Haptoglobin ELISA Kit

Produktnummer ABIN612684

Produktdetails Haptoglobin ELISA Kit

Target: Haptoglobin (HP)

Reaktivität: Hund

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 0.313-20 ng/mL

Untere Nachweisgrenze: 0.313 ng/mL

Applikation: ELISA

Verwendungszweck: The AssayMax Canine Haptoglobin ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of canine haptoglobin in plasma and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures canine haptoglobin in approximately 4 hours. A polyclonal antibody specific for canine haptoglobin has been pre- coated onto a 96-well microplate with removable strips. Haptoglobin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for haptoglobin, 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, Plasma

Analytische Methode: Quantitative

Spezifität: 10% FBS in culture media will not affect the assay.

Bestandteile: Canine Haptoglobin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against canine haptoglobin. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Canine Haptoglobin Standard: Canine haptoglobin in a buffered protein base (40 ng, lyophilized). Biotinylated Canine Haptoglobin Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against canine haptoglobin (120 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

Probenmenge: 50 μL

Testdauer: 4 h

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 10 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 10-fold with reagent grade water. Store for up to 30 days at 2-8 °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. A 200000-fold sample dilution is suggested into MIX Diluent, however, user should determine proper dilutions 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). Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris and collect supernatants. Store the remaining samples at -20 °C or below. Avoid repeated freeze-thaw cycles. Refer to Sample Dilution Guidelines below for further instruction. Guidelines for Dilutions of 100-fold or Greater (for reference only, please follow the insert for specific dilution suggested) 100x 10000x 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. 1000x 100000x 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 Canine Haptoglobin Standard or sample per well. Cover wells 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 Canine Haptoglobin 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 the 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.

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.

Testpräzision: Intra-assay and inter-assay coefficients of variation were 5.0% and 7.1 % respectively.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: This product is for Research Use Only and is not intended for use in diagnostic procedures. 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 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

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 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.

Details zu Haptoglobin

Target: Haptoglobin (HP)

Andere Bezeichnung: Haptoglobin (HP Produkte)

Synonyme: HP ELISA Kit, wu:fb64e01 ELISA Kit, BP ELISA Kit, HP2ALPHA2 ELISA Kit, HPA1S ELISA Kit, HP-1 ELISA Kit, HPR ELISA Kit, Zonulin ELISA Kit, haptoglobin ELISA Kit, haptoglobin-like ELISA Kit, HP ELISA Kit, hp ELISA Kit, Hp ELISA Kit, LOC479668 ELISA Kit, LOC101102413 ELISA Kit

Hintergrund: Haptoglobin (HP, Zonulin) 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 (1).

UniProt: P19006

Pathways: Transition Metal Ion Homeostasis