Description

The SHBG [¹²⁵I] IRMA system provides a direct quantitative determination of sex hormone binding globulin in human serum. SHBG can be assayed in the range of 0-250 nmol/l using 10 µl serum samples. Each kit contains materials sufficient for 100 assay tubes permitting the construction of one standard curve and the assay of 42 unknowns in duplicate.

Introduction

Sex hormone binding globulin (SHBG), also known as Testosterone-estradiol binding globulin (TEBG) and sex steroid binding protein (SBP), is a circulating glycoprotein with a molecular weight of around 86000. It is thought to be synthesized in the liver, and in the circulation its biological function is the transport of sex steroid hormones. It has a high binding affinity for testosterone, dihydrotestosterone and estradiol.

Serum SHBG levels are relatively low at birth, increase to high levels during infancy, then decrease during puberty. The highest physiologic levels of SHBG are observed in pregnant, near-term maternal serum. Abnormal serum SHBG levels have been reported in number of conditions, including obesity and female hyperandrogenism (including polycystic ovary disease). Serum SHBG levels are inversely related to free, presumably bioactive, testosterone concentracions, a “free testosterone index” based on the ratio of total testosterone to SHBG has been characterized.

Principle of method

The ¹²⁵I labelled signal-antibody binds to an epitope of the SHBG molecule, which is different from that recognised by the unlabelled capture-antibody. The two antibodies react simultaneously with the antigen present in standards or samples which leads to the formation of a capture antibody - antigen - signal antibody complex, also referred to as a “sandwich”.

During a 2 hour incubation period with continuous agitation immuno-complex is immobilized on the reactive surface of test tubes. Reaction mixture is then discarded, test tubes are washed exhaustively, and radioactivity is measured in a gamma counter.

The concentration of antigen is directly proportional to the radio-activity measured in test tubes. By constructing a calibration curve plotting binding values against a series of standards containing known amount of SHBG, the unknown concentration of SHBG in patient samples can be determined.

Contents of the kit

Materials, tools and equipment required

Test tube rack
Precision pipettes with disposable tip (10 µl, 300 µl, 2 ml)
Repeating pipette, 300 µL and 2.0 mL
Shaker
Plastic foil
Gamma counter
Absorbent tissue

Recommended tools and equipment

Repeating pipettes
Dispenser with reservoir (instead of the 2 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 48 hours, otherwise aliquots should be prepared and stored deep frozen (-20 °C). Do not store serum samples longer than 4 months. Do not use lipemic, hemolyzed or turbid specimens. Frozen samples should be thawed and thoroughly mixed before assaying. Repeated freezing and thawing should be avoided.

Preparation of reagents, storage

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

Add 0.5 ml distilled water to the lyophilised control serum, and 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.

Add the wash buffer concentrate to 700 ml distilled water. The diluted solution can be stored at 2-8 °C until expiry date of the kit.

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

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

Calculation of results

Calculate the average CPM for each pair of assay tubes. Draw the standard curve by plotting mean CPM of each standard level (ordinate) against the respective concentration, except for 0 standard (abscissa) using log-log graph paper.

Obtain sample concentration by interpolation of sample counts on the standard curve.

For computerized calculations and/or quality assessment normalized specific binding values, rather than cpm values are used. Specific binding values can be calculated for each standard and sample according to the following equation:

Characterization of the assay

Calibration

Standards are calibrated against the WHO Standard, Code 95/560.

Assay parameters

Sensitivity

Analytical sensitivity: 0.11 nmol/l

It is defined as the concentration of SHBG equivalent to the mean CPM of 20 replicates of the zero standard.

Functional sensitivity: 0.22 nmol/l

It is defined as the value extra-polated to 20% of the inter-assay imprecision profile obtained from 10 independent runs on patient samples with low endogenous SHBG concentration.

Hook effect

There is no high dose “hook effect” up to a SHBG concentration of 835 nmol/l.

Specificity

Cross reactivity of the SHBG antiserum has been measured against human IgG (10 g/l) and human serum albumin (50 g/l). In both cases cross reactivity is non-detectable.

Precision and reproducibility

6 samples with 20 replicates in 1 assay run, and 8 samples with duplicates in 12 runs were measured to determine intra-assay and inter-assay precision, respectively. Values obtained are shown below.

Recovery

Recovery was defined as the measured increase expressed as per cent of expected increase upon spiking serum samples with known amount of SHBG. Values for 8 serum pools spiked with SHBG at 3 levels were as follows: 94.52 ± 5.21%

Dilution test

4 samples were measured in a series of dilution (2, 4, 8, 16-fold) with zero-standard. The following equation obtained for measured (Y) versus expected (X) concentration demonstrates the good linearity:

y = 0.9301x – 0.5552; R = 0.9962; n = 16

Expected values

It is recommended that each laboratory establish its own reference intervals.

In a population (n = 134) of adult female blood donors the mean (± SD) serum concentration of SHBG was 85 ± 65 (range 10–330) nmol/l.
In a population (n = 139) of adult male blood donors the mean (± SD) serum concentration of SHBG was 32 ± 12 (range 7.7–81) nmol/l.

Results obtained should only be interpreted in the context of the overall clinical picture. None of in vitro diagnostic kits can be used as the one and only proof of any disease or disorder.

Additional information

Components from various lots or from 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 75 mg.