IL1A ELISA Kit

Catalog No. ABIN612717

Product Details IL1A ELISA Kit

Target: IL1A (Interleukin 1 alpha (IL1A))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.977-250 pg/mL

Minimum Detection Limit: 0.977 pg/mL

Application: ELISA

Purpose: The AssayMax Human Interleukin-1 alpha ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human IL-1 alpha in plasma, serum, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique which measures IL-1 alpha in less than 5 hours. A murine monoclonal antibody specific for IL-1 alpha has been pre-coated onto a 96-well microplate with removable strips. IL-1 alpha in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for IL-1 alpha, 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.

Brand: AssayMax™

Sample Type: Cell Culture Cells, Plasma, Serum

Analytical Method: Quantitative

Cross-Reactivity (Details): No significant cross-reactivity or interference was observed.

Components: Human IL-1 alpha Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a murine monoclonal antibody against IL-1 alpha. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human IL-1 alpha Standard: Human IL-1 alpha in a buffered protein base (250 pg, lyophilized). Biotinylated Human IL-1 alpha Antibody (100x): A 100-fold biotinylated polyclonal antibody against human IL-1 alpha (60 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)

Application Details

Sample Volume: 50 μL

Assay Time: 5 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 2 hours.
• 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 20 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 and assay. 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, remove serum, and assay. 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 IL-1 alpha 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 IL-1 alpha Antibody to each well and incubate for 2 hours. Wash the microplate as described above. 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 20 minutes or until the optimal blue color density develops. Gently tap the plate to 5 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.

Assay Precision: Intra-assay and inter-assay coefficients of variation were 4.5 % and 7.1% respectively.

Restrictions: For Research Use only

Handling

Handling Advice: This product is for Research Use Only and is Not 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. 2 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. 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. 3

References for IL1A ELISA Kit (ABIN612717)

Niyazoglu, Baykara, Koc, Aydoğdu, Onaran, Dellal, Tasan, Sultuybek: "Association of PARP-1, NF-κB, NF-κBIA and IL-6, IL-1β and TNF-α with Graves Disease and Graves Ophthalmopathy." in: Gene, Vol. 547, Issue 2, pp. 226-32, (2014) (PubMed).

March, Mosley, Larsen, Cerretti, Braedt, Price, Gillis, Henney, Kronheim, Grabstein: "Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs." in: Nature, Vol. 315, Issue 6021, pp. 641-7, (1985) (PubMed).

Target Details for IL1A

Target: IL1A (Interleukin 1 alpha (IL1A))

Alternative Name: Interleukin-1-alpha (IL-1-alpha) (IL1A Products)

Synonyms: IL-1A ELISA Kit, IL1 ELISA Kit, IL1-ALPHA ELISA Kit, IL1F1 ELISA Kit, IL-6 ELISA Kit, Il-1a ELISA Kit, IL-1 alpha ELISA Kit, IL-1alpha ELISA Kit, L1A ELISA Kit, interleukin 1 alpha ELISA Kit, interleukin 6 ELISA Kit, IL1A ELISA Kit, IL6 ELISA Kit, Il1a ELISA Kit

Background: Interleukin-1 alpha (IL-1 alpha) is a member of the IL-1 superfamily containing IL-1 alpha, IL-1 beta, and IL-1 Ra receptor antagonist. IL-1 alpha is known as hematopoietin (IL-1 F1) and IL-1 beta as catabolin (IL-1 F2). IL-1 alpha and IL-1 beta correspond to two different isoelectric forms, acidic pI 5 and neutral pI 7, respectively. IL-1 alpha has a molecular mass of 17 kDa and consists of 159 amino acids having 26 - 33 % homology with IL-1 beta (1 - 6). They are produced mainly by macrophages and monocytes, processed and released during apoptosis, and bind with high affinity to specific receptors on target cells. While only the mature form of IL-1 beta has biological activity, both the pro and mature forms of IL-1 alpha are active (7). IL-1 alpha and IL-1 beta are pro-inflammatory cytokines involved in immune responses, inflammatory reactions, and hematopoiesis (8, 9). IL-1 alpha is an epidemal cytokine that is constitutively produced by epithelial cells and plays an important role in the maintenance of skin barrier function (10).

Gene ID: 3552

UniProt: P01583

Pathways: NF-kappaB Signaling, Autophagy, Cancer Immune Checkpoints