Description

The ¹²⁵I-hFSH IRMA system provides a direct quantitative determination of human follicle stimulating hormone (h FSH) in human serum. h FSH can be assayed in the range of 0-150 mIU/ml using 100 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

The follicle stimulating hormone (follitropin or h FSH) is a glycoprotein with a molecular weight of 30000, secreted by the adenohypophysis. Like other glycoprotein hormones (LH, TSH and HCG), hFSH contains two different subunits, an a- and a ß-chain, linked by noncovalent bounds. The primary structures of the a subunits of h FSH and of those mentioned are virtually identical, whilst their ß subunits are different. The ß subunits are responsible for the immunological and biological specificity of these hormones.

Follicle stimulating hormone synthesis and release is stimulated by the hypothalamic gonadotropin releasing hormone (GnRH), whereas the ovarian steroids secreted from the corpus luteum control further secretions of h FSH by negative feedback.

The measurement of follicle stimulating hormone concentrations is an important part of the investigation of disorders of the hypothalamic-pituitary-gonadal axis. It is recommended to measure both h FSH and hLH to discriminate between hypothalamic and pituitary dysfunction.

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 h FSH molecule different from that recognised by the unlabelled capture-antibody. The two antibodies react simultaneously with the h FSH molecule forming a “sandwich”.

Standards and samples are incubated with a mixture of the antibodies at room temperature. At the end of a one hour incubation period (no need of a shaker), magnetic immunosorbent (MIS) is added in excess. MIS particles selectively bind the h FSH – 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 follicle stimulating hormone concentration can be determined.

Contents of the kit

Materials, tools and equipment required

Test tube rack
Precision pipettes with disposable tip (100, 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 hFSH concentration higher than that of the most concentrated standard should be diluted and reassayed.

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 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)

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:

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 FSH.

Determine the FSH 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.

Table 2. Typical Assay Data

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

Characterization of the assay

Assay parameters

Sensitivity

A detection limit of 0.05 mIU/ml has been obtained by assaying 20 replicates of the zero standard. The sensitivity has been determined as the concentration corresponding to the sum of the mean cpm and its double standard deviation.

Hook effect

There is no high dose “hook effect” up to a hFSH concentration of 1000 mIU/ml.

Specificity

No cross reactivity with hLH, hCG and hTSH can be detected in normal physiological concentrations.

Precision

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

Reproducibility

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

Recovery

Recovery was defined as the measured increase expressed as percent of expected increase upon spiking serum samples with known amount of hFSH. The average percent recovery for 10 serum pools spiked with hFSH at 2 levels was: 97.13 ± 5.8%

Expected Values

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 from 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