Haptoglobulin ELISA Kit

Antigen: Haptoglobulin

Reaktivität: Schwein

Applikation: ELISA

Produktnummer: ABIN612758

Produktbeschreibung

Produktmerkmale: The minimum detectable dose of Haptoglobulin is typically ~1.8 ng/ml. Intra-assay and inter-assay coefficients of variation were 5.2% and 7.3 % respectively.

Beschreibung: Haptoglobulin (Hpt) is a plasma protein with hemoglobin-binding capacity, and a plasma glycoprotein that forms a stable complex with hemoglobin to aid the recycling of heme iron. It is a well-known marker of hemolysis (1). High haptoglobulin levesl in plasma has been associated with an increased cardiovascular risk in obese men (2), inflammation (3), atherosclerosis (4), and systemic sclerosis (5). Principal of the Assay The AssayMax Swine Haptoglobulin ELISA kit is designed for detection of swine haptoglobulin in plasma, urine, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures haptoglobulin in less than 4 hours. A polyclonal antibody specific for haptoglobulin has been pre-coated onto a microplate. Haptoglobulin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for haptoglobulin, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured. Caution and Warning: Prepare all reagents (working diluent buffer, wash buffer, standards, 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 protocol. 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. This kit is for research use only. The kit should not be used beyond the expiration date. The Stop Solution is an acid solution.

Spezifität: Cross Reactivity: Beagle, Bovine, Monkey, Mouse, Rat, Human, Swine. 10% FBS in culture media will not affect the assay.

Anwendungen

Protokoll: Sample collection and storage:
Cell Culture Supernatants: Centrifuge cell culture media at 2000 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 Urine: Collect urine using sample pot. Centrifuge samples at 600 x g for 10 minutes. Urine dilution is suggested at 1:50 in MIx Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes. Dilute samples 1:40000 with MIx Diluent. Store the remaining samples at -20°C or below. Avoid repeated freeze-thaw cycles. (EDTA or Heparin can also be used as anticoagulant.)
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 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 240 ng of Swine Haptoglobulin Standard with 2 ml of MIx Diluent to generate a solution of 120 ng/ml. 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 solution (120 ng/ml) 1:2 with MIx Diluent to produce 60, 2 30, 15, 7.5, 3.75, and 1.875 ng/ml solutions. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at < -20°C. Standard Point Dilution [Haptoglobulin] (ng/ml) P1 Standard (120 ng/ml) 120.0 P2 1 part P1 + 1 part MIx Diluent 60.00 P3 1 part P2 + 1 part MIx Diluent 30.00 P4 1 part P3 + 1 part MIx Diluent 15.00 P5 1 part P4 + 1 part MIx Diluent 7.500 P6 1 part P5 + 1 part MIx Diluent 3.750 P7 1 part P6 + 1 part MIx Diluent 1.875 P8 MIx Diluent 0.000 Biotinylated Haptoglobulin 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.
Assay procedure:
Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-300C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µl of Standard or sample per well. Cover wells and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µl of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µl of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µl of Biotinylated Haptoglobulin Antibody to each well and incubate for one hour. Wash the microplate as described above. Add 50 µl 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 µl of Chromogen Substrate per well and incubate for about 20 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 µl 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. 3

Applikationshinweise: Data Analysis: 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 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. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed. Human PAI-1 Standard Curve 1.0 OD 450 nm 0.1 10 -1 10 0 10 1 [hPAI-1] (ng/ml)

Bestandteile: Swine Haptoglobulin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against swine Haptoglobulin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Swine Haptoglobulin Standard: Swine Haptoglobulin in a buffered protein base (240 ng, lyophilized). 1 Biotinylated Haptoglobulin Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against swine Haptoglobulin (140µl). 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 (80µl). 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 450 nm. Pipettes (1-20 l, 20-200 l, 200-1000 l and multiple channel). Deionized or distilled reagent grade water

Lagerung: Store components of the kit at 2-8°C or -20°C upon arrival 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 Opened unused microplate wells may be returned to the foil pouch with the desiccant packs. Reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator. Diluent (1x) may be stored for up to 1 month at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Forschungsgebiet: Serum/Plasma Proteine