Description

The cortisol [¹²⁵I] assay system provides the quantitative in vitro determination of cortisol in human serum. Cortisol can be assayed in the range 0-1600 nmol/l (0-580 ng/ml). Each kit contains materials sufficient for 100 assay tubes, permitting the construction of one standard curve and assay of 42 unknowns and 1 control in duplicate.

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 (10, 100 and 500 µl)
Vortex mixer
Magnetic separator (or, alternatively, centrifuge)
Decanting racks
Gamma counter

Recommended tools and equipment

Orbital shaker
Repeating pipettes

Preparation of reagents

Add 500 µl distilled water to the lyophilised 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.

For repeated use the rest of reagent can be stored at 4-8 ^oC for two months.

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 ^oC 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.

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:

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 / B₀% for each standard versus the corresponding concentration of cortisol. Figure 1 shows a typical standard curve.

Determine the cortisol concentration of the unknown samples by interpolation from the standard curve. Do not extrapolate values beyond the standard curve range. Automated data processing systems are also available.

Table 2. Typical Assay Data

Cortisol 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 = 0.362 ng/ml

Characterization of the assay

Assay parameters

Specificity

Cross reactivity of the cortisol antiserum in the kit with various substances are shown below:

Sensitivity

10 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 percent of expected increase upon spiking serum samples with known amount of cortisol. Values for 11 serum samples spiked with cortisol at 3 levels were: 101 ± 10 %

Expected reference values

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

morning values: 160-620 nmol/l (58-225 ng/ml)
evening values: 90-400 nmol/l (33-145 ng/ml)

was obtained from normal patients.

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 80.5 mg.