Hemopexin ELISA Kit

Catalog No. ABIN5564608

Product Details Hemopexin ELISA Kit

Target: Hemopexin (HPX)

Reactivity: Mouse

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.188-12 ng/mL

Minimum Detection Limit: 0.188 ng/mL

Application: ELISA

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

Analytical Method: Quantitative

Components: Mouse Hemopexin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against mouse hemopexin. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes, which can be cut to fit the format of the individual assay. Mouse Hemopexin Standard: Mouse hemopexin in a buffered protein base (24 ng, lyophilized). Biotinylated Mouse Hemopexin Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against mouse hemopexin (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).

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 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 10 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 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C. Mouse Hemopexin Standard: Reconstitute the 24 ng of Mouse Hemopexin Standard with 1 mL of MIX Diluent to generate a 24 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 (24 ng/mL) 1:2 with MIX Diluent to produce 12, 6, 3, 1.5, 0.75, 0.375, and 0.188 ng/mL solutions. MIX Diluent serves as the zero standard (0 ng/mL). Any remaining solution should be frozen at -20 °C and use within 30 days. 4 Standard Point Dilution [Mouse Hemopexin] (ng/mL) P1 1 part Standard (24 ng/mL) + 1 part MIX Diluent 12.00 P2 1 part P1 + 1 part MIX Diluent 6.000 P3 1 part P2 + 1 part MIX Diluent 3.000 P4 1 part P3 + 1 part MIX Diluent 1.500 P5 1 part P4 + 1 part MIX Diluent 0.750 P6 1 part P5 + 1 part MIX Diluent 0.375 P7 1 part P6 + 1 part MIX Diluent 0.188 P8 MIX Diluent 0.000 Biotinylated Mouse Hemopexin Antibody (50x): Spin down the 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.

Sample Collection: Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris. Collect supernatants and assay. Samples can be stored at -20 °C or below. Avoid repeated freeze-thaw cycles. 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 1:200000 into MIX Diluent or within the range of 50000x - 500000x, 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 1:200000 into MIX Diluent or within the range of 50000x - 500000x, 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. Dilute samples 1:5 into MIX Diluent or within the range of 2x - 20x, 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 Mouse Hemopexin 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 Mouse Hemopexin Antibody to each well and incubate for 1 hour. Wash the microplate as described above. 5 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 10 minutes or till 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. 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 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. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date. 2

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 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 for Hemopexin

Target: Hemopexin (HPX)

Alternative Name: Hemopexin (HPX Products)

Synonyms: HPX ELISA Kit, HX ELISA Kit, Hpxn ELISA Kit, hx ELISA Kit, wu:fi17f11 ELISA Kit, wu:fi19a10 ELISA Kit, hemopexin ELISA Kit, HPX ELISA Kit, Mmar10_1124 ELISA Kit, Hpx ELISA Kit, hpx ELISA Kit

Background: Hemopexin is a heme-binding plasma glycoprotein which, after haptoglobin, forms the second line of defense against hemoglobin-mediated oxidative damage during intravascular hemolysis. A decrease in plasma hemopexin concentration reflects a recent release of heme compounds in the extracellular compartment. Heme-hemopexin complexes are delivered to hepatocytes by receptor-mediated endocytosis after which hemopexin is recycled to the circulation (1).

Gene ID: 15458

UniProt: Q91X72

Pathways: Transition Metal Ion Homeostasis, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response