Description

The progesterone [¹²⁵I] assay system provides the quantitative determination of progesterone in human serum or plasma. Progesterone can be assayed in the range of 0-120 nmol/I (0-37.7 ng/ml) using 50 µI serum samples. Each kit contains materials sufficient for 100 assay tubes, permitting the construction of one standard curve and assay of 41 unknowns and 1 control in duplicate.

Introduction

Progesterone is one of the C₂₁-steroids (Mw = 314.5) secreted by the corpus luteum in females during the menstrual cycle, and in a much higher amount by the placenta during pregnancy. It is also secreted in a minor quantity by the adrenal cortex in both males and females. Majority of circulating progesterone is bound to albumin and corticosteroid binding globulin (CBG), the bioactive free hormone represents only 2.5-3% of the total progesterone. Measurement of serum progesterone is of diagnostic value in menstrual disorders and infertility. Measurement of progesterone in the first 10 weeks of gestation have been suggested in the diagnosis and treatment of patients with threatened abortion and ectopic pregnancy.

Principle of method

This assay is based on the competition between unlabelled progesterone and a fixed quantity of ¹²⁵I -labelled progesterone for a limited number of binding sites on progesterone specific antibody. Allowing to react a fixed amount of tracer and antibody with different amounts of unlabelled ligand the amount of tracer bound by the antibody will be inversely proportional to the concentration of unlabelled ligand. Upon addition of magnetizable immunosorbent the antigen-antibody complex is bound on solid particles which are then separated by either magnetic sedimentation or centrifugation. Counting the radioactivity of solid phase enables a standard curve to be constructed and samples to be quantitated.

Contents of the kit

Materials and equipment required

Round bottom polystyrene or polypropylene assay tubes (about 12 x 75 mm)
Plastic film to cover tubes
Precision pipettes (50, 100 and 500 µl)
Vortex mixer
Magnetic separator (or, alternatively, centrifuge)
Decanting racks
Gamma counter

Recommended tools and equipment

Orbital shaker
Repeating pipettes

Specimen collection and storage

Serum samples can be prepared according to common procedures used routinely in clinical laboratory practice. Sera can be stored at 2-8 °C for two days after collection. For later analysis they should be stored deep-frozen. Repeated freezing and thawing should be avoided.

Do not use lipemic, hemolyzed or turbid specimens. Samples with a progesterone concentration higher than that of the most concentrated standard should be diluted and reassayed. Use the zero standard as diluent.

Assay procedure

(For a quick guide refer to Table 1)

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

Calculation of results

The assay data collected should be similar to those shown in Table 2. Calculate the average counts per minute (CPM) for each pair of assay tubes. Calculate the percent B₀ / T for zero standard (S₁) by using the following equation:

Calculate the normalized percent binding for each standard, control and sample respectively by using the following equation:

Using semi-logarithmic graph paper plot B / B₀ % for each standard versus the corresponding concentration of progesterone. Figure 1 shows a typical standard curve.

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

Table 2.    Typical Assay Data

progesterone concentration nmol/l

Figure 1.
A typical standard curve
(Do not use to calculate sample values)

Conversion of SI units can be performed according to the following formula:
1 nmol/l = 0.3145 ng/ml
1 ng/ml = 3.18 nmol/l

Characterization of the assay

Assay parameters

Specificity

Cross reactivity was defined by weight at the 50% displacement level in per cent.

Sensitivity

0.44 ± 0.12 nmol/l, defined as the concentration 2 standard deviations from the zero standard.

Precision, reproducibility

Recovery

Recovery was defined as the measured increase expressed as per cent of expected increase upon spiking serum samples with known amount of progesterone.

The mean recovery for added progesterone was 97.3 ± 5.7% in the range 0.79-54.25 nmol/l.

Expected reference values

It is recommended that each laboratory establish its own reference intervals. As a guide,

follicular phase: 0.6-3.8 nmol/l (0.2-1.2 ng/ml)
luteal phase: 10.5-58 nmol/l (3.3-18.2 ng/ml)

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

Additional information

Storage

Store the reagents between 2 and 8 °C. At this temperature each reagent is stable until expiry date. Pay special attention to preventing magnetic immunosorbent suspension from freezing.

Availability

From stock.

Shelf life

The shelf life of kit reagents is 8 weeks from the date of manufacturing. To make maximum benefit of long-term stability it is recommended to adjust the date of ordering to labelling calendar issued each year. The actual expiry date is given on package label and in the quality certificate. Components from various lots or from kits of different manufacturers should not be mixed or interchanged.

Precautions and warnings

This kit should only be used for in vitro diagnostic purposes.

Radioactivity

This kit contains radioactive material. Receipt, acquisition, possession, or use of radioactive materials are subject to regulations, and a licence of (inter)national authorizing bodies. It is the responsibility of the user to ensure that local regulations or codes of practice are satisfied.

Potentially infectious materials

Human blood products provided as components of this product have been obtained from donors tested individually and found negative for Human Immunodeficiency Virus antibody (HIV-Ab) as well as for Hepatitis B surface Antigen (HBsAg) using approved EIA methods.

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), or other infectious agents are absent, and all human blood samples should be considered potentially infectious.

Chemical and other hazard

Some components contain sodium azide (0.1% w/v) as an antimicrobial agent. Dispose the waste by flushing it with copious amounts of water to avoid build up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 81 mg.