Journal of Clinical Laboratory Analysis 1 :238-242 (1987)

Sensitive Enzyme lmmunoassay of Human Growth Hormone for Clinical Application:

A Review Eiji Ishikawa,’ Seiichi Hashida,’ Yuzuru Kato,2 and

Hiroo Imura2 Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki, ’

and Second Division, Department of Medicine, Kyoto University School of Medicine, Kyoto, Japan

Radioimmunoassay, a useful tool to mea- the development and applicability of sen- sure human growth hormone (hGH) in sitive enzyme immunoassay technique for serum, cannot measure very low hGH lev- hGH, which can measure very low levels els in serum and urine under some condi- of hGH in serum and urine that have not tions. This work reviews recent studies on been measured by radioimmunoassay.

Key words: Peroxidase, fi-D-galactosidase, radioimmunoassay, dwarfism, acromegaly, Sheehan’s syndrome, craniopharyngioma

IN T ROD U CT I0 N The detection limit of 0-D-galactosidase from Eschevichia

Radioimmunoassay has been a useful tool to measure hu- man growth hormone (hGH) in serum and aid the diagnosis of hypothalamic and pituitary disorders. However, the de- tection limit of hGH by radioimmunoassay is not sufficient to measure very low levels in serum and urine under various conditions (1,2). The detection limit of hGH by competitive radioimmunoassay, which has been most widely used, is 500-1,000 ng/l. The detection limit of hGH by a recently in- troduced immunoradiometric assay (SUCROSEP, Boots- Cclltech Diagnostics Limited, Berkshire, UK) is 75 ng/l. In normal subjects, the serum level of hGH fluctuates with time to a great extent with the lowest level of 50 ngil ( 3 ) and is lowered to as low as 7 ng/l after the administration of the so- matostatin analogue SMS 201-995 (4). The hGH levels in urine are similarly low or even lower (2,5,6).

ENZYME IMMUNOASSAY FOR hGH

Radioimmunoassay is sensitive, since radioisotopes can be detected with high sensitivity. The detection limit of radio- isotopes can be assessed from the specific radioactivity that can be calculated from the half life. The half life of 1251, which has been most frequently used for radioimmunoassay, is 60.2 days, and the specific radioactivity of carrier-free lZ5I

is 4.8 dpm/amol (7). Therefore, the detection limit of car- rier-free 1251 by a conventional gamma counter is 5-10 amol.

By contrast, some enzymes can be detected with higher sensitivities. The detection limit by a 100 min fluorimetric assay of horseradish peroxidase, which has been most widely used as a label in enzyme immunoassay, is 0.5 amol (7,8).

0 1987 Alan R. Liss, Inc.

coli by a 100 min fluorimetric assay is even lower (0.02 amol) (7,8). Therefore, enzyme immunoassay is potentially more sensitive than radioimmunoassay, and sensitive en- zyme immunoassays that can measure macromolecular antigens at attomole levels have been described since 1977

In 1982, a two-site binding (immunoenzymometric or sandwich) enzyme immunoassay for hGH was described us- ing 0-D-galactosidase from Escherichia coli as a label of anti-hGH antibodies (14). However, the detection limit of hGH by this assay was similar to that by competitive ra- dioimmunoassay (4 pg/tube and 400 ng/l of serum). In 1983, the detection limit of hGH was improved 67-fold using af- finity-purified anti-hGH Fab‘ labeled with horseradish per- oxidase (9,lO). Moreover, serum interference was less severe with the peroxidase-labeled anti-hGH Fab’ than with the 0-D-galactosidasc-labeled anti-hGH Fab’ (15). The de- tection limit of hGH has been recently further improved (20 fg/tube, 1 ng/l of serum using 20 pI of serum and 0.1 ng/l of urine using 0.15 ml of dialyzed urine) (16). In terms of fg/ tube, this is 375-fold less than that by a recently introduced immunoradiometric assay (SUCROSEP, Boots-Celltech Di- agnostics Limited, Berkshire, UK) (Fig. 1). Variation coef- ficients for within-assay and between-assay over the range of 0 . 5 4 4 0 pg/tube were less than 10%. In this enzyme im-

(7-13).

~~

Address reprint requests to Dr. Eiji Ishikawa, Department of Biochemis- try, Medical College of Miyazaki, Kiyotake, Miyazaki 889-16, Japan.

Enzyme lmmunoassay of Growth Hormone 239

were 12, 16, 17, 65, and 103 ng/l, and little response to in- sulin-induced hypoglycemia was observed (16).

The sensitive enzyme immunoassay for hGH will be use- ful for further investigation of serum hGH levels under var- ious conditions.

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Fig. 1. Standard curves of hGH by sensitive two-site binding (immu- noenzymometric or sandwich) enzyme immunoassay using affinity-puri- fied anti-hGH Fab’-horseradish peroxidase conjugate (EIA) and immunoradiometric assay using 1251-labeled anti-hGH IgG (IRMA) (SUC- ROSEP, Boots-Celltech Diagnostics Limited, Berkshire, UK).

munoassay, there is no cross reaction with 0.16 IU/tube of human luteinizing hormone, 0.16 IU/tube of human follicle- stimulating hormone, 86 pU/tube of human thyrotropin, 22 ng/tube of human prolactin, or 1 ng/tube of human placental lactogen (6,16). The specificity remains to be improved for the measurement of hGH in pregnant women with high lev- els of placental lactogen.

Details of this sensitive two-site binding enzyme immu- noassay for hGH is described in Appendix.

hGH IN SERUM

When blood was collected before breakfast early in the morning, serum levels of hGH in normal male and female adults were 312 f 284 (SD) ng/l (range: 53-940 ng/l; n = 10) and 662 f 597 (SD) ng/l (range: 112-2,195 ng/l; n = 13), respectively (10). This wide variation of serum hGH levels in normal subjects is due to the fact that the serum hGH level fluctuates with time to a great extent with no ap- parent stimuli (3). Using the sensitive two-site binding en- zyme immunoassay, the basal serum hGH level was measured to be 50-200 ng/l (3), while it has not been mea- sured by radioimmunoassay (1).

Much lower serum levels of hGH were observed under different conditions. After the administration of the somato- statin analogue SMS 201-995 to normal adults, the serum level of hGH was persistently lowered, and the lowest level was 7 ng/l(4). In Sheehan’s syndrome, serum levels of hGH

hGH IN DRIED BLOOD ON FILTER PAPER DISCS

It is not easy to collect enough blood from the neonates for the separation of serum, but it is easy to collect a small vol- ume of blood and dry it on filter paper. When a 30 p1 aliquot of blood is dried on filter paper that has been used for mass screening of neonatal metabolic disorders, the diameter of blood spot is about 10 mm, and a filter paper disc of 3 mm in diameter contains 2.7 pl of blood. Using a single filter pa- per disc of 3 mm in diameter, on which 2.7 pI of blood had been dried, the detection limit of hGH was 7.5 ng/l of blood, and blood levels of hGH in normal neonates 5-7 days after birth were 7.91 k 4.17 (SD) pgll (range: 1.30-21.3 pg/l, n = 34). It may be possible to detect hGH deficiency in the neonates.

hGH IN URINE

Since serum hGH level fluctuates with time to a great ex- tent with no apparent stimuli (1,3), the determination of serum hGH level by a single collection of blood provides no reliable information on the secretion of hGH, and blood samples have to be collected every 15-20 min for 24 h to as- sess the secretion of hGH under physiological conditions.

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Fig. 2. Elution profile of hGH in urine from a column of Sephadex G- 75. Arrows 1-5 indicate the elution volumes of blue dextran 2,000 (MW: 2,000,000), egg albumin (MW: 43,000), soybean trypsin inhibitor (MW: 21,500), myoglobin (MW: 17,200) and cytochrome C (MW: 12,400), respectively.

240 lshikawa et al.

And it is difficult to collect blood samples frequently during physical exercises. By contrast, urine samples can be more easily collected than serum samples, and hGH level in urine may reflect serum hGH level during the same time period for the accumulation of urine and provide useful information on the secretion of hGH. However, radioimmunoassay is not sufficiently sensitive to measure hGH levels without concen- tration of urine samples (2).

The sensitive two-site binding enzyme immunoassay for hGH was used to demonstrate and measure hGH in urine. Urine samples were concentrated 50-fold and subjected to gel filtration on a column of Sephadex G-75 (5). A major peak of hGH corresponded to a molecular weight of 22,000 dalton, although a small peak with a larger molecular weight of approximately 70,000 dalton was observed (Fig. 2). And hGH was measured in unconcentrated urine samples from normal subjects (5,6,16). When urine samples were col- lected from normal subjects before breakfast immediately after awakening early in the morning, urine hGH level in terms of ng/g of creatinine decreased with increasing age (6,16) (Fig. 3). The highest level observed was 113 nglg of creatinine in a normal female aged 0.7 yr. In normal adults aged 26-35 yr, urine hGH levels ranged from 1.0 to 6.7 ng/

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is useful as an aid to the diagnosis of various disorders, it was examined whether the urine hGH level reflected the serum hGH level (16). Normal subjects and patients with Shee-

- % b @ han's syndrome, craniopharyngioma, and acromegaly were intravenously injected with insulin, growth hormone-releas- ing factor (GRF), arginine, or thyrotropin-releasing hor- mone (TRH). Blood was collected at 0, 0.5, 1.0, 1.5, and 2 h. Serum hGH levels were measured, and the mean values of 5 serum hGH levels were calculated. Urine was voided at 0 h and collected at 2 h, and urine hGH levels were mea- sured. Urine hGH levels were well correlated to the maxi- ma1 serum hGH levels (r = 0.79, n = 35) and the mean serum hGH levels (r = 0.86, n = 35), when the maximal serum hGH level was above 0.2 pg/l (Fig. 5) . Below this serum level, hGH was not detectable in urine.

The measurement of urine hGH levels by the sensitive en-

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Enzyme lmmunoassay of Growth Hormone 241

mol/l NaCl, 1 g/l NaN3 and 1 g/l bovine serum albumin and 0.05 ml of 10 mmol/l sodium phosphate buffer, pH 7.0, con- taining 1 mol/l NaCl, 1 g/l NaN3 and 1 g/l bovine serum al- bumin. Urine samples (5 ml) were mixed with 0.05 ml of 100 g/l bovine serum albumin and dialyzed against 10 mmol/ 1 sodium phosphate buffer, pH 7.0, containing 0.4 mol/l NaCl and 1 g/l NaN3 at 4°C overnight. The dialyzed urine samples (0.05-0.15 ml) were mixed with 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.4 mol/l NaCI, 1 g/l NaN3 and 1 g/l bovine serum albumin to adjust the final vol- ume to 0.15 ml.

After removal of the incubation mixture, the polystyrene ball was washed twice by addition and aspiration of 2 ml of 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaC1. Then, the polystyrene ball was incubated with affinity-purified anti-hGH Fab’-peroxidase conjugate (50 ng/ tube) and nonspecific rabbit F(ab’)2 (100 pgltube) in a total volume of 0.15 ml of 10 mmol/l sodium phosphate buffer, pH 7.0, cottaining 1 g/l bovine serum albumin and 0.1 mol/ 1 NaCl at 4°C for 16 h without shaking and at 20°C for 6 h with continuous shaking. The incubation in the presence of nonspecific F(ab’):! improved the detection limit of hGH 2- fold.

After removal of the conjugate solution, the polystyrene ball was washed once as described above and incubated with 2 ml of the same buffer as that for washing at 30°C for 10 min and then washed once as described above. The 10-min incubation only slightly improved the detection limit of hGH. Bound peroxidase activity was assayed by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate (18). The polystyrene ball was incubated with 0.1 ml of 6 g/l 3-(4-hy- droxypheny1)propionic acid in 0.1 mol/l sodium phosphate buffer, pH 7.0, at 30°C for 5 min. The enzyme reaction was started by addition of 0.05 ml of 0.15 g/l hydrogen peroxide followed by incubation at 30°C for 90 min. The enzyme re- action was stopped by addition of 2.5 ml of 0.1 mol/l gly- cine-NaOH buffer, pH 10.3. Fluorescence intensity was measured relative to 0.2 mg/l quinine in 0.05 mol/l H2S04 using a Shimadzu fluorophotometer (RF-5 10, Shimadzu Sei- sakusho, Ltd., Kyoto, Japan). The wavelengths used for ex- citation and emission analysis were 320 nm and 405 nm, respectively.

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Fig. 5. Correlation of urine hGH level to the mean serum hGH level after the injection of insulin, GRF, arginine, or TRH. Blood was collected at 0, 0.5, 1.0, 1.5, and 2 h after the injection. Urine was voided at 0 h of the injection and collected at 2 h.

APPEND I X

Preparation of anti-hGH IgG-coated polystyrene balls: Polystyrene balls (3.2 mm in diameter, Precision Plastic Ball Co., Chicago, IL) were coated by physical adsorption with rabbit anti-hGH IgG, which had been passed through a col- umn of human placental lactogen-Sepharose 4B (6,16).

Preparation of affinity-purified anti-hGH Fab’-horserad- ish peroxidase: Rabbit anti-hGH F(ab’);! was affinity-puri- fied by elution from a column of hGH-Sepharose 4B at pH 2.5, reduced with 2-mercaptoethylamine, and conjugated to horseradish peroxidase (Grade I, RZ = 3.0, Boehringer Mannheim GmbH, Mannheim, FRG) using N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (Zieben Chemical Co., Ltd., Tokyo, Japan) (9,17).

Two-site binding enzyme immunoassay for hGH: An anti- hGH IgG-coated polystyrene ball was incubated with hGH standards or test samples in a total volume of 0.15 ml at 37°C for 6 h with continuous shaking. hGH standards were di- luted with 10 mmol/l sodium phosphate buffer, pH 7.0, con- taining 0.1 mol/l NaC1, 1 g/l NaN3 and 1 g/l bovine serum albumin to a final volume of 0.1 ml and mixed with 0.05 ml of 10 mmol/l sodium phosphate buffer, pH 7.0, containing 1 mol/l NaCI, 1 g/l NaN3 and 1 g/l bovine serum albumin. Serum samples (0.02 ml) were mixed with 0.08 ml of 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1

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