Description

The ¹²⁵I-hFSH IRMA system provides a direct quantitative in vitro determination of human follicle stimulating hormone (hFSH) in human serum. hFSH 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 hFSH) 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 hFSH 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.

The hFSH 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 hFSH by negative feedback.

The measurement of hFSH concentrations is an important part of the investigation of disorders of the hypothalamic-pituitary-gonadal axis. It is recommended to measure both hFSH 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.

The ¹²⁵I labelled signal-antibody binds to an epitope of the FSH molecule spatially different from that recognised by the biotin-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 shaking immuno-complex is immobilized to the reactive surface of streptavidin coated test tubes. Reaction mixture is then discarded, test tubes washed exhaustively, and the radioactivity is measured in a gamma counter.

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

Contents of the kit

Materials, tools and equipment required

Test tube rack
Precision pipettes with disposable tip (100, 200 and 2000 µl)
Distilled water
Vortex mixer
Shaker
Plastic foil
Gamma counter
Absorbent tissue

Recommended tools and equipment

Repeating pipettes (e.g. Eppendorf)
Dispenser with 1-L 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 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 700 ml distilled water to obtain 720 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)

Table 2. Typical Assay Data

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

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:

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.

Characterization of the assay

Assay parameters

Hook effect

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

Specificity

No cross reactivity with hLH hTSH and hCG 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 per cent of expected increase upon spiking serum samples with known amount of hFSH. The average percent recover for 10 serum pools spiked with hFSH at 3 levels was: 97.3 ± 3.5%

Expected Values

It is recommended that each laboratory determine a reference range for its own patient population.

Procedural notes

1) Source of error! Reactive test tubes packed in plastic boxes are not marked individually. Care should be taken of not mixing them with common test tubes. To minimize this risk, never take more tubes than needed out of plastic box, and put those left after work back to the box. It is recommended to label assay tubes by a marker pen.

2) Source of error! To ensure the efficient rotation, tubes should be firmed tightly inside the test tube rack. Never use a rack type with open hole. An uneven or incomplete shaking may result in a poor assay performance.

3) Addition of wash buffer. For the addition of wash buffer the use of a common laboratory dispenser equipped with a 1-L 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 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 74 mg.

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