Search, Find and Buy Antibodies, ELISA Kits and Proteins.
Loading Loading
Order hotline:
phone +1 404 474 4654
fax +1 888 205 9894 (TF)
Details for Product No. ABIN612685

Elastase, Neutrophil Expressed (ELANE) ELISA Kit

Request
Want additional data for this product?

The Independent Validation Initiative strives to provide you with high quality data. Find out more

Target Name (Antigen)
Synonyms ELA2, GE, HLE, HNE, NE, PMN-E, SCN1, Ela2, F430011M15Rik, ela2, ELANE
Reactivity
»Alternatives Human
Kits with alternative reactivity to: (4), (5), (7), (4), (4), (13), (5), (10), (4), (6), (8), (3)
Methode Type Sandwich ELISA
Application
ELISA
Pubmed 9 references available
Catalog no. ABIN612685
Quantity 96 tests
Price
379.50 $   Plus shipping costs $45.00
Shipping to
Availability Will be delivered in 2 to 3 Business Days
Purpose The AssayMax Human Neutrophil Elastase ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human Neutrophil Elastase in plasma, serum, urine, saliva, milk, and cell culture supernatants
Sample Type Plasma, Cell Culture Supernatant
Detection Method Colorimetric
Cross-Reactivity Dog (Canine), Cow (Bovine), Monkey, Mouse (Murine), Rat (Rattus), Pig (Porcine), Rabbit
Components Human Neutrophil Elastase Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human Neutrophil Elastase. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Neutrophil Elastase Standard: Human Neutrophil Elastase in a buffered protein base (20 ng, lyophilized). Biotinylated Neutrophil Elastase Antibody (80x): A 80-fold concentrated biotinylated polyclonal antibody against Neutrophil Elastase (100µl). EIA 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).
Material not included Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water.
Alternative Name Neutrophil Elastase (ELA-2)
Background Human Neutrophil Elastase (NE), also known as ELA2, is a subfamily of serine proteinase elastases. It consists of 218 amino acid residues with a molecular weight of 23 kDa and two asparagine-linked carbohydrate side chains. NE is stored in neutrophil lysosomes azurophil granules during neutrophil differentiation, and is involved in a variety of immune defense reactions and degenerative and inflammatory diseases (2-3). Upon infection, the activated neutrophils release NE which then hydrolyzes azurophil granule proteins and extracellular matrix proteins: elastin, collagen, and proteoglycan. As a host defense protein, NE degrades bacterial outer membrane and virulence proteins (5-6). When expressed abnormally, NE causes the development of pulmonary emphysema. NE mutations are associated with cyclic neutropenia and severe congenital neutropenia (8-9).
Sample Volume 50 µL
Assay Time < 4 h
Plate Pre-coated,Strips (12 x 8)
Protocol This assay employs a quantitative sandwich enzyme immunoassay technique that measures human Elastase in less than 4 hours. A polyclonal antibody specific for human Elastase has been pre-coated onto a 96-well microplate with removable strips. Elastase in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for Elastase, 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.
Reagent Preparation Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. EIA Diluent Concentrate (10x): Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 20 ng of Neutrophil Elastase Standard with 2 ml of EIA Diluent to generate a solution of 10 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 (10 ng/ml) 1:2 with EIA Diluent to produce 5, 2.5, 1.25, 0.625, 0.313, and 0.156 ng/ml solutions. EIA Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [Elastase] (ng/ml) P1 Standard (10 ng/ml) 10.000 P2 1 part P1 + 1 part EIA Diluent 5.000 P3 1 part P2 + 1 part EIA Diluent 2.500 P4 1 part P3 + 1 part EIA Diluent 1.250 P5 1 part P4 + 1 part EIA Diluent 0.625 P6 1 part P5 + 1 part EIA Diluent 0.313 P7 1 part P6 + 1 part EIA Diluent 0.156 P8 EIA Diluent 0.000 Biotin Elastase Antibody (80x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:80 with EIA Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): 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 EIA Diluent. Any remaining solution should be frozen at -20°C.
Sample Collection 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 and assay. Dilute samples 1:50 into EIA Diluent. 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 2000 x g for 10 minutes. Remove serum and assay. Dilute samples 1:50 into EIA Diluent. 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 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. Dilute Urine 1:8 with EIA Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. 2 Saliva: Collect saliva using sample tube. Centrifuge samples at 600 x g for 10 minutes. Dilute Saliva 1:1000 with EIA Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Milk: Collect milk using sample tube. Centrifuge samples at 600 x g for 10 minutes. Dilute Milk 1:200000 with EIA Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.
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 - 30 °C). 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 Neutrophil Elastase standard or sample per well. Cover wells with a sealing tape 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 Neutrophil Elastase Antibody to each well and incubate for one hour. Wash the microplate as described above. Add 50 µL 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 µL of Chromogen Substrate per well and incubate for about 8 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 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.
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 on the y-axis. The best-fit line can be determined by regression analysis using four-parameter or log-log 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.
Restrictions For Research Use only
Handling Advice 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. The kit should not be used beyond the expiration date.
Storage 4 °C/-20 °C
Storage Comment 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.
Product cited in: Zimmer, Medcalf, Fink et al.: "Three human elastase-like genes coordinately expressed in the myelomonocyte lineage are organized as a single genetic locus on 19pter." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 89, Issue 17, pp. 8215-9, 1992 (PubMed).

Lindmark, Persson, Olsson: "Biosynthesis and processing of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937." in: Blood, Vol. 76, Issue 11, pp. 2374-80, 1991 (PubMed).

Takahashi, Nukiwa, Yoshimura et al.: "Structure of the human neutrophil elastase gene." in: The Journal of biological chemistry, Vol. 263, Issue 29, pp. 14739-47, 1988 (PubMed).

Sinha, Watorek, Karr et al.: "Primary structure of human neutrophil elastase." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 84, Issue 8, pp. 2228-32, 1987 (PubMed).

Belaaouaj, Kim, Shapiro: "Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase." in: Science (New York, N.Y.), Vol. 289, Issue 5482, pp. 1185-8, 2000 (PubMed).

Dale, Person, Bolyard et al.: "Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia." in: Blood, Vol. 96, Issue 7, pp. 2317-22, 2000 (PubMed).

Weinrauch, Drujan, Shapiro et al.: "Neutrophil elastase targets virulence factors of enterobacteria." in: Nature, Vol. 417, Issue 6884, pp. 91-4, 2002 (PubMed).

Guarnieri, Spencer, Lucey et al.: "A human surfactant peptide-elastase inhibitor construct as a treatment for emphysema." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, Issue 23, pp. 10661-6, 2010 (PubMed).

van de Vosse, Verhard, Tool et al.: "Severe congenital neutropenia in a multigenerational family with a novel neutrophil elastase (ELANE) mutation." in: Annals of hematology, Vol. 90, Issue 2, pp. 151-8, 2011 (PubMed).

Alternatives for antigen "Elastase, Neutrophil Expressed (ELANE)", type "Kits"
Reactivities (4), (5), (7), (4), (4), (13), (5), (10), (4), (6), (8), (3)
Beacon