Description

The ¹²⁵I-hGH IRMA system provides a direct quantitative in vitro determination of human growth hormone (hGH) in human serum. hGH can be assayed in the range of 0-100 µIU/ml using 50 µl serum samples.

Introduction

The human growth hormone (somatotropin, hGH) is a protein hormone with a molecular weight of 22000, secreted by the pituitary gland. The structure of the molecule is similar to that of prolactin (hPRL) and placental lactogen (hPL).

The secretion of human growth hormone is under double regulation: in certain neurones of the hypothalamus a growth hormone releasing hormone (GH-RH) and growth hormone inhibiting hormone, somatostatin (GH-RIH) are produced.

Several effects of hGH have been known. It does not only regulate protein synthesis, the growth of skeleton, the muscles and the viscera but has lipolytic and lactogenic effects and influences glycogen storage in the liver as well. In clinical practice the diabetogenic effect of human growth hormone has also been well-known.

hGH mean serum level progressively decreases post natally. Its level increases again significantly during puberty to further decrease with ageing. Because human growth hormone secretion is pulsative, a single measurement of hGH concentration does not reflect endogenous hGH secretion. About 50% of the population has a very low, sometimes undetectable growth hormone concentration. Standardised stimulatory tests are therefore necessary to asses pituitary hGH secretion.

The measuring of human growth hormone can widely be used in clinical practice for diagnosing hypo- and hypersecretion.

Principle of method

The technology uses two high affinity monoclonal antibodies in an immunoradiometric assay (IRMA) system. It offers an increased level of sensitivity and specificity compared with conventional RIA methods.

The ¹²⁵I labelled signal-antibody binds to an epitope of the hGH molecule different from that recognised by the unlabelled capture-antibody. The two antibodies react simultaneously with the hGH molecule forming a “sandwich”.

Standards and samples are incubated with a mixture of the antibodies at room temperature. At the end of a two hours incubation period (no need of a shaker), magnetic immunosorbent (MIS) is added in excess. MIS particles selectively bind the hGH – signal antibody – capture antibody complex and settle out in a magnetic field. A wash step is critical to reducing non-specific binding to a minimum for increased low end precision. By measuring the radioactivity of the magnetic immunosorbent pellet in a gamma counter the human growth hormone concentration can be determined.

Contents of the kit

Materials, tools and equipment required

Round bottom polystyrene or polypropylene assay tubes, about 12 x 75 mm
Test tube rack
Precision pipettes with disposable tip (50, 200, 500 and 1000 µl)
Distilled water
Vortex mixer
Shaker
Plastic foil
Gamma counter
Absorbent tissue

Recommended tools and equipment

Repeating pipettes (e.g. Eppendorf)
Dispenser with 300 ml reservoir (instead of the 1 ml pipette)

Specimen collection and storage

Serum samples can be prepared according to common procedures used routinely in clinical laboratory practice. Samples can be stored at 2-8 °C if the assay is carried out within 24 hours, otherwise aliquots should be prepared and stored deep frozen (-20 °C). Frozen samples should be thawed and thoroughly mixed before assaying. Repeated freezing and thawing should be avoided. Do not use lipemic, hemolyzed or turbid specimens. Samples with a hGH concentration higher than that of the most concentrated standard should be diluted and assayed.

Preparation of reagents, storage

Add the wash buffer concentrate (20 ml) to 200 ml distilled water to obtain 220 ml wash solution. Upon dilution store at 2-8 °C until expiry date.

Add 1000 µl distilled water to the lyophilized standards and control serum. Mix gently with shaking or vortexing (foaming should be avoided).

Ensure that complete dissolution is achieved, and allow the solution to equilibrate at room temperature for at least 20 minutes. Store at -20 °C until expiry date.

Store the rest of reagents between 2-8 °C after opening. At this temperature each reagent is stable until expiry date. The actual expiry date is given on the package label and in the quality certificate.

CAUTION! Equilibrate all reagents and serum samples to room temperature. Mix all reagents and samples thoroughly before use. Avoid excessive foaming.

Assay procedure

(For a quick guide, refer to Table 1.)

Table 1. Assay Protocol, Pipetting Guide (all volumes in microliters)

Table 2. Typical Assay Data

Calculation of results

The calculation is illustrated using representative data. The assay data collected should be similar to those shown in Table 2.

Calculate the average CPM for each pair of assay tubes. Calculate the normalized percent binding for each standard, control and sample respectively by using the following equation:

	S2-6 [C, Mx] (cpm) - S1 (cpm)
B / T % =	———————————	x 100
	T (cpm)

For simplicity, these values are uncorrected for non-specific binding (NSB). This is enabled by low NSB being less than 3% of total count.

Using semi-logarithmic graph paper plot B/T (%) for each standard versus the corresponding concentration of GH.

Determine the GH concentration of the unknown samples by interpolation from the standard curve. Do not extrapolate values beyond the standard curve range.

Out of fitting programs applied for computerized data processing logit-log, or spline fittings can be used. Automated data processing systems are also available.

Figure 1
A typical standard curve
(Do not use to calculate unknown samples)

Characterization of the assay

Assay parameters

NSB / T	< 0.05%
Bmax / B0	> 800

Sensitivity

A detection limit of 0.03 µIU/ml has been obtained by assaying 20 duplicates of the zero standard. The sensitivity has been determined as the concentration corresponding to the sum of the mean cpm of the zero standard and its double standard deviation.

Hook effect

There is no high dose “hook effect” up to a hGH concentration of 2000 µIU/ml.

Specificity

Cross reactivities with hPL 0.2% and hPRL 1.0%; no other can be detected in normal physiological concentration.

Precision

4 patient samples were assayed in 15 replicates to determine intra-assay precision. Values obtained are shown below.

Sample	Number of replicates	Mean value µIU/ml	SD µIU/ml	CV %
1	15	0.74	0.046	6.2
2	15	2.98	0.12	4.0
3	15	8.30	0.17	2.1
4	15	24.44	0.81	3.3
5	15	84.33	2.45	2.9

Reproducibility

To determine inter-assay precision 4 patient samples were measured in duplicates in 15 independent assays by 2 operators using different kit batches. Values obtained are shown below.

Sample	Number of runs	Mean value µIU/ml	SD µIU/ml	CV %
1	15	0.75	0.042	5.6
2	15	2.85	0.137	4.8
3	15	8.26	0.37	4.5
4	15	22.58	1.06	4.7
5	15	65.29	4.37	6.7

Recovery

Recovery was defined as the measured increase expressed as percent of expected increase upon spiking serum samples with known amount of hGH. The average percent recovery for 10 serum pooles spiked with hGH at 3 levels was: 85.0-100.6%

Expected Values

Healthy adults: 0-14 µIU/ml (mean 0.79 µIU/ml SD = 1.94 µIU/ml)
It is recommended that each laboratory determine a reference range for its own patient population.

Procedural notes

Addition of wash buffer. For the addition of wash buffer the use of a common laboratory dispenser equipped with a 300 ml glass bottle, and a flexible outlet tubing end is recommended. In lack of this tool a large volume syringe attached to a repeating pipette can be used.

Additional information

Components from various lots or kits of different manufacturers should not be mixed or interchanged.

Precaution

Radioactivity

This product contains radioactive material. It is the responsibility of the user to ensure that local regulations or code of practice related to the handling of radioactive materials are satisfied.

Biohazard

Human blood products used in the kit have been obtained from healthy human donors. They were tested individually by using approved methods (EIA, enzyme immunoassay), and were found to be negative, for the presence of both Human Immunodeficiency Virus antibody (Anti-HIV-1) and Hepatitis B surface Antigen (HBsAg).

Care should always be taken when handling human specimens to be tested with diagnostic kits. Even if the subject has been tested, no method can offer complete assurance that Hepatitis B Virus, Human Immunodeficiency Virus (HIV-1), or other infectious agents are absent. Human blood samples should therefore be handled as potentially infectious materials.

Chemical hazard

Components contain sodium azide as an antimicrobial agent. Dispose of waste by flushing with copious amount of water to avoid build-up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 74 mg.

 

	Use by		In vitro diagnostic medical device		Control
	Batch code		Manufacturer		Standard
	Caution, consult accompanying documents		Radioactive material		Magnetic immunosorbent
	Biological risk		Temperature limitation Store between 2-8 °C		Tracer
	Consult instructions for use		Catalogue number		Wash buffer

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