PRTN3 ELISA Kit

Catalog No. ABIN5564550

Product Details PRTN3 ELISA Kit

Target: PRTN3 (Proteinase 3 (PRTN3))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1.875-120 ng/mL

Minimum Detection Limit: 1.875 ng/mL

Application: ELISA

Purpose: The AssayMax™ Human Proteinase 3 ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of PR3 in human plasma, serum, saliva, urine, milk, CSF, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures PR3 in approximately 4 hours. A polyclonal antibody specific for PR3 has been pre- coated onto a 96-well microplate with removable strips. PR3 in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for PR3, which is recognized by a streptavidin- peroxidase conjugate. All unbound material is washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Brand: AssayMax™

Sample Type: Cell Culture Cells, Cerebrospinal Fluid, Milk, Plasma, Saliva, Serum, Urine

Analytical Method: Quantitative

Components: Human Proteinase 3 Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human PR3. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes, which can be cut to fit the format of the individual assay. Human Proteinase 3 Standard: Human PR3 in a buffered protein base (180 ng, lyophilized). Biotinylated Human Proteinase 3 Antibody (50x): A 50-fold biotinylated polyclonal antibody against PR3 (120 l). MIX Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 l). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).

Material not included: Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water Incubator (37 °C)

Application Details

Plate: Pre-coated

Protocol:

• Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
• Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 1 hour.
• Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
• Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 25 minutes.
• Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. MIX Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the MIX Diluent Concentrate 10-fold with reagent grade water. Store for up to 30 days at 2-8 °C. Human Proteinase 3 Standard: Reconstitute the 180 ng of Human Proteinase 3 Standard with 1.5 mL of MIX Diluent to generate a 120 ng/mL standard stock solution. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution (120 ng/mL) 2-fold with MIX Diluent to produce 60, 30, 15, 7.5, 3.75, and 1.875 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Any remaining stock solution should be frozen at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. Standard Point Dilution [PR3] (ng/mL) P1 1 part Standard (120 ng/mL) 120 P2 1 part P1 + 1 part MIX Diluent 60 P3 1 part P2 + 1 part MIX Diluent 30 P4 1 part P3 + 1 part MIX Diluent 15 P5 1 part P4 + 1 part MIX Diluent 7.5 P6 1 part P5 + 1 part MIX Diluent 3.75 P7 1 part P6 + 1 part MIX Diluent 1.875 P8 MIX Diluent 0.0 Biotinylated Human Proteinase 3 Antibody (50x): Spin down the biotinylated antibody briefly and dilute the desired amount of the antibody 50-fold with MIX Diluent. The undiluted antibody should be stored at -20 °C. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 20-fold with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent. The undiluted conjugate should be stored at -20 °C. 5

Sample Collection: Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes and collect plasma. A 10-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as an anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes and remove serum. A 10-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using samples tube. Centrifuge samples at 800 x g for 10 minutes. A 50-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes. A 5-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Urine: This kit can be used to detect high PR3 levels in human urine. Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes. Samples can be stored at -20 ° C or below for up to 3 months. Avoid repeated freeze-thaw cycles. CSF: Collect cerebrospinal fluid (CSF) using sample pot. Centrifuge samples at 3000 x g for 10 minutes. A 2-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -80 °C for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes at 4 °C to remove debris and collect supernatants. Samples can be stored at -20 °C or below. Avoid repeated freeze-thaw cycles. 4

Assay Procedure:

Prepare all reagents, standard solutions, and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-25 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Human Proteinase 3 Standard or sample to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 l of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid. Add 50 l of Biotinylated Human Proteinase 3 Antibody to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 1 hour. Wash the microplate as described above. Add 50 l of Streptavidin-Peroxidase Conjugate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 l of Chromogen Substrate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 25 minutes or until the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings. 6

Calculation of Results:

• Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
• To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance (OD) on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit.
• Determine the unknown sample concentration from the standard curve and multiply the value by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (diluent buffer, wash buffer, standard, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this insert. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents. The Stop Solution is an acidic solution. 2 The kit should not be used beyond the expiration date.

Storage: 4 °C,-20 °C

Storage Comment: Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. Unused microplate wells may be returned to the foil pouch with the desiccants and resealed. May be stored for up to 30 days in a vacuum desiccator. Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Target Details for PRTN3

Target: PRTN3 (Proteinase 3 (PRTN3))

Alternative Name: Proteinase 3 (PR3, cANCA) (PRTN3 Products)

Synonyms: ela2 ELISA Kit, MGC132250 ELISA Kit, ACPA ELISA Kit, AGP7 ELISA Kit, C-ANCA ELISA Kit, CANCA ELISA Kit, MBN ELISA Kit, MBT ELISA Kit, NP-4 ELISA Kit, NP4 ELISA Kit, P29 ELISA Kit, PR-3 ELISA Kit, PR3 ELISA Kit, mPR3 ELISA Kit, proteinase 3 L homeolog ELISA Kit, proteinase 3 ELISA Kit, prtn3.L ELISA Kit, PRTN3 ELISA Kit, Prtn3 ELISA Kit

Background: Proteinase 3 (PR3, PRTN3), a neutral serine proteinase, is also known as myeloblastin, Wegener autoantigen, azurophil granule protein 7, and neutrophil protease p29 (1). PR3 is produced and packaged into azurophil granules during neutrophil differentiation. The mature PR3 consists of 228 amino acids and has a molecular mass of approximately 29 kDa (2). PR3 degrades connective-tissue proteins, particularly elastin, fibronectin, type IV collagen, and laminin (3). It has potent antimicrobial activity and is involved in a variety of immune defense reactions that contribute to the destruction of ingested microorganisms (4).

Gene ID: 5657

UniProt: P24158