Description

The ¹²⁵I-PSA IRMA system provides direct quantitative in vitro determination of human Prostate Specific Antigen (PSA) in human serum. PSA can be assayed in the range of 0-50 ng/ml using 100 µl serum samples.

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 PSA 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 PSA has long been the subject to scientific criticism, due to special analytical difficulties encountered. Immunoassays may detect two forms of PSA in different molar ratio, a fact that may result in significiant differences of values assigned. Ideally, a reliable total PSA immunoassay is characterized by an equimolar response to both free and complex forms of PSA, contrary to those showing different immunoreactivity towards these forms ("skewed response assays"). The lack of international reference material makes it even more difficult to assign true values to PSA in serum.

The current IRMA system is characterized with an equimolar response to these two forms of PSA, and has been functionally calibrated against FDA-approved immunoassays according to recommendations of international standardization committees.

Another prominent feature of total PSA immunoassays is the sensitivity which determines their suitability for monitoring cancer therapy, particularly surgical prostatectomy, associated with a virtually zero-level of PSA. The outstanding sensitivity, < 0.01 ng/ml, of present IRMA system makes it particularly suitable for assaying ultra-low concentrations of PSA.

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

Standards and samples are incubated with a mixture of the antibodies at room temperature. At the end of a two hours incubation period (no need of a shaker), magnetic immunosorbent (MIS) is added in excess. MIS particles selectively bind the PSA-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 PSA concentration can be determined.

Contents of the kit

Materials, tools and equipment required

Round bottom polystyrene or polypropylene assay tubes, about 12 x 75 mm
Test tube rack
Precision pipettes with disposable tip (100 µl, 200 µl, 500 µl and 1000 µl)
Distilled water
Vortex mixer
Shaker
Plastic foil
Gamma counter
Absorbent tissue

Recommended tools and equipment

Repeating pipettes (e.g. Eppendorf or other)
Dispenser with 300 ml reservoir (instead of the 1000 µl 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 200 ml distilled water to obtain 220 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.

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 count per minute (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 PSA.

Determine the PSA 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 to calculate unknown samples)

Characterization of the assay

Typical assay parameters

Sensitivity

Sensitivity For the analytical sensitivity 0.007 ng/ml has been obtained by assaying 15 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.

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 endogenous PSA concentration, lower than 0.06 ng/ml was obtained.

Hook effect

There is no high dose “hook effect” up to a PSA concentration of 1400 ng/ml.

Specificity

The monoclonal antibodies used in this IRMA kit are specific for PSA. 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.

Dilution test (linearity)

2 samples were measured in a series of dilution with zero-standard. The following equation obtained for measured (Y) versus expected (X) concentration demonstrates the good linearity:

y = 1.0055x + 0.0444
R = 0,9997
n =10

Expected Values

Healthy adult males: < 3.5 ng/ml
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 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 96 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 operating instructions		Catalogue number		W