Free PSA ELISA Kit

Produktnummer ABIN996906

Das Human Free PSA ELISA Kit (ABIN996906) ist ein Colorimetric ELISA Kit zur Detektion von Human Free PSA.

Kurzübersicht für Free PSA ELISA Kit (ABIN996906)

Target: Free PSA (fPSA) (Free Prostate Specific Antigen (fPSA))

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 0-10 ng/mL

Applikation: ELISA

Proben: Serum

Produktdetails Free PSA ELISA Kit

Untere Nachweisgrenze: 0 ng/mL

Verwendungszweck: The f-PSA Enzyme Immunoassay test kit is intended for the quantitative determination of f-PSA in human serum.

Analytische Methode: Quantitative

Spezifität: 98.7%

Sensitivität: 0.05 ng/mL

Benötigtes Material: 1. Precision pipettes: 0.04, 0.4 mL , and 0.4 mL .
2. Disposable pipette tips.
3. Distilled water.
4. Vortex mixer or equivalent.
5. Absorbent paper or paper towels.
6. Graph paper.
7. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-2 OD or greater at 450 nm

Anwendungsinformationen

Kommentare:

Limitations of procedure:
1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence - good laboratory practice.
2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
3. Heterophilic antibodies such as human anti-mouse antibodies (HAMA) are frequently found in the serum of human subjects. Those antibodies can cause severe interference in many immunodiagnostic procedures. This assay has been designed - minimize that kinds of interference. Nevertheless, complete elimination of this interference from all patient specimens cannot be guaranteed. A test result that is inconsistent with the clinical picture and patient history should be interpreted with caution.

Probenmenge: 100 μL

Testdauer: 2 - 3 h

Plattentyp: Pre-coated

Aufbereitung der Reagenzien:

1. All reagents should be brought to room temperature (18-22 °C) and mixed by gently inverting or swirling prior to use. Do not induce foaming.
   2. Dilute 1 volume of Wash Buffer (50x) with 49 volumes of distilled water. For example, Dilute 15 mL of Wash Buffer (50x) into distilled water to prepare 750 mL of washing buffer (1x). Mix well before use.

Aufbereitung der Proben:

1. Blood should be drawn using standard venipuncture techniques and the serum should be separated from the red blood cells as soon as possible. Avoid grossly hemolytic, lipemic, or turbid samples.
   2. Plasma samples collected in tubes containing EDTA, heparin, or oxalate may interfere with the test procedures and should be avoided.
   3. Specimens should be capped and may be stored up to 48 hours at 2-8 °C, prior to assaying. Specimens held for a longer time can be frozen at -20 °C. Thawed samples must be mixed prior to testing. coated antibody-antigen- antibody-enzyme complex, the unbound antibody-enzyme tracers are removed by washing. The horseradish peroxidase activity bound in the wells is then assayed by a colorimetric reaction. The intensity of the color formed is proportional to the concentration of f-PSA present in the sample. Test Free PSA ELISA Method Enzyme Linked Immunosorbent Assay Principle Sandwich Complex
   Detection Range 0-10n/mL
   Sample 100μL Serum
   Specificity 98.7 %

Testdurchführung:

1. Secure the desired number of coated wells in the holder.
   2. Dispense 100 mL of standards, specimens, and controls into appropriate wells.
   3. Dispense 100 mL of sample diluent into each well.
   4. Thoroughly mix for 10 seconds. It is very important to have a complete mixing in this step.
   5. Incubate at 37 °C for 60 minutes
   6. Remove the incubation mixture by emptying plate contents into a suitable waste container.
   7. Rinse and empty the microtiter wells 5 times with washing buffer (1X).
   8. Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets.
   9. Dispense 200 mL of Enzyme Conjugate Reagent into each well. Gently mix for 5 seconds.
   10. Incubate at 37 °C for 60 minutes.
   11. Remove the incubation mixture by emptying plate contents into a suitable waste container.
   12. Rinse and empty the microtiter wells 5 times with washing buffer (1X).
   13. Strike the wells sharply onto absorbent paper to remove residual water droplets.
   14. Dispense 100 mL TMB solution into each well. Gently mix for 5 seconds.
   15. Incubate at room temperature for
   2. minutes in the dark.
   16. Stop the reaction by adding 100 mL of stop solution to each well.
   17. Gently mix for 30 seconds to make sure that the blue color changes completely to yellow.
   18. Using a microtiter plate reader, read the optical density at 450 nm within 2 minutes.
   
   Important Notes:
   1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
   2. It is recommended that if manual pipetting is used, no more than 32 wells be used for each assay run, since pipetting of all standards, specimens and controls should be completed within 3 minutes. A full plate of 96 wells may be used if automated pipetting is available.
   3. Duplication of all standards and specimens, although not required, is recommended.

Ergebnisberechnung:

Calculate the mean absorbance value (A450) for each set of reference standards, controls, and patient samples. Construct a standard curve by plotting the mean absorbance obtained from each reference standard against its concentration in ng/mL on graph paper. The absorbance values are placed on the vertical, or Y-axis, and concentrations on the horizontal, or X-axis. Use the mean absorbance values for each specimen to determine the corresponding concentration of f-PSA in ng/mL from the standard curve. Example of Standard Curve Results of typical standard run with optical density reading at 450nm shown in the Y-axis against f-PSA concentrations shown in the X-axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own standard curve and data.

1. Precision. 1]. Intra-Assay: Concentrations Replicates Mean S.D. % CV Level I 20 0.038 0.005 1
3.1 Level II 20 0.250 0.011
4.4 Level III 20
4.00 0.128
3.2 2]. Inter-Assay: Concentrations Replicates Mean S.D. % CV Level I 20 0.041 0.006 15.8 Level II 20 0.280 0.019
6.8 Level III 20
4.10 0.209
5.1 II. Linearity Two patient sera were serially diluted with 0 ng/mL standard. The average recovery was 108.7 %. f-PSA (ng/mL) Absorbance (450nm) 0 0.006 0.1 0.032 0.5 0.155
2.0 0.597
5.0
1.302
1.0
2.361 Sample A Dilution Expected Observed % Recov. undiluted
8.691
8.691 100 2X
4.346
4.455 10
2.5 4X
2.173
2.290 105.4 8X
1.086
1.258 115.8 16X 0.543 0.617 11
3.6 32X 0.272 0.294 108.1 64X 0.136 0.147 108.1 Average Recovery: 107.6 % Sample B Dilution Expected Observed % Recov. undiluted
7.015
7.015 100 2X
3.508
3.516 100.2 4X
1.754
1.834 104.6 8X 0.877 0.970 110.6 16X 0.438 0.509 116.2 32X 0.219 0.249 11
3.7 64X 0.110 0.136 12
3.6 Average Recovery: 109.8 % III. Recovery Equal parts of diluted patient sera were mixed to test for interference by unknown materials, such as drugs or hormones, in the assay. Concentrations of Free PSA were determined before (original and added) and after (observed). The average recovery was 99.2 %. Sample 1 Sample Orig. Conc Added Expected Observed %Recov A
9.802 0.141
4.972
4.540 9
1.3 B
9.802 0.281
5.042
4.638 9
2.0 C
4.699
2.168
3.434
3.342 97.3 D
2.168
1.170
1.669
1.661 99.5 E 0.563 0.281 0.422 0.423 100.2 F
4.699
1.125
2.912
2.727 9
3.6 G 0.563 0.141 0.352 0.399 11
3.4 Average Recovery: 98.2 % Sample 2 Sample Orig. Conc Added Expected Observed % Recov A
6.825 0.098
3.462
3.301 95.3 B
6.825 0.195
3.510
3.156 89.9 C
3.342
1.563
2.453
2.503 10
2.0 D
1.706 0.781
1.244
1.205 96.9 E 0.391 0.195 0.293 0.327 11
1.6 F
3.342 0.781
2.062
1.964 95.2 G 0.391 0.098 0.245 0.271 110.6 Average Recovery: 100.2 % IV.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Haltbarkeit: 12-14 months

Details zu Free PSA

Target: Free PSA (fPSA) (Free Prostate Specific Antigen (fPSA))

Andere Bezeichnung: Free-PSA

Hintergrund: Human Prostate Specific Antigen (PSA) is a 33 kD serine proteinase which, in human serum, is predominantly bound to alpha 1-antichymotrypsin (PSA-ACT) and alpha 2-macroglobulin (PSA-AMG). Trace amounts of alpha 1-antitrypsin and inter-alpha trypsin inhibitor bound to PSA can also be found. Any remaining PSA is in the free form (f-PSA). Current methods of screening men for prostate cancer utilize the detection of the major PSA- ACT form. Levels of 4.0 ng/ml or higher are strong indicators of the possibility of prostatic cancer. However, elevated serum PSA levels have also been attributed to benign prostatic hyperplasia and prostatitis, leading to a large percentage of false positive screening results. A potential solution to this problem involves the determination of free PSA levels. Preliminary studies have suggested that the percentage of free PSA is lower in patients with prostate cancer than those with benign prostatic hyperplasia. Thus, the measurement of free serum PSA in conjunction with total PSA, can improve specificity of prostate cancer screening in selected men with elevated total serum PSA levels, which would subsequently reduce unnecessary prostate biopsies with minimal effects on cancer detection rates.