Total Prostate Specific Antigen (tPSA) ELISA Kit

Catalog No. ABIN625079

Product Details

Target: Total Prostate Specific Antigen (tPSA)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 8-2000 pg/mL

Minimum Detection Limit: 8 pg/mL

Application: ELISA

Purpose: Human PSA-total ELISA Kit for cell culture supernatants, plasma, and serum samples.

Sample Type: Plasma, Cell Culture Supernatant, Serum

Analytical Method: Quantitative

Specificity: This ELISA kit shows no cross-reactivity with the following cytokines tested: human Angiogenin, BDNF, BLC, CNTF, ENA- 78, FGF-4, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, I-309, IP-10, FGF-4, FGF-6, FGF- 7, G-CSF, GDNF, GM-CSF, IFN-gamma, IGFBP-2, IGFBP-3, IGFBP-4, Leptin (OB), MCP-1, MCP-2, MCP-3, MDC, MIF, MIG, MIP-1 alpha, MIP-1 beta, MIP-1 delta, PARC, PDGF, RANTES, SCF, SDF-1 alpha, TARC, TGF-beta, TIMP-1, TIMP-2, TNF-alpha, TNF-beta, TPO, VEGF.

Sensitivity: < 8 pg/mL

Characteristics:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Components:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Material not included:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis

Application Details

Application Notes: Recommended Dilution for serum and plasma samples2 fold

Sample Volume: 100 μL

Plate: Pre-coated

Protocol:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Reagent Preparation:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: Assay Diluent A (Item D) should be used for dilution of serum/plasma samples, 1x Assay Diluent B (Item E) should be used for dilution of cell culture supernates/urine. Suggested dilution for normal serum/plasma: 2 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
   3. Assay Diluent B should be diluted 5-fold with deionized or distilled water before use.
   4. Preparation of standard: Briefly spin the vial of Item C and then add 400 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture supernates/urine) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 50 µL PSA-total standard (50 ng/mL) from the vial of Item C, into a tube with 950 µL Assay Diluent A or 1x Assay Diluent B to prepare a 2,500 pg/mL standard solution. Pipette 300 µL Assay Diluent A or 1x Assay Diluent B into each tube. Use the 2,500 pg/mL standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent A or 1x Assay Diluent B serves as the zero standard (0 pg/mL). 50 µL standard + 950 µL 200 µL 200 µL 200 µL 200 µL 200 µL 200myl 2,500 1,000 400 160 64 25.6 10.24 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
   5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
   6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent B into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 500-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 20 µL of HRP-Streptavidin concentrate into a tube with 10 Ml 1x Assay Diluent B to prepare a 500-fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.

Assay Procedure:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
   5. Discard the solution. Repeat the wash as in step
   6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
   7. Discard the solution. Repeat the wash as in step
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Calculation of Results:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent A Human PSA-total concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 n m 0.01 0.1 1 10 Assay Diluent B Human PSA-total concentration (pg/mL) 1 10 100 1000 10000 O D =4 50 n m 0.01 0.1 1 10
Sensitivity: The minimum detectable dose of PSA-total is typically less than 8 pg/mL.
Recovery: Recovery was determined by spiking various levels human PSA-total into human serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 105.4 94-112 Plasma 80.63 72-94 Cell culture media 95.25 81-108
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 112.5 107.3 84.77 Range ( %) 103-120 100-115 76-92 1:4 Average % of Expected 87.91 97.14 72.89 Range ( %) 77-95 79-107 67-78
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Assay Precision: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Restrictions: For Research Use only

Handling

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: -20 °C

Storage Comment: The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Expiry Date: 6 months

References

Fawzy, Elfayoumi, Mohamed, Fatah, Saadawy: "Cyclooxygenase 2 (rs2745557) Polymorphism and the Susceptibility to Benign Prostate Hyperplasia and Prostate Cancer in Egyptians." in: Biochemical genetics, Vol. 54, Issue 3, pp. 326-36, (2017) (PubMed).

Garcia-Cordero, Maerkl: "A 1024-sample serum analyzer chip for cancer diagnostics." in: Lab on a chip, Vol. 14, Issue 15, pp. 2642-50, (2014) (PubMed).

Target Details

Target: Total Prostate Specific Antigen (tPSA)

Abstract: tPSA Products

Synonyms: APS ELISA Kit, KLK2A1 ELISA Kit, PSA ELISA Kit, hK3 ELISA Kit, KLK3 ELISA Kit, Klk1c10l2 ELISA Kit, RSGK-50 ELISA Kit, RSKG-50 ELISA Kit, rGK-3 ELISA Kit, kallikrein related peptidase 3 ELISA Kit, kallikrein 1-related peptidase C3 ELISA Kit, KLK3 ELISA Kit, Klk1c3 ELISA Kit

Background: The Human PSA-total (Prostate Specific Antigen, total) ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human PSA-total in serum, plasma, cell culture supernatants and urine. This assay employs an antibody specific for human PSA-total coated on a 96-well plate. Standards and samples are pipetted into the wells and PSA-total present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human PSA-total antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of PSA-total bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.

Gene ID: 354

UniProt: P07288