Lactoferrin Antibody IgG ELISA Kit

Produktnummer ABIN5564547

Dieses Colorimetric ELISA kit dient der quantitativen Messung von Human Lactoferrin Antibody IgG.

Kurzübersicht für Lactoferrin Antibody IgG ELISA Kit (ABIN5564547)

Target: Lactoferrin Antibody IgG

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 1.563-100 U/mL

Applikation: ELISA

Proben: Plasma, Serum

Produktdetails

Untere Nachweisgrenze: 1.563 U/mL

Verwendungszweck: The AssayMax™ Human Lactoferrin Autoantibody ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative determination of autoimmune response (IgG) to a target antigen (lactoferrin). The kit detects autoantibodies in plasma and serum samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures autoantibodies (anti-Lactoferrin IgG) in less than 4 hours. A lactoferrin antigen has been pre-coated onto a 96-well microplate with removable strips. Autoantibody specific for lactoferrin in standards and samples is sandwiched by the immobilized antigen and an antibody-HRP conjugate. All unbound 2 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™

Analytische Methode: Quantitative

Bestandteile: Human Lactoferrin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with lactoferrin. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Lactoferrin Standard: Plasma standard (100 AU, lyophilized). HRP Conjugate (50x): A 50-fold concentrated HRP-antibody conjugate (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, 1 bottle). 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

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 HRP Conjugate per well. Incubate 1 hour.
• Step 3. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 30 minutes.
• Step 4. 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 1:10 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. Dilute samples 1:40 into MIX 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 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 1:40 into MIX Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles.

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 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 HRP Conjugate to each well and incubate for 1 hour. Wash the microplate as described above. Add 50 l of Chromogen Substrate per well and incubate for 30 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 5 570 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.

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 (working diluent buffer, wash buffer, standard, HRP 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 HRP conjugate vial before opening and using contents. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date.

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 HRP Conjugate 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. 3 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.

Antigendetails

Target: Lactoferrin Antibody IgG

Andere Bezeichnung: Lactoferrin Autoantibody

Substanzklasse: Antibody

Hintergrund: Lactoferrin is an 80 kDa iron-binding glycoprotein produced by many exocrine glands with a major constituent in the secondary granules of neutrophilic leukocytes. Serum lactoferrin concentration is much higher during inflammation (1). Lactoferrin is known to be an immune modulator or enhancer due to specific receptors for lactoferrin that are found on many key immune cells such as lymphocytes, monocytes, and macrophages. Lactoferrin is known to be directly involved in the up-regulation of natural killer (NK) cell activity (2). Lactoferrin is present in maternal milk, saliva, tears, vaginal secretions, semen, bronchoalveolar lavage fluid, and specific granules of polymorphonuclear leukocytes (PMNs) (3). Lactoferrin is found mainly in the oral cavity where it can come into direct contact with pathogens such as viruses, bacteria, etc. Lactoferrin directly inhibits viruses by binding to viral receptor sites, thus preventing the virus from infecting healthy cells. Lactoferrin has a direct bactericidal function to certain bacteria such as Streptococcus mutans, Vibrio cholerae, Escherichia coli, Actinobacillus actinomycetemcomitans, and Legionella pneumophila (2-4). Also, it has a bacteriostatic effect that deprives iron-requiring bacteria of this essential growth nutrient (4). Lactoferrin is also considered an antioxidant that scavenges free iron, helping to prevent uncontrolled iron based free radical reactions, thus protecting certain cells from peroxidation (2). Autoantibodies against lactoferrin belong to the pANCA class (Perinuclear Anti-Neutrophil Cytoplasmic Antibodies).

Gen-ID: 4057

UniProt: P02788