Description

The free PSA [¹²⁵I] IRMA system provides a direct quantitative in vitro determination of human free prostate specific antigen (fPSA) in human serum. Free PSA can be assayed in the range of 0-50 ng/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 43 (42) unknowns in duplicate.

Introduction

Prostate specific antigen (PSA) is a tissue-specific serine protease similar to the chymotrypsin- like glandular kallikreins. The active enzyme is a single chain glycoprotein of 237 amino acids (approximately 30 kDa). PSA is mainly responsible for gel dissolution in freshly ejaculated semen by proteolysis of the major gel forming proteins. The major part (70-90%) of prostate specific antigen in serum is complexed to alpha₁-antichymotrypsin (ACT). Total PSA (free + ACT-complex) is increased in both benign prostate hyperplasia and malignant prostate cancer.

Reliable determination of fPSA has long been the subject to scientific criticism, due to special analytical difficulties encountered. The prominent feature of free PSA immunoassays is their suitability for diagnostic and staging the prostate cancer.

Principle of method

The technology uses two monoclonal antibodies of high affinity in an immunoradiometric assay (IRMA) system.

The ¹²⁵I labelled signal-antibody binds to an epitope of the fPSA molecule different from that recognised by the un-labelled 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 the immuno-complex is immobilized on 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 fPSA, the unknown concentration of fPSA in patient samples can determined.

Contents of the kit

Materials, tools and equipment required

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

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.

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.

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:

Using semi-logarithmic graph paper plot B/T (%) for each standard versus the corresponding concentration of fPSA.

Determine the fPSA 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.
Typical standard curve
(Do not use to calculate sample values)

Characterization of the assay

Typical assay parameters

Calibration

Free PSA standards are callibrated against the WHO ECBS 96/668.

Sensitivity

For the analytical sensitivity 0.02 ng/ml has been obtained by assaying 15 duplicates 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.

For the functional sensitivity, determined as the value extrapolated to 22% of the inter-assay imprecision profile obtained in 15 independent runs on patient samples with low endogeneous fPSA concentration, 0.05 ng/ml was obtained.

Hook effect

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

Specificity

The monoclonal antibodies used in this IRMA kit are specific for fPSA. Less than 0.04% cross reactivity was found with human kallikreins and human prostate acid phosphatase.

Precision

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

Reproducibility

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

Expected Values

The optimal free/total PSA cut-off value is 25%, it was determined by ROC analysis.
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 not to mix 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 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 41 mg.