Description

The PSA ELISA system provides a direct quantitative determination of PSA in human serum. PSA can be assayed in the range of 0-50 ng/ml using 100 µl serum samples. Each kit contains materials sufficient for 96 determinations permitting the construction of one standard curve and the assay of 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 majority (70-90%) of PSA in serum is complexed to alpha₁-antichymotrypsin (ACT). Total PSA (free PSA + PSA-ACT-complex) is increased in both benign prostate hyperplasia and malignant prostate cancer.

The 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 characterised 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 microtiter plate immuno-enzymometric assay (“sandwich” ELISA) system is characterised with an equimolar response to these two forms of PSA, and has been functionally calibrated against FDA-approved immunoassays according to recommendations of international standardisation committees.

Principle of the method

The technology uses two high affinity monoclonal antibodies in an immunometric assay system. The two antibodies react simultaneously with the antigen present in standards or samples This reaction leads to the formation of a capture antibody - antigen - signal antibody complex, also referred to as a “sandwich”. In the standard solid-phase ELISA system the reaction is carried out in a microtiter plate (12 strips) which acts as the binder of sandwich complex.

In the present product standards and samples are incubated with the tracer which contains the horseradish peroxidase (HRPO) labelled antibody at 37°C for 2 hours, then washed repeatedly. After the addition of a ready-to-use tetramethyl-benzidine (TMB)/peroxide substrate the signal is measured in an ELISA photometer at 450 nm wavelength.

The concentration of antigen is directly proportional to the optical density measured in the wells. The unknown concentration of PSA in patient samples is read off a calibration curve constructed by plotting binding values against a series of calibrators containing known amount of PSA.

Contents of the kit

Materials, and equipment required

Precision micropipettes (100 and 200 µl)
Multi-channel ELISA pipettes
Disposable plastic reagent-trays (separate for each reagent)
ELISA thermostate
ELISA photometer
Shaker
Vortex mixer
distilled water

Preparation of reagents

Except the wash buffer, each reagent is supplied in ready to use form.
Add the wash buffer concentrate (20 ml) to 600 ml distillated water to obtain 620 ml washing solution.
For use in the assay allow all regents to equilibrate to room temperature.

Specimen collection and storage

Serum samples can be prepared according to common proce-dures 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 of measured, or expected, concentration > 100 ng/ml can be (re)assayed after dilution with physiologic saline.

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

Calculation of results

The calculation is illustrated using representative data. Data obtained should be similar to those shown in Table 2.

Manual calculation

Calculate the average OD for each pair of duplicates. Substract the mean NSB from all (standars and samples) mean ODs. Draw the standard curve on a lin-lin graph paper by plotting calculated OD of each standard level (ordinate) against the respective concentration (abscissa). Obtain sample values by interpolation of sample counts on the standard curve.

Data evaluation using normalised binding

For computerised calculations and/or quality assessment normalised specific binding values, rather than A.U values are used. Specific binding values can be calculated for each standard and sample according to the following equation:

S₀ is the zero-binding, or non-specific binding (NSB).

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

Characterisation of the assay

Assay parameters

B₀ / B_max           < 5 %

Sensitivity

For the analytical sensitivity 0.5 ng/ml has been obtained by assaying 15 replicates of the zero standard. It is defined as the concentration corresponding to the sum of the mean cpm and its double standard deviation.

For the functional sensitivity 1 ng/ml has been obtained by measuring low-level sera in 27 independent run. This value is defined as the concentration intercept at 22% CV of the inter-assay imprecision curve.

Specificity

Monoclonal antibodies do not cross-react (< 0.04 %) with prostate acidic phosphatase and human kallikrein.

Hook effect

There is no high dose “hook effect” up to the PSA concentration of 2000 ng/ml.

Precision and reproducibility

6 samples were assayed with 15 replicates within one run, and with duplicates in 15 runs, to determine the intra-assay, and inter-assay precision, respectively. Values obtained are shown below.

Normal values

Healthy male: =< 4 ng/ml.

It is recommended that each laboratory establish its own reference intervals.
The results obtained should only be interpreted in the context of the overall clinical picture. None of 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 2-8 °C. At this temperature each reagent is stable until expiry date. Unused reagents left from a partial working up can be stored for a later use under the same conditions.

Availability

From stock.

Shelf life

The minimum shelf life of kit reagents is usually 16 weeks from the date of manufacturing. The actual expiry date is given on the 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.

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.