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  • 产品名称：APP ELISA Kit(Amyloidbeta(A4)PrecursorProtein)

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  • 产品厂商：半导体
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APP ELISA Kit(Amyloidbeta(A4)PrecursorProtein)
详情介绍：
Purpose The AssayMax™ Amyloid Precursor Protein (APP) ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human APP in plasma, serum, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human APP in less than 4 hours. A polyclonal antibody specific for human APP has been pre-coated onto a 96-well microplate with removable strips. APP in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for APP, 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. 2
Brand AssayMax™
Sample Type Cell Culture Cells, Plasma, Serum
Analytical Method Quantitative
Detection Method Colorimetric
Components Human APP Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human APP. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human APP Standard: Human APP in a buffered protein base (19.2 ng, lyophilized). Biotinylated Human APP Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against APP (120 l). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (20 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)
Alternative Name Amyloid Precursor Protein (APP) (APP ELISA Kit Abstract)
Background Amyloid Precursor Protein (APP), also known as Alzheimer disease amyloid protein, cerebral vascular amyloid peptide (CVAP), protease nexin-II (PN-II) and preA4, is a multidomain glycosylated cell-surface receptor. Generated by alternative splicing, APP has several isoforms with 695 (APP 695), 751 (APP 751) and 770 (APP 770) amino acids. The latter two isoforms contain a 56- amino acid domain that is homologous to Kunitz-type serine protease inhibitors (KPI). APP 695 is a predominant form in neuronal tissues, whereas APP 751 is the predominant variant elsewhere (1-3). Alternative cleavage of APP results in generation and secretion of both soluble APP and a 39-42 amino acid beta-amyloid. By acting as an anticoagulant to inhibit coagulation factors XIa and IXa serine proteases, the KPI-containing isoforms of secreted soluble APP may play a role in regulating hemostasis. Beta-amyloid-42 directly activated neutral sphingomyelinase and downregulated sphingomyelinase levels, whereas beta-amyloid-40 reduced de novo cholesterol synthesis by inhibition of HMG-CoA reductase. These processes were dependent on gamma-secretase activity, suggesting that a proteolytic APP fragment is involved in lipid homeostasis. APP plays important roles in the cellular growth, metabolic responses, cell-cell and cell-surface adhesion in neural and non-neural cells (4-8).
Gene ID 351
UniProt P05067
Research Area Alzheimer's Disease, Neurology
Pathways Caspase Cascade in Apoptosis, EGFR Signaling Pathway, Transition Metal Ion Homeostasis, Skeletal Muscle Fiber Development, Toll-Like Receptors Cascades, Feeding Behaviour
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 12 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. 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 plasma samples 1:2 with 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 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 serum samples 1:2 with 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. 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.
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 APP 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 APP Antibody to each well and incubate for 1 hour. 5 Wash the microplate as described above. Add 50 l of Streptavidin-Peroxidase Conjugate to each 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 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 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, 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.
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. 3 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.
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