gratefully acknowledged. THE IMMUNOLOGICAL ASSAY OF GROWTH HORMONE IN HUMAN SERUM Department of Medicine, Postgraduate Medical School,

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1 THE IMMUNOLOGICAL ASSAY OF GROWTH HORMONE IN HUMAN SERUM M. HARTOG and RUSSELL FRASER Department of Medicine, Postgraduate Medical School, London, W.12 (Amended manuscript received 7 October 1960) SUMMARY An immunological method for the assay of growth hormone in human serum is described. The method, based on that of Read, has a reproducibility of 20%; the normal mean level for adults was found to be 172 \g=m\g./l.,and the range of two standard deviations was \g=m\g.l.serum. The assay has shown raised levels in seven out of ten acromegalic patients and lowered ones in four out of seven hypopituitary patients. INTRODUCTION The standard biological methods for the assay of growth hormone (GH) are insufficiently sensitive for application to body fluids. The most popular methods are based either on the increase in width of the epiphysial cartilage of the tibia, or on the increase of body weight produced in the hypophysectomized rat by the administration of GH (Greenspan, Li, Simpson & Evans, 1950). The tibial epiphysis method has been applied by Gemzell (1959) to the assay of GH in human serum, but he was able to detect only raised levels such as those found in acromegalie patients. Recently Salmon & Daughaday (1957) and Daughaday, Salmon & Alexander (1959) have described a method for the assay of a ' sulphation factor ' in serum, and the results of this assay appear to be correlated with the expected level of circulating GH. The method measures the in vitro uptake of sulphate by cartilage from hypophysecto mized rats, and while it is one of great promise, further studies will have to be done to determine its exact specificity. Since GH is a protein, an assay procedure could be based upon the use of an anti body prepared, in animals, in response to GH used as an antigen. Read (1960) and his collaborators (Read & Stone, 1958; Read & Bryan, 1960) have already announced the successful application of an immunological method to the assay of GH in human serum. We have used a very similar procedure and, in this paper, we describe our results which confirm the practicability of such a method. The human growth hormone (HGH) has been prepared by Prof. Young and his colleagues in the Depart ment of Biochemistry, Cambridge University, from pituitaries supplied to the Clinical Endocrinology Committee of the Medical Research Council by the kind permission of many pathologists throughout the United Kingdom, whose help is gratefully acknowledged. * Saltwell Research Scholar, Royal College of Physicians. 7-2

2 102 M. Hartog and Russell Fraser PRELIMINARY STUDIES This work was begun in 1958 in collaboration with Dr J. H. Humphrey of the National Institute for Medical Research, Mill Hill, who immunized two rabbits with a preparation of HGH kindly given to us by Dr Ottoway. On testing the antiserum obtained against HGH on an Ouchterlony plate (Ouchterlony, 1949), the antiserum was found to contain sufficient antibody to produce lines of precipitation. Instead of a single line which would have been expected if the original preparation of HGH had contained only one antigen, three lines of precipitation were produced indicating that the HGH had contained more than one antigen. On testing the antiserum against normal human serum several lines were formed. One of the impurities was identified as y-globulin and, after adsorption of the antiserum with small amounts of serum from a hypophysectomized patient, all the lines previously seen against human serum were removed, and only one or possibly two lines against the HGH prepara tions remained. When Read's reports (Read, 1959, 1960; Read & Bryan, 1960) suggested that HGH prepared by the Raben technique (Raben, 1957) was purer, HGH was made by this method in Prof. Young's Department, and this has been used for all our subsequent work which is described below. When antiserum obtained by immunizing rabbits with this material was tested against the antigen on an Ouchterlony plate, only one line of precipitation was visible.. j.. j METHODS Immunization procedure Two rabbits were immunized with three weekly intramuscular injections of 2 mg. HGH together with Freund's adjuvant (Freund, 1951). They were both sub sequently given two intravenous injections of 1 mg. HGH in suspension adsorbed on alumina, making a total of 8 mg. for each rabbit, and were bled from an ear vein 5 days later. One of the two rabbits produced satisfactory levels of antibody in the serum; this animal, after an interval of 6 weeks, was given a further 2 mg. HGH in suspension adsorbed on alumina in three separate intravenous injections, and then bled again 5 days after its final injection. The assay (a) Tanned red cell test. Sheep red blood cells were treated with fannie acid and coated with HGH, using the technique described by Boyden (1951). A solution containing 1 mg. HGH in 10 ml. phosphate buffered saline, ph 6-4, was employed and it was found possible to use the same solution about five times. The exact titre of the antiserum was determined. With the successfully immunized rabbit, the titre of the serum obtained from the first bleeding was approx. 1/4000 and that of the serum from the second bleeding approx. 1/14,000. These titres are variable and have to be determined after each coating. The assay depends upon the inhibition of this agglutination. (b) Inhibition procedure. The assay is performed by comparing the inhibition produced by the sera to be assayed with that produced by of HGH. To each tube are added 0-1 ml. of diluted antiserum (see below), a set of standard solutions 0 1 ml. of

3 Growth hormone in human serum 103 inhibiting fluid (i.e. standard HGH or unknown serum), and then 0-02 ml. of red cell suspension (Fig. 1). The antiserum is diluted in a fluid consisting of a 1/80 solution of horse serum in saline, and the dilution used depends on its measured titre. We have found it convenient to use a dilution of antiserum of about two-thirds of the titre (i.e. 1/4000 for a titre of 1/6000), which will detect amounts of GH down to 6-10 µg. \. The standard HGH solutions routinely used are 20, 15, 12, 10, 8, 6, 4, 2 /xg./l. in the same diluting fluid as is used for antiserum and for the dilutions of serum. The series of dilutions of each serum vary, to a certain extent, with the value expected for the serum, but usually include 1/6, 1/8, 1/10, 1/12, 1/15, 1/20, 1/25, 1/30, 1/40, 1/50. The tubes are left at room temperature and read after 3 hr. The end point is read as the first tube in which there is complete agglutination (i.e. no inhibition). The tubes are set up in triplicate to check the consistency of the end points, which sometimes may Fig. 1. Assay of human growth hormones. be difficult to read accurately. In about 25 % of the experiments the control tube showed the assay to be unsatisfactory, and no result was recorded. The antiserum and the sera to be assayed are heated at 56 C for 30 min. in order to inactivate complement, and are adsorbed with an equal volume of washed sheep red cells. RESULTS We have assayed sera from eighteen normal adult subjects, ten patients with acromegaly and seven who had been hypophysectomized. With the exception of that of one acromegalie, each serum was assayed, on different days, at least twice, and many were done several times. The normal subjects comprised ten healthy volunteers and eight patients convalescing from non-endocrinological conditions. The other patients studied were chosen as unequivocal examples of either acromegaly or hypopituitarism. The acromegaly was considered to be active by the clinical criteria of symptoms of enlargement of the extremities, change in facial appearance, headache and paraesthesiae, as well as by findings of insulin resistance and sometimes overt or latent diabetes. The hypopituitary patients were all suffering from metastatic breast carcinoma; three had had their pituitaries removed by surgeons with special 7-3

4 104 M. Hartog and Russell Fraser experience of the operation, and in four the pituitaries had been ablated by implants of 90Yt. All the Yt-implanted patients had signs of impaired pituitary function as shown by dependence on cortisone, abnormal water diuresis tests and lowered uptakes of 131I. The completeness of hypophysectomy may have varied in these patients. We have no results of tests of endocrine function in the surgically hypophys ectomized patients. Table 1. Concentration of growth hormone in sera of eighteen normal adult subjects (µ^.ß. serum) Mean + s.d.* (24%) Range Í2 s-*>* \ Individual estimations * Using mean value of all estimations carried out on each serum. Acromegalie (10) Normal Hypophysectomy (7) Normal range (2S.D.'s) S.D. of estimation Fig. 2. Results of growth hormone assays in serum. The results for the normal sera give a mean value of 172 µg. \. normal serum, and a normal range, on the basis of two standard deviations, of µg. \. serum (see Table 1). Our results in the three groups of subjects are set out in Fig. 2. This shows that seven out of ten acromegalie patients had raised values ( µg. \.), and that four out of seven hypophysectomized patients were found to have subnormal values (45-76 jug./l); the results in the remaining six patients were within the normal range. The variation of serial assays on each serum indicates a reproducibility of 20 % for duplicate estimations (Table 2), which is possibly lower for the higher levels of acro megalie sera. The results of three recovery experiments are shown in Table 3 and vary between 106 and 139%; this corresponds approximately to the estimates of the accuracy of the procedure given above.

5 Growth hormone in human serum 105 Serum from Table 2. Normal subjects Acromegalie subjects Hypophysectomized subjects Accuracy of growth hormone assay No. of subjects* 18 No. of esti mations Mean s.d. of single estimation («) * Single serum from each subject. (%) S.E. of duplicate estimations (%) Table 3. Initial assay («? /! ) 133 ( + 7-1%) ( %) Recovery of growth hormone added to serum Expected = = = 246 Final assay (pg.ß.) Observed 415 (= 139%) 270 (= 106%) 302 (= 123%) DISCUSSION The validity of this immunological method for the assay of growth hormone (GH) depends upon whether the antibody produced was pure and specific for GH, i.e. neither wholly nor partially a response to a contaminant, nor showing affinity for any protein other than HGH. The Raben preparation of HGH used as the basis of the assay has chemical properties suggesting that it is a pure protein (Raben, 1957), and has been found to have the metabolic actions of GH in man (Clinical Endocrinology Committee M.R.C., personal communication); thus HGH was the likely antigen. The antiserum prepared was found to give only one line on the Ouchterlony plate against the HGH and therefore, by this relatively insensitive test, the antiserum appears to be pure. More extensive investigations of an antiserum prepared in response to Raben HGH have been done by Grumbach, Kaplan & Solomon (1960). Their investigations comprised agar gel diffusion studies, immuno-electrophoresis, and quantitative precipitation, all of which indicated a pure antibody. Other workers have shown that an antiserum to HGH will inhibit the widening, by GH, of the tibial epiphysial cartilage of hypophysectomized rats (Hayashida & Li, 1958; Read, 1960; Read & Bryan, 1960). Furthermore, the clinical correlations of both Read's (1960), Read & Bryan's (1960) and our data provide circumstantial evidence that GH is being measured, as we have usually been able to detect raised levels in the serum in conditions in which it was expected to be raised, e.g. in acromegalics, and lowered levels in hypophysectomized patients. Our data suggest that the method can be performed with a reproducibility of 20 % when the assays are done in duplicate; indeed, much of the spread of the normal range from the mean might depend merely on the error of the method. As already noted, seven out of ten of the acromegalie subjects had raised serum GH values, and four out of seven of the hypophysectomized patients had lowered values, whilst three acromegalics and three hypophysectomized patients (two who had been surgically hypophysectomized and one who had had90yt), had values falling

6 106 M. Hartog and Russell Fraser within the normal range. From the clinical state of the patients it seems to us likely that these normal values represent considerable errors of estimation in sera that were only slightly abnormal. We wish to thank Prof. F. G. Young and his colleagues of the Department of Biochemistry, Cambridge University, for supplying the growth hormone, and Dr J. H. Humphrey of the National Institute for Medical Research, Mill Hill, for his active support in the early stages of the work, and for his constant advice and encouragement since then. One of us (M.H.) is in receipt of a grant from the Royal College of Physicians, for which he would like to take this opportunity of expressing his thanks. REFERENCES Boyden, S. V. (1951). J. exp. Med. 93, 107. Daughaday, W. H Salmon, W. D. & Alexander, F. (1959). J. clin. Endocrin. 19, 743. Freund, J. (1951). Amer. J. clin. Path. 21, 645. Gemzell, C. A. (1959). J. din. Endocrin. 19, Greenspan, F. S., Li, C. H., Simpson, M. E. & Evans, H. M. (1950). In Hormone assay, p Ed. C. W. Emmens. New York: Academic Press. Grumbach, M. M., Kaplan, S. L. & Solomon, S. (1960). Nature, Lond., 185, 172. Hayashida, T. & Li, C. H. (1958). Science, 128, Ouchterlony, O. (1949). Arlciv. Kemi, Mineral, Geol (Stockholm), 26B. Paper No. 14. Raben, M. S. (1957). Science, 125, 883. Read, C. H. (1959). Recent Progr. Hormone Res. 15, 107. Read, C. H. (I960). Recent Progr. Hormone Res. 16, 187. Read, C. H. & Bryan, G. T. (1960). Ciba Foundation Colloquia on Endocrinology, 13, 68. Read, C. H. & Stone, D. B. (1958). Amer. J. Dis. Child. 96, 538. Salmon, W. D. & Daughaday, W. H. (1957). J. Lab. clin. Med. 49, 825. Note added in proof Subsequently during similar assays two troubles have been encountered : (a) at times, apparent inhibition of the agglutination has occurred with various protein solutions (e.g. with rabbit serum), unless the diluent used is rabbit serum and the tubes are read early (at 3rd-5th hr.) and not overnight; (b) although normal sera have inhibited as before, the recent HGH standards have only inhibited down to the lesser dilutions ; temporarily, therefore, we are using as standard a pool of normal serum.