONTHE

IMMUNOPRECIPITATION OF HGH-811. The assay withbovine and porcine growth hormones was performed ata different time from that with monkey growth hormone,but the standard curves with HGHof the two assays

were comparable.

PoUnextroctedPlasma

E-Plasma Extract(Gemzell)

ANIMAL HUMANBeef

SheepPig

FIG. 6. REMOVAL OF INHIBITOR OF HGH RADIO-IM-

MUNOASSAY: ASSAY RESULTS WITH UNEXTRACTED (P)AND EXTRACTED (E) PLASMA. The results on humanplasma include samples from normal, acromegalic, andhypopituitary subjects.

relation of the apparent HGHconcentration andthe state of pituitary function of the patient (Fig-ure 6). In addition, ovine, porcine, and bovinesera inhibited the precipitation of HGH-I131 to amajor extent (Figure 6), making it appear thatthese sera contained HGH.

Numbers of observations have been made todefine the nonspecific inhibitor of HGH-I131 im-munoprecipitation in unextracted plasma. Theinhibitor is not inactivated by heating to 60° C for60 minutes or by prolonged dialysis against wateror phosphate-saline buffer. Treating the plasmawith 0.01 Mperiodate according to the method ofBurnet and Lind (23), a technique which removesthe mucoprotein inhibitor of influenza hemagglu-tination, failed to remove the nonspecific inhibitorof HGH-I3' precipitation.

The possibility has been considered that they-globulin of the test samples might cross reactwith the antirabbit y-globulin immune serum, sothat there would be insufficient goat antisera toprecipitate all the rabbit y-globulin present. Al-though cross reactivity between human y-globulinand goat anti-RGG serum does exist, as demon-strated by a precipitin zone in a double diffusionagar system, human y-globulin, when added tocontrol assay tubes in amounts comparable to thatin undiluted serum, failed to inhibit HGH-Il'3precipitation. Furthermore, neither increasing theamount of anti-RGG serum as much as fourfoldnor diluting the test serum as much as tenfoldabolished the nonspecific inhibitor.

258

HUMANGROWTHHORMONERADIO-IMMUNOASSAY

There is clear evidence that albumin does notinterfere with the immunoreactions used for HGHassay, in that bovine albumin solutions are used asdiluents throughout the procedure and the Gem-zell extraction procedure yields a product whichis almost exclusively composed of albumin.

The possibility that the nonspecific inhibitordegrades HGH-I'31 is made unlikely by two ob-servations. First, at the completion of the im-munologic reactions no increase in TCA-solubleradioactivity could be demonstrated in tubes con-taining serum showing nonspecific inhibition.Second, when HGHwas added to unextractedserum, the increment could be measured almostquantitatively by radio-immunoassay.

Extraction of HGHfrom plasma. The findingthat plasma from human beings and other animalspecies contained a nonspecific inhibitor of theradio-immunoassay prompted an examination ofseveral methods of extracting HGHfrom plasma.Fortunately, the procedure of Gemzell (4) com-pletely excluded the nonspecific inhibitor of ovine,bovine, and porcine sera (Figure 6). When thismethod, using unconcentrated extracts, was appliedto 30 plasma samples from normal adults, HGHactivity was detected in 12. All plasma ex-tracts contained less than 50 mLg of HGHevenwhen corrected for losses incurred in the extrac-tion procedure (Figure 7). Extracts from plasmaof 22 untreated acromegalic patients had from 15to 260 mjug per ml, with a mean of 112 mug perml. Included also in Figure 7 are the results ofassays on plasma extracts from a few acromegalicpatients after completion of radiation or surgicaltherapy, whose disease was considered clinicallyinactive. At the present level of sensitivity littleor no HGHwas found in the plasma of hypo-pituitary patients. An exception, however, wasone adult patient who had had a craniotomy forcraniopharyngioma and who had a serum HGHassay of 32 m~ug per ml.

DISCUSSION

The iodination of growth hormone has pres-ented special problems. With the low concentra-tion of hormone protein employed and the highspecific activities required for millimicrogramsensitivity of the assay, instability of the labeledhormone has frequently been encountered with

direct methods of iodination. It is likely thattrace impurities in the original iodine solution orother reagents led to difficulty in using the meth-ods of Yalow and Berson (11). Most of thesedifficulties are obviated by the distillation tech-nique. It has been observed that much of thedegraded hormone exists as relatively small frag-ments which can be removed by prolonged dialy-sis. For this reason, the dialysis following iodina-tion has been continued for 20 to 24 hours. Sincethe labeled hormone has been used solely as ananalytical reagent, we have made no attempt todetermine its biological activity in experimentalanimals, but have been content with the demon-stration of its reactivity with specific antibody.It is likely that further refinements of existingmethods will permit preparation of labeled hor-mone of higher specific activities.

The sensitivity of the assay system is dependentnot only on the specific activity of the labeledgrowth hormone, but also on the careful adjust-ment of the concentrations of rabbit anti-HGH andgoat anti-RGG sera. A slight excess of HGH-I131in respect to anti-HGH must be in the systembefore added HGHwill effectively compete forthe limited number of specific binding sites onthe anti-HGH serum. The limit of sensitivity ofthe assay to date is about 3 mpg. When labeledHGHof higher specific activity becomes available

Treated

RESULTS OF ASSAYS OF EXTRACTS OF HUMANPLASMA.

259

R. D. UTIGER, M. L. PARKERAND W. H. DAUGHADAY

and improved methods of counting are devised,greater sensitivities should be obtainable. Theanti-RGG must be in excess in respect to the anti-HGHto insure precipitation of all rabbit anti-bodies and, therefore, of all bound HGH-I'1 .

The use of immunoprecipitation rather thanchromatoelectrophoresis to separate free frombound HGH-I'31 offers certain technical advan-tages. Large numbers of samples can be assayedsimultaneously with a series of tubes containingstandard HGHdilutions, and the free HGH-I131is easily sampled for counting after centrifugation.However, the major obstacle to the applicationof radio-immunoassay to the measurement ofHGH in plasma has been the existence of thenonspecific inhibitor. It is recognized that theextraction procedure used is not a completelysatisfactory solution to this problem because itintroduces a relatively tedious operation and issubject to significant and variable losses. Moreinformation concerning the properties of the non-specific inhibitor is needed before a simpler pro-cedure for its removal can be designed.

The antigenic specificity of HGH has beenstudied and extensively described by previousworkers (7, 9, 24, 25). Our observations withradio-immunoprecipitation confirm the informa-tion obtained with more orthodox immunologicprocedures showing that antisera induced in rab-bits by HGH reacts only with other primategrowth hormones and not with growth hormonesof other species. Antisera produced by Rabenhuman growth hormone may contain traceamounts of antibodies reacting with serum pro-teins (6, 9, 26, 27). Our results confirm thisgeneral experience. A faint precipitin zone wasobserved when serum from a hypophysectomizedpatient was tested against undiluted antiserum inthe highly sensitive double diffusion technique.It is doubtful whether the presence of contaminat-ing antibodies in our antisera participated sig-nificantly in our assay system, because the concen-trations of non-growth hormone antigenic proteinswhich might be iodinated in our preparations ofgrowth hormone is probably very low. Only asingle precipitin zone was evident in the reactionbetween growth hormone and antisera. Also, inboth starch and paper electrophoretic studies theradioactivity of our labeled growth hormone mi-grated as a single component with a mobility of

a-globulin. Trace contamination of the humangrowth hormone used for iodination would lead tosignificant error in our assay only if selectiveheavy labeling of the contaminants in preferenceto the hormone occurred.

The levels of circulating HGHfound in normalindividuals by this method have been from 0 to50 m1ug per ml. Further improvement in thesensitivity of the assay and concentration of ex-tracts will be required before the normal level ofhormone can be accurately measured. Neverthe-less, it is clear that the concentrations of HGHdetected by this method are less than one-fourththose reported by Read and Bryan (9). Thereasons for this discrepancy are unknown. Thepossibility exists that nonspecific inhibition mayoperate in the hemagglutination inhibition systemand contribute to difficulties with its use (10, 28).The current lack of accord of insulin assays maybe due in part to differences in the physical stateof insulin in the blood (29). Perhaps the di-vergent results obtained with growth hormoneassays may be due to the measurement of differentcomponents of the total growth hormone activityof plasma.

SUMMARY

Methods for the radioiodination of humangrowth hormone which yield labeled hormoneof both high specific radioactivity and reactivitywith specific antibodies are described. With thesepreparations a radio-immunoassay for humangrowth hormone, capable of detecting 3 to 6 m/xghuman growth hormone, has been developed.

Bovine, ovine, and porcine growth hormone didnot prevent the immunoprecipitation of iodinatedhuman growth hormone in this assay, but monkeygrowth hormone inhibited iodinated humangrowth hormone precipitation.

Human and animal sera were found to containa nonspecific inhibitor of the immunoassay sys-tem which can be excluded by an extraction pro-cedure that permits the extraction of growthhormone.

Circulating levels of growth hormone deter-mined by radio-immunoassay in normal adultsranged from 0 to 50 m~ug per ml. Levels of 15 to250 m~ug per ml were found in a series of un-treated acromegalic subjects while values aftertreatment ranged from 0 to 27 m/Ag per ml.

260

HUMANGROWTHHORMONERADIO-IMMUNOASSAY

ACKNOWLEDGMENT

The authors wish to thank Dr. Joseph D. Fisher ofArmour Laboratories for his contributions to thisproj ect.

REFERENCES

1. Kinsell, L. W., Michaels, G. D., Li, C. H., and Lar-sen, W. E. Studies in growth. I. Interrelationshipbetween pituitary growth factor and growth-pro-moting androgens in acromegaly and gigantism;quantitative evaluation of bone and soft tissuegrowth in acromegaly and gigantism. J. clin. En-docr. 1948, 8, 1013.

2. Segaloff, A., Komrad, E. L., Flores, A., Segaloff,A., and Hardesty, M. The growth hormone con-

tent of human plasma. Endocrinology 1955, 57,527.

3. Gemzell, C. A., Heijkenskj6ld, F., and Str6m, L.A method for demonstrating growth hormone ac-

tivity in human plasma. J. clin. Endocr. 1955, 15,537.

4. Gemzell, C. A. Demonstration of growth hormonein human plasma. J. clin. Endocr. 1959, 19, 1049.

5. Forsman, O., and Gemzell, C. A. Plasma levels ofgrowth hormone in patients with diabetes mellitus,hypercholesterolaemia and liver diseases. Actaendocr. (Kbh.) 1959, 32, 480.

6. Daughaday, W. H., Salmon, W. D., Jr., and Alex-ander, F. Sulfation factor activity of sera frompatients with pituitary disorders. J. clin. Endocr.1959, 19, 743.

7. Li, C. H., Moudgal, N. R., and Papkoff, H. Im-munochemical investigations of human pituitarygrowth hormone. J. biol. Chem. 1960, 235, 1038.

8. Greenspan, F. S., Cofell, J. C., Lew, W., and Peng,C. T. Assay of human growth hormone with I"3labeled antibody. Clin. Res. 1961, 9, 99.

9. Read, C. H., and Bryan, G. T. The immunologicalassay of human growth hormone. Recent Progr.Hormone Res. 1960, 16, 187.

10. Hartog, M., and Fraser, R. The immunological as-

say of growth hormone in human serum. J. En-docr. 1961, 22, 101.

11. Yalow, R. S., and Berson, S. A. Immunoassay ofendogenous plasma insulin in man. J. clin. In-vest. 1960, 39, 1157.

12. Skom, J. S., and Talmage, D. W. Nonprecipitatinginsulin antibodies. J. clin. Invest. 1958, 37, 783.

13. Unger, R. H., Eisentraut, A. M., McCall, M. S.,Keller, S., Lanz, H. C., and Madison, L. L. Glu-cagon antibodies and their use for immunoassayfor glucagon. Proc. Soc. exp. Biol. (N. Y.) 1959,102, 621.

14. Raben, M. S. Preparation of growth hormone frompituitaries of man and monkey. Science 1957, 125,883.

15. Salmon, S., Utiger, R., Parker, M., and Reichlin, S.The fate of I`~-labeled human growth hormonein the rabbit. In preparation.

16. Staub, A., Springs, V., and Elrick, H. An improvedtechnique for labelling proteins with I31. Int. J.appl. Radiat. 1957, 2, 59.

17. Berson, S. A., and Yalow, R. S. Radiochemical andradiobiological alterations of P'31-labeled proteinsin solution. Ann. N. Y. Acad. Sci. 1957, 70, 56.

18. Hill, J. B. The adsorption of I13. insulin to glass.Endocrinology 1959, 65, 515.

19. Ramon, G., Lemetayer, E., and Richou, R. Del'influence de diverses substances ajoutes 'a 'anti-gene anatoxique dans la production de l'immuniteantitoxique. Rev. Immunol. 1935, 1, 199.

20. Freund, J., Lipton, M. M., and Thompson, G. E.Aspermatogenesis in the guinea pig induced bytesticular tissue and adjuvants. J. exp. Med. 1953,97, 711.

21. Preer, J. R., Jr. A quantitative study of a tech-nique of double diffusion in agar. J. Immunol.1956, 77, 52.

22. Gitlin, D. Use of ultraviolet absorption spectroscopyin the quantitative precipitin reaction. J. Immunol.1949, 62, 437.

23. Burnet, F. M. and Lind, P. E. Recombination of in-fluenza viruses in the de-embryonated egg. Aust.J. exp. Biol. med. Sci. 1954, 32, 145.

24. Fishman, J., McGarry, E. E., and Beck, J. C. Stud-ies using anterior pituitary hormones as antigens.Proc. Soc. exp. Biol. (N.Y.) 1959, 102, 446.

25. Hayashida, T., and Li, C. H. A comparative im-munological study of pituitary growth hormonefrom various species. Endocrinology 1959, 65, 944.

26. Grumbach, M. M., Kaplan, S. L., and Solomon, S.Immunochemical observations on antisera to puri-fied human growth hormone. Nature (Lond.)1960, 185, 170.

27. Boucher, B. J. Immunoelectrophoresis of humansera with antiserum to Raben growth hormone.Nature (Lond.) 1960, 188, 1025.

28. McGarry, E. E., Ballantyne, A., and Beck, J. C.Effect of human serum on the reaction betweenhormones and their specific antisera (abstract no.159). Endocrine Society, 43rd Annual Meeting,New York, 1961.

29. Antoniades, H. N. Studies on the state of insulin inblood: The state and transport of insulin in blood.Endocrinology 1961, 68, 7.

261