DRG PSA Total ELISA Instructions

DRG PSA Total ELISA

Please use only the valid version of the Instructions for Use provided with the kit.

INTENDED USE

The DRG PSA Total ELISA is an enzyme immunoassay for the quantitative in vitro diagnostic measurement of total Prostate-specific Antigen concentration (t-PSA) in serum or plasma (EDTA, lithium heparin or citrate plasma). The determination of PSA levels is used to estimate the risk of prostate carcinoma in men in conjunction with digital rectal examination (DRE) or to monitor the effectiveness of prostate carcinoma treatment in patients.

Summary and Explanation

Prostate-specific antigen (PSA), also known as gamma-seeming protean or kallikrein-3 (KLK3), is a glycoprotein enzyme of the kallikrein-related peptidase family. PSA is secreted by the epithelial cells of the prostate gland in very high concentrations to the ejaculate, where it liquefies semen in the seminal coagulum and dissolves cervical mucus, allowing the entry of sperm into the uterus (1,2). PSA circulates in blood in much lower concentrations. The main form of immunoreactivity PSA is bound by alpha-1 ant chymotrypsin (PSA-ACT), representing approximately 70-80% of the total PSA in the circulation, while the free (uncompleted) PSA (fossa; enzymatically inactive) represents 20-30% in serum (3,4). Furthermore, PSA bound to alpha-2 macroglobulin exists in less than 0.1% (undetectable by commercial tests). In male serum, the normal PSA concentration range is < 4 ng/mL while elevated concentrations of PSA are found in many carcinomas (5). However, increased PSA levels are not only found in patients with prostate cancer, but also in those with a diagnosis of benign prostatic hyperplasia (BPH), acute, subclinical or chronic prostatitis and urinary retention. Analysis of PSA levels in combination with digital rectal examination (DRE) further increase the chance of early detection of prostate cancer. In addition to total PSA, the most useful diagnostic index for distinguishing benign hypertrophy from prostate cancer is the free to total PSA ratio. In order to achieve even better specificity in early detection of prostate cancer, the following indexes may be determined: age-specific PSA, PSA density, acceleration of PSA, and PSA density of the transition zone (6-11). The determination of PSA serum levels is not only important for the screening of patients for prostate cancer, but also for
monitoring patients who have been treated for this disease. Here regular PSA measurements are an important tool to examine the potential and actual effectiveness of surgery or other therapies. An increase of PSA in patients after radical prostatectomy or radiotherapy may allow an earlier discovery of residual or recurrent carcinoma (12-14). The American Cancer Society recommends to offer PSA blood test and the digital rectal examination annually, beginning at age 50, to men who are at average risk of prostate cancer and who have a life expectancy of at least 10 years. Men at high risk of developing prostate cancer (African Americans or men with a close relative diagnosed with prostate cancer before age 65) should be tested beginning at age 45 (15,16).

PRINCIPLE OF THE TEST

The DRG PSA Total ELISA is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. The microtiter wells are coated with a monoclonal (mouse) antibody directed towards a unique antigenic site of the PSA molecule. During incubation, the PSA molecules in the added sample bind to the immobilized antibody. The added enzyme conjugate, which contains an anti- PSA antibody conjugated to horseradish peroxidase, binds to the PSA forming a sandwich complex. After a washing step to remove all unbound substances, the solid phase is incubated with the substrate solution. The colorimetric reaction is stopped by addition of stop solution, and optical density (OD) of the resulting yellow product is measured. The intensity of color is proportional to the concentration of the analyte in the sample. A standard curve is constructed by plotting OD values against concentrations of standards, and concentrations of unknown samples are determined using this standard curve.

WARNINGS AND PRECAUTIONS

1. This kit is for in vitro diagnostic use only. For professional use only.
2. All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal.
3. Before starting the assay, read the instructions completely and carefully. Use the valid version of instructions for use provided with the kit. Be sure that everything is understood.
4. The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil pouch and used in the frame provided.
5. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
6. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution cultured. Do not pour reagents back into vials as reagent contamination may occur.
7. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse micro wells.
8. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
9. Allow the reagents to reach room temperature (20 °C to 26 °C) before starting the test. Temperature will affect the optical density readings of the assay. However, values for the patient samples will not be affected.
10. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
11. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
12. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
13. Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
14. Do not use reagents beyond expiry date as shown on the kit labels.
15. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiter plate readers.
16. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different.
17. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
18. Some reagents contain Proclaim 300, BND and/or MIT as preservatives. In case of contact with eyes or skin, flush immediately with water.
19. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open air.
20. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation.
21. For information on hazardous substances included in the kit please refer to Safety Data Sheets. Safety Data Sheets for this product are available upon request directly from DRG.

REAGENTS

Reagents provided

1. Microtiter wells, 12 x 8 (break apart) strips, 96 wells; Wells coated with anti-PSA antibody (monoclonal).
2. Zero Standard, 1 vial, 10.0 mL, ready to use. (Sample Diluent) Contains non-mercury preservative.
3. Standard (Standard 1 – 5), 5 vials, 0.5 mL each, ready to use; Concentrations: 1.56 – 3.12 – 6.25 – 12.5 – 25.0 ng/mL The standards are calibrated against the following reference material: WHO International Standard Prostate Specific Antigen NIBSC code: 96/670 Contain non-mercury preservative.
4. Control Low & High, 2 vials, 0.5 mL each, ready to use; For control values and ranges please refer to vial label or Certificate of Analysis. Contain non-mercury preservative.
5. Enzyme Conjugate, 1 vial, 12 mL, ready to use; Anti-PSA antibody conjugated with horseradish peroxidase; Contains non-mercury preservative.
6. Substrate Solution, 1 vial, 12 mL, ready to use; Tetramethylbenzidine (TMB).
7. Stop Solution, 1 vial, 14 mL, ready to use; Contains 0.5 M H2SO4, Avoid contact with the stop solution. It may cause skin irritations and burns.

Materials required but not provided

• A calibrated microtiter plate reader (450 nm, with reference wavelength at 620 nm to 630 nm)
• Calibrated variable precision micropipettes
• Absorbent paper
• Distilled water
• Timer
• Graph paper or software for data reduction

Storage Conditions

When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.
Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once the foil bag has been opened, care should be taken to close it tightly again. Opened kits retain activity for 8 weeks if stored as described above.

Reagent Preparation

Bring all reagents and required number of strips to room temperature (20 °C to 26 °C) prior to use.

Disposal of the Kit

The disposal of the kit and all used materials/reagents must be performed according to the national regulations. Special information for this product is given in the Safety Data Sheet, section 13.

Damaged Test Kits

In case of any damage to the test kit or components, DRG must be informed in writing, at the latest one week after receiving the kit. Damaged single components should not be used for a test run. They have to be stored until a final solution has been found. After this, they should be disposed of according to the official regulations.

SPECIMEN COLLECTION AND PREPARATION

Serum or plasma (EDTA, lithium heparin or citrate plasma) can be used in this assay.
Note: Samples containing sodium aside should not be used in the assay. In general, it should be avoided to use hemolytic, icteric, or lipemic specimens. For further information refer to chapter “Interfering Substances”.

Important notes before blood drawing for PSA determination:
As different factors could influence the PSA level in blood, doctors should ensure that the patient has avoided the following conditions before taking the blood sample.

The following conditions may lead to an increase of PSA levels

• Manipulation of the prostate during medical examinations like digital rectal examination (DRE), transrectal prostatic ultrasound, etc.
• Prostatitis
• Biking
• Sexual intercourse (ejaculation)
• Liver dysfunction

The following conditions may lead to a decrease of PSA levels

• Intake of 5-alpha-reductase-inhibitors, antiandrogens, or GnRH analog

Specimen Collection

Serum:
Collect blood by venipuncture (e.g. Sestet Monolete for serum), allow to clot, and separate serum by centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Patients receiving anticoagulant therapy may require increased clotting time.

Plasma:
Whole blood should be collected into centrifuge tubes containing anticoagulant (e.g. Sestet Monolete with the appropriate plasma preparation) and centrifuged immediately after collection.

Specimen Storage and Preparation

Specimens should be capped and may be stored for up to 7 days at 2 °C to 8 °C prior to assaying. Specimens stored for a longer time (up to 12 months) should be frozen only once at -20 °C prior to assay. Thawed samples should be inverted several times prior to testing.

Specimen Dilution

If in an initial assay, a specimen is found to contain more analyte than the highest standard, the specimen can be diluted with Zero Standard and re- assayed as described in “Assay Procedure”. For the calculation of the concentrations this dilution factor has to be taken into account.

Example:

1. dilution 1:10: 10 µL sample + 90 µL Zero Standard (mix thoroughly)
2. dilution 1:100: 10 µL dilution a) 1:10 + 90 µL Zero Standard (mix thoroughly).

ASSAY PROCEDURE

General Remarks

• All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming.
• Once the test has been started, all steps should be completed without interruption.
• Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination.
• Optical density is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.
• As a general rule the enzymatic reaction is linearly proportional to time and temperature.

Test Procedure

Each run must include a standard curve.
Note: It is highly recommended to perform all measurements as duplicates.

1. Secure the desired number of Microtiter wells in the frame holder.

2. Dispense 25 µL of each Standard, Control and sample with new disposable tips into appropriate wells.

3. Incubate for 5 minutes at room temperature.

4.  Dispense 100 µL Enzyme Conjugate into each well.
   Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

5. Incubate for 60 minutes at room temperature.

6. Rinse the wells 5 times with 400 µL distilled water per well, if a plate washer is used. – OR – Briskly shake out the contents of the wells. Rinse the wells 5 times with 300 µL distilled water per well for manual washing. Strike the wells sharply on absorbent paper to remove residual droplets.
   Important note:
   The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure!

7. Add 100 µL of Substrate Solution to each well.

8. Incubate for 20 minutes at room temperature.

9. Stop the enzymatic reaction by adding 100 µL of Stop Solution to each well.

10. Determine the optical density of the solution in each well at 450 nm (reading) and at 620 nm to 630 nm (background subtraction, recommended) with a microtiter plate reader. It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

Calculation of Results

1. Calculate the average optical density (OD) values for each set of standards, controls and patient samples.
2. Using graph paper, construct a standard curve by plotting the mean OD obtained from each standard against its concentration with OD value on the vertical (Y) axis and concentration on the horizontal (X) axis.
3. Using the mean OD value for each sample determine the corresponding concentration from the standard curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a 4-Parameter curve fit. (4-Parameter Rodbard or 4-Parameter Marquardt are the preferred methods.) Other data reduction functions may give slightly different results.
5. The concentration of the samples can be read directly from this standard curve. Samples with concentrations higher than that of the highest standard have to be further diluted or reported as > 25 ng/mL. For the calculation of the concentrations this dilution factor has to be taken into account.

Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the time of assay

EXPECTED NORMAL VALUES

It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with men, using the DRG PSA Total ELISA the following data were observed:

The generally recommended threshold for follow-up examinations is:

Cut-off value PSA: 4.0 ng/mL
Healthy men generally have a PSA concentration lower than 4.0 ng/mL.
If the PSA concentration is equal or higher than 4.0 ng/mL, follow-up examinations are highly recommended.
This PSA concentration indicates an elevated risk for prostate cancer but might also be caused by benign prostatic hyperplasia (BPH).
Please note that the 4 ng/mL threshold is only a guideline value.
In the literature it is discussed that modifications according to age and ethnological background might be useful e.g. that for younger men the threshold should be lower than for older men.
It is important to keep in mind that some prostate tumors do not cause elevated PSA levels so that PSA measurements should never replace DRE but should only be used in conjunction with digital rectal examination (DRE).
As elevated PSA levels might also be caused by non-cancerous conditions follow-up examinations might try to increase the diagnostic specificity of total PSA values.
In the literature PSA density, PSA velocity and the ratio of free PSA to total PSA (f-PSA ∕ t-PSA) are discussed to improve discrimination between cancerous and non-cancerous conditions and might be used to reduce unnecessary prostate biopsies.
But only a prostate biopsy can finally show if a prostate carcinoma is present or not.
Note: PSA values can only be used to estimate the cancer risk.
They should always be interpreted in conjunction with other clinical findings and should not be used as a sole basis for prostate cancer diagnosis.
The results alone should not be the only reason for any therapeutic consequences. The results should be correlated to other clinical observations and diagnostic tests.

QUALITY CONTROL

Good laboratory practice requires that controls be run with each calibration curve. A statistically significant number of controls should be assayed to establish mean values and acceptable ranges to assure proper performance. It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day-to-day validity of results. Use controls at both normal and pathological levels. The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results. It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results. Employ appropriate statistical methods for analyzing control values and trends. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid. In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above-mentioned items without finding any error contact your distributor or DRG directly.

PERFORMANCE CHARACTERISTICS

Assay Dynamic Range

The range of the assay is between 0.2 ng/mL – 25.0 ng/mL.

Specificity of Antibodies (Cross-Reactivity)

The following substances were tested for cross-reactivity of the assay. No interference with the assay was found for:

Additional data for specificity of antibodies were obtained from verification study of the DRG: Hybrid- XL PSA (HYE-5730). This assay uses the same antibodies and reagents as EIA-3719 but was adapted to the fully automated system DRG :Hybrid- XL. For method comparison studies, please refer to chapter 9.7.

The following substance was tested for cross-reactivity. No interference with the assay was found for:

Sensitivity

The analytical sensitivity of the DRG ELISA was calculated by adding 2 standard deviations to the mean of 20 replicate analyses of the Zero Standard and was found to be 0,054 ng/mL.

• The Limit of Blank (LoB) is 0.045 ng/mL.
• The Limit of Detection (LoD) is 0.216 ng/mL.
• The Limit of Quantification (LoQ) is 1.172 ng/mL.

Reproducibility

Intra-Assay
The within-assay variability was determined by measuring each sample 24 or 32 times per run:

Inter-Assay
The between-assay variability was determined by measuring each sample with 3 different lots:

Inter-Lot
The inter-assay (between-lots) variation was determined by measuring each sample 6 times with 3 different kit lots:

Inter-Lot
The inter-assay (between-lots) variation was determined by measuring each sample 6 times with 3 different kit lots:

Linearity
Samples were measured undiluted and in serial dilutions with standard 0. The recovery (%) was calculated by multiplying the ratio of expected and measured values with 100.

Comparison Studies

A comparison of the DRG PSA Total ELISA (EIA-3719) (y) and the reference method Access Hygrotech PSA (x) using clinical samples gave the following correlation:

• y = 1.108x – 0.054, R2 = 0.952, n = 57

A comparison of the DRG PSA Total ELISA (EIA-3719) (y) and the reference method DRG: Hybrid- XL PSA (HYE-5370) (x) using clinical samples gave the following correlation:

• y = 1.172 + 0.583, R2 = 0.992, n = 57

A comparison of the DRG: Hybrid- XL PSA (HYE-5370) (y) and the reference method Roche cobas ECLIA (x) using clinical samples gave the following correlation:

• y = 0.955x + 0.434, R2 = 0.990, n = 62

LIMITATIONS OF USE

Reliable and reproducible results will be obtained when the assay procedure is performed with a complete understanding of the package insert instruction and with adherence to good laboratory practice. Any improper handling of samples or modification of this test might influence the results.

Interfering Substances

Hemoglobin (up to 0.1 mg/mL), Bilirubin (up to 0.2 mg/mL) and Triglyceride (up to 15 mg/mL) have no influence on the assay results.

Drug Interferences

The following cytostatic drugs were tested. No interference with the assay was found for:

Furthermore, the following hypertension drugs were tested. No interference with the assay was found for:

In addition, the following antimicrobial agent was tested.

The antimicrobial agent Benzalkonium Chloride (0.5%) shows no interference with the assay.

Additional data were obtained from verification study of the Hybrid PSA (HYE-5730). This assay uses the same reagents as EIA-3719 but was adapted to the fully automated system DRG: Hybrid- XL. For method comparison studies, please refer to chapter 9.7.

The following cytostatic drug were tested. No interference with the assay was found for:

Until today no other substances (drugs) are known to us, which have an influence on the measurement of PSA in a sample.

High-Dose-Hook Effect

Hook effect was not observed in this test up to a concentration of 2000 ng/mL of PSA.

LEGAL ASPECTS

Reliability of Results

The test must be performed exactly as per the manufacturer’s instructions for use. Moreover, the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws. This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of
controls for validating the accuracy and precision of the test. The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. In case of any doubt or concern please contact DRG.

Therapeutic Consequences

Therapeutic consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 11.1. Any laboratory result is only a part of the total clinical picture of a patient. Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient should therapeutic consequences be derived. The test result itself should never be the sole determinant for deriving any therapeutic consequences.

Liability

Any modification of the test kit and/or exchange or mixture of any components of different lots from one test kit to another could negatively affect the intended results and validity of the overall test. Such modification and/or exchanges invalidate any claim for replacement. Claims submitted due to customer misinterpretation of laboratory results subject to point 11.2 are also invalid. Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit. Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer.

References

• Analyticon® Biotechnologies GmbH
• It's Uptime | International® Trucks
• DRG Diagnostics GmbH | Home
• Medical Supplies & Diagnostic Products | DRG International, Inc.

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