Proteinase 3 Antineutrophil Cytoplasmic Antibody (PR3 ANCA) ELISA Kit

Catalog No. ABIN5564549

Product Details

Target: Proteinase 3 Antineutrophil Cytoplasmic Antibody (PR3 ANCA)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1.875-30 U/mL

Minimum Detection Limit: 1.875 U/mL

Application: ELISA

Purpose: The AssayMax™ Human Proteinase 3 Autoantibody ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative determination of autoimmune response (IgG) to a target antigen (Proteinase 3). The kit detects autoantibodies in plasma and serum samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures autoantibodies (anti-Proteinase 3 IgG) in less than 4 hours. A proteinase 3 antigen has been pre-coated onto a 96-well microplate with removable strips. Autoantibody specific for proteinase 3 in standards and samples is sandwiched by the immobilized antigen and an antibody-HRP 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: Plasma, Serum

Analytical Method: Quantitative

Components: Human Proteinase 3 Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with proteinase 3. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Proteinase 3 Standard: Plasma standard (15 AU, lyophilized). HRP Conjugate (50x): A 50-fold concentrated HRP-antibody conjugate (120 l). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 1 bottle). 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

Assay Time: 4 h

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 HRP Conjugate per well. Incubate 1 hour.
• Step 3. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 30 minutes.
• Step 4. 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. EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C.

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. Dilute samples 1:40 into EIA Diluent and assay. 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. Dilute samples 1:40 into EIA Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles.

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 Standard or sample per well. 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 HRP Conjugate to each well and incubate for 1 hour. Wash the microplate as described above. Add 50 l of Chromogen Substrate per well and incubate for 30 minutes or till the optimal blue color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. 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.

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 (working diluent buffer, wash buffer, standard, HRP 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. 2 Spin down the HRP conjugate vial before opening and using contents. The Stop Solution is an acidic solution. 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 HRP Conjugate 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 desiccant packs 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

Target: Proteinase 3 Antineutrophil Cytoplasmic Antibody (PR3 ANCA)

Alternative Name: Proteinase 3 (PR3, cANCA) Autoantibody

Target Type: Antibody

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, in particular 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. Autoantibodies to PR3 are involved in the pathogenesis of autoimmune-mediated vasculitis in granulomatosis with polyangiitis (4).

Gene ID: 5657

UniProt: P24158