DPP4 Kit ELISA

N° du produit ABIN5564599

Détail du produit DPP4 Kit ELISA

Antigène: DPP4 (Dipeptidyl-Peptidase 4 (DPP4))

Reactivité: Humain

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 6.25-400 ng/mL

Seuil minimal de détection: 6.25 ng/mL

Application: ELISA

Fonction: The AssayMax™ Human DPP4 ELISA (Enzyme-Linked Immunosorbent Assay) Kit is designed for detection of DPP4 in human plasma, serum, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human DPP4 in approximately 4 hours. A polyclonal antibody specific for human DPP4 has been pre-coated onto a 96-well microplate with removable strips. DPP4 in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human DPP4, which is recognized by a streptavidin-peroxidase (SP) 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.

Marque: AssayMax™

Type d'échantillon: Cell Culture Cells, Plasma, Serum

Analytical Method: Quantitative

Ingrédients: Human DPP4 Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human DPP4. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human DPP4 Standard: Human DPP4 in a buffered protein base (1200 ng, lyophilized). Biotinylated Human DPP4 Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against human DPP4 (120 l). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (20 ml). Standard Diluent (1x): A buffered protein base with stabilizer (2 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). SP Conjugate (100x): A 100-fold concentrate (80 l). Chromogen Substrate (1x): A stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution (1x): A 0.5 N hydrochloric acid solution to stop the chromogen substrate reaction (12 ml).

Matériel non inclus: 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)

Information d'application

Plaque: Pre-coated

Protocole:

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

Préparation des réactifs:

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 10-fold with reagent grade water to produce a 1x solution. Store for up to 30 days at 2-8 °C. Human DPP4 Standard: Reconstitute the Human DPP4 Standard (1200 ng) with 0.75 mL of Standard Diluent to generate a 1600 ng/mL standard stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to making dilutions. From the standard stock solution (1600 ng/mL), dilute 4-fold with EIA Diluent to produce a 400 ng/mL standard working solution. Prepare duplicate or triplicate standard points by serially diluting the standard working solution (400 ng/mL) 2-fold with equal volume of EIA Diluent to produce 200, 100, 50, 25, 12.5, and 6.25 ng/mL solutions. EIA Diluent serves as the zero standard (0 ng/mL). Aliquot remaining stock solution to limit repeated freeze-thaw cycles. This solution should be stored at -20 °C and used within 30 days. Standard Point Dilution [DPP4] (ng/mL) P1 1 part Standard (1600 ng/mL) + 3 parts EIA Diluent 400 P2 1 part P1 + 1 part EIA Diluent 200 P3 1 part P2 + 1 part EIA Diluent 100 P4 1 part P3 + 1 part EIA Diluent 50 P5 1 part P4 + 1 part EIA Diluent 25 P6 1 part P5 + 1 part EIA Diluent 12.5 P7 1 part P6 + 1 part EIA Diluent 6.25 P8 EIA Diluent 0.0 Biotinylated Human DPP4 Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 50-fold with EIA Diluent to produce a 1x solution. The undiluted antibody should be stored 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 20-fold with reagent grade water to produce a 1x solution. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with EIA Diluent to produce a 1x solution. The undiluted conjugate should be stored at -20 °C. 5

Prélèvement de l'échantillon: 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 and collect plasma. A 5-fold sample dilution is suggested into EIA Diluent, however, user should determine optimal dilution factor depending on application needs. 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. A 5-fold sample dilution is suggested into EIA Diluent, however, user should determine optimal dilution factor depending on application needs. 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 at 4 °C to remove debris and collect supernatants. Samples can be stored at -20 °C or below. Avoid repeated freeze-thaw cycles.

Procédure de l'essai:

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 DPP4 Standard or sample to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. 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 DPP4 Antibody to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 1 hour. Wash the microplate as described above. Add 50 l of SP Conjugate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape 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 to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 15 minutes or until the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. 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. 6

Calcul des résultats:

• 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

Stockage

Conseil sur la manipulation: This product is for Research Use Only and is not intended for use in diagnostic procedures. 2 Prepare all reagents (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, the biotinylated antibody vial, and the standard diluent vial before opening and using contents. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date.

Stock: 4 °C,-20 °C

Stockage commentaire: Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store Standard, SP Conjugate, and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Standard Diluent (1x), 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. 3

Détail du antigène

Antigène: DPP4 (Dipeptidyl-Peptidase 4 (DPP4))

Autre désignation: Dipeptidyl Peptidase IV (DPP4) (DPP4 Produits)

Synonymes: ADABP Kit ELISA, ADCP2 Kit ELISA, CD26 Kit ELISA, DPPIV Kit ELISA, TP103 Kit ELISA, MGC81966 Kit ELISA, MOP9.8 Kit ELISA, MOP9_8 Kit ELISA, DPP4 Kit ELISA, si:ch73-2d23.3 Kit ELISA, Cd26 Kit ELISA, Dpp-4 Kit ELISA, THAM Kit ELISA, dipeptidyl peptidase 4 Kit ELISA, dipeptidyl-peptidase 4 S homeolog Kit ELISA, prolyl oligopeptidase family protein Kit ELISA, dipeptidyl-peptidase 4 Kit ELISA, dipeptidylpeptidase 4 Kit ELISA, DPP4 Kit ELISA, dpp4.S Kit ELISA, AT5G24260 Kit ELISA, dpp4 Kit ELISA, Dpp4 Kit ELISA

Sujet: Dipeptidyl peptidase 4 (DPP4), also known as adenosine deaminase complexing protein 2 (ADCP-2), T-cell activation antigen CD26, or CD26, belongs to the peptidase S9B family and DPPIV subfamily. It is an intrinsic membrane glycoprotein that comprises 766 amino acids and weighs 110 kDa (1-2). Its extracellular cysteine-rich region is necessary for association with collagen, dimer formation, and optimal dipeptidyl peptidase activity. Under certain stimuli and chronic inflammation, DPP4 can be released from the membrane, constituting a soluble form by matrix metalloproteases (MMPs). As a serine exopeptidase, DPP4 cleaves and inactivates N-terminal X-proline dipeptides of cytokines, chemokines, and neuropeptides involved in inflammation, immunity, and vascular function. Circulating soluble DPP4 is a novel adipokine that may impair insulin sensitivity in an autocrine and paracrine fashion. DPP4 plays a role in glucose homeostasis through proteolytic inactivation of the intestinal peptide incretins, such as glucose- dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP- 1), which are major regulators of post-prandial insulin secretion (3-4).

ID gène: 1803

UniProt: P27487

Pathways: Peptide Hormone Metabolism, Regulation of Leukocyte Mediated Immunity