Myeloperoxidase ELISA Kit

Produktnummer ABIN5564581

Produktdetails Myeloperoxidase ELISA Kit

Target: Myeloperoxidase (MPO)

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 0.063-4 ng/mL

Untere Nachweisgrenze: 0.063 ng/mL

Applikation: ELISA

Verwendungszweck: The AssayMax™ Human Myeloperoxidase ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of myeloperoxidase in human plasma, serum, urine, saliva, milk, CSF, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures myeloperoxidase in less than 4 hours. A polyclonal antibody specific for myeloperoxidase has been pre-coated onto a 96-well microplate with removable strips. Myeloperoxidase in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for myeloperoxidase, 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.

Marke: AssayMax™

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

Analytische Methode: Quantitative

Bestandteile: Human Myeloperoxidase Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human myeloperoxidase. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes, which can be cut to fit the format of the individual assay. Human Myeloperoxidase Standard: Human myeloperoxidase in a buffered protein base (2.4 ng, lyophilized). Biotinylated Human Myeloperoxidase Antibody (50x): A 50-fold biotinylated polyclonal antibody against myeloperoxidase (140 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).

Benötigtes Material: 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)

Anwendungsinformationen

Plattentyp: Pre-coated

Protokoll:

• 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 12 minutes.
• Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.

Aufbereitung der Reagenzien:

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 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C. Human Myeloperoxidase Standard: Reconstitute the 2.4 ng of Human Myeloperoxidase Standard with 0.6 mL of MIX Diluent to generate a 4 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 the standard stock solution (4 ng/mL) 1:2 with MIX Diluent to produce 2, 1, 0.5, 0.25, 0.125, and 0.0625 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Any remaining solution should be frozen at -20 °C and used within 30 days. Standard Point Dilution [Myeloperoxidase] (ng/mL) P1 1 part Standard (4 ng/mL) 4.000 P2 1 part P1 + 1 part MIX Diluent 2.000 P3 1 part P2 + 1 part MIX Diluent 1.000 P4 1 part P3 + 1 part MIX Diluent 0.500 P5 1 part P4 + 1 part MIX Diluent 0.250 P6 1 part P5 + 1 part MIX Diluent 0.125 P7 1 part P6 + 1 part MIX Diluent 0.063 P8 MIX Diluent 0.000 5 Biotinylated Human Myeloperoxidase Antibody (50x): Spin down the biotinylated antibody briefly and dilute the desired amount of the antibody 1:50 with MIX Diluent. Any remaining solution should be frozen 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 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with MIX Diluent. Any remaining solution should be frozen at -20 °C.

Probennahme: 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 1:200 into MIX 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 1:200 into MIX Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store the remaining samples at -20 °C or below. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using samples tube. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:2000 into MIX Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes and assay. If necessary, dilute samples within the range of 1x- 10x into MIX Diluent, and assay. 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 pot. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:400000 into MIX Diluent and assay. The undiluted 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. Dilute samples 1:50 into MIX Diluent and assay. The undiluted samples can be stored at -80 °C for up to 3 months. Avoid repeated freeze-thaw cycles. Refer to Sample Dilution Guidelines below for further instruction. 4 Guidelines for Dilutions of 1:100 or Greater (for reference only, please follow the insert for specific dilution suggested) 1:100 1:10000 A) 4 μL sample: 396 μL buffer(100x) = 100 fold dilution Assuming the needed volume is less than or equal to 400 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) = 10000 fold dilution Assuming the needed volume is less than or equal to 400 μL. 1:1000 1:100000 A) 4 μL sample : 396 μL buffer (100x) B) 24 μL of A : 216 μL buffer (10x) = 1000 fold dilution Assuming the needed volume is less than or equal to 240 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) C) 24 μL of B : 216 μL buffer (10x) = 100000 fold dilution Assuming the needed volume is less than or equal to 240 μL.

Testdurchführung:

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 Myeloperoxidase 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 Biotinylated Human Myeloperoxidase Antibody to each well and incubate for 1 hour. Wash the microplate as described above. Add 50 l of Streptavidin-Peroxidase Conjugate per well 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 per well and incubate for 12 minutes or until the optimal blue color density develops. Gently tap 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. 6 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.

Ergebnisberechnung:

• 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.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: This product is for Research Use Only and is Not For Use In Diagnostic Procedures. Prepare all reagents (working 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. 2 The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date.

Lagerung: 4 °C,-20 °C

Informationen zur Lagerung: 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.

Details zu Myeloperoxidase

Target: Myeloperoxidase (MPO)

Abstract: MPO Produkte

Synonyme: mKIAA4033 ELISA Kit, MPO ELISA Kit, LOC100335032 ELISA Kit, POX2' ELISA Kit, XPOX2' ELISA Kit, mpo ELISA Kit, mpo-A ELISA Kit, pmr-1 ELISA Kit, pmr1 ELISA Kit, pox2 ELISA Kit, xpox2 ELISA Kit, myeloperoxidase ELISA Kit, myeloperoxidase L homeolog ELISA Kit, MPO ELISA Kit, Mpo ELISA Kit, LOC100335032 ELISA Kit, mpo.L ELISA Kit

Hintergrund: Myeloperoxidase (MPO), a member of the heme peroxidase superfamily, is secreted by activated neutrophils, monocytes, and some macrophages. The 150 kDa MPO is a tetrameric protein with 2 light subunits and 2 glycosylated heavy subunits bound to a prosthetic heme group (1). This enzyme possesses both peroxidase and chlorination activities which catalyze the production of a potent oxidant hypochlorous acid central to immune defenses (2). However, under pathological conditions, MPO-derived oxidants can also lead to cell and tissue damage (3).

Gen-ID: 4353

UniProt: P05164

Pathways: Chromatin Binding