Interleukin 1 (IL1) ELISA Kit

Details for Product No. ABIN612716
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Target Name (Antigen)
Reactivity
Alternatives Human
Kits with alternative reactivity to:
(2), (2), (1), (2), (1), (1), (2), (1), (2), (1), (1)
Methode Type Sandwich ELISA
Minimum Detection Limit 3 pg/mL
Application
ELISA
Pubmed 8 references available
Quantity 96 tests
Options
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Catalog No. ABIN612716
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Purpose The AssayMax Human IL-1beta ELISA kit is designed for detection of human IL-1beta in plasma, serum, saliva and cell culture supernatants
Sample Type Plasma, Cell Culture Supernatant
Detection Method Colorimetric
Components IL-1beta Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody against IL-1beta. 1 Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. IL-1beta Standard: Human IL-1beta in a buffered protein base (375 pg, lyophilized). Biotinylated IL-1beta Antibody (100x): A 100-fold biotinylated polyclonal antibody against human IL-1beta (80µl). 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 (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 Interleukin-1 (IL-1 )
Background Interleukin-1beta (IL-1beta) has a wide spectrum of inflammatory, metabolic, haemopoietic, and immunological properties. IL-1 beta plays a significant role in hippocampal synaptic function , and is a potential genetic marker as indicator of gastric cancer risk. High plasma level of Interleukin-1 beta (IL-1b) is associated with rheumatoid and osteoarthritic joint disease , infectious gastroenteritis , neurodegeneration , and breast cancer. High gingival crevicular fluid levels of interleukin-1beta is related to type 2 diabetes.
Sample Volume 50 µL
Assay Time < 5 h
Plate Pre-coated,Strips (12 x 8)
Protocol This assay employs a quantitative sandwich enzyme immunoassay technique, which measures IL-1beta in less than 5 hours. A murine monoclonal antibody specific for IL-1beta has been pre-coated onto a microplate. IL-1beta in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for IL-1beta, 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. MIx Diluent Concentrate (10x): Dilute the MIx Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. 2 Standard Curve: Reconstitute the 375 pg of human IL-1beta Standard with 1.5 ml of MIx Diluent to generate a standard solution of 250 pg/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 IL-1beta standard solution (250 pg/ml) twofold with equal volume of MIx Diluent to produce 125, 62.5, 31.3, 15.7, 7.8, and 3.9 pg/ml solutions. MIx Diluent serves as the zero standard (0 pg/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [IL-1beta] (pg/ml) P1 1 part Standard (250 pg/ml) 250.0 P2 1 part P1 + 1 part MIx Diluent 125.0 P3 1 part P2 + 1 part MIx Diluent 62.5 P4 1 part P3 + 1 part MIx Diluent 31.3 P5 1 part P4 + 1 part MIx Diluent 15.7 P6 1 part P5 + 1 part MIx Diluent 7.8 P7 1 part P6 + 1 part MIx Diluent 3.9 P8 MIx Diluent 0.0 Biotinylated IL-1beta Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute Wash Buffer 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 MIx 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. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. 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. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using sample tube. Centrifuge samples at 2000 x g for 10 minutes and assay. 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.
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 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 IL-1beta Antibody to each well and incubate for two hours. Wash a microplate as described above. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash a microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 10 minutes or until 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 µL of Stop Solution to each well. The color will change from blue to yellow. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. 3 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 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. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.
Assay Precision Intra-assay and inter-assay coefficients of variation were 5.1 % and 7.5% respectively.
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 kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened MIx Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
Product cited in: Lynch: "Interleukin-1 beta exerts a myriad of effects in the brain and in particular in the hippocampus: analysis of some of these actions." in: Vitamins and hormones, Vol. 64, pp. 185-219, 2002 (PubMed).

Palmi, Meini: "Role of the nitric oxide/cyclic GMP/Ca2+ signaling pathway in the pyrogenic effect of interleukin-1beta." in: Molecular neurobiology, Vol. 25, Issue 2, pp. 133-47, 2002 (PubMed).

Troost, Hold, Smith et al.: "The role of interleukin-1beta and other potential genetic markers as indicators of gastric cancer risk." in: Canadian journal of gastroenterology = Journal canadien de gastroenterologie, Vol. 17 Suppl B, pp. 8B-12B, 2003 (PubMed).

Mettler, Salmassi, Heyer et al.: "Perioperative levels of interleukin-1beta and interleukin-6 in women with breast cancer." in: Clinical and experimental obstetrics & gynecology, Vol. 31, Issue 1, pp. 20-2, 2004 (PubMed).

Enocksson, Lundberg, Weitzberg et al.: "Rectal nitric oxide gas and stool cytokine levels during the course of infectious gastroenteritis." in: Clinical and diagnostic laboratory immunology, Vol. 11, Issue 2, pp. 250-4, 2004 (PubMed).

Fan, Bau, Yang et al.: "IL-1beta induction of IL-6 and LIF in normal articular human chondrocytes involves the ERK, p38 and NFkappaB signaling pathways." in: Cytokine, Vol. 28, Issue 1, pp. 17-24, 2004 (PubMed).

Engebretson, Hey-Hadavi, Ehrhardt et al.: "Gingival crevicular fluid levels of interleukin-1beta and glycemic control in patients with chronic periodontitis and type 2 diabetes." in: Journal of periodontology, Vol. 75, Issue 9, pp. 1203-8, 2004 (PubMed).

Allan, Tyrrell, Rothwell: "Interleukin-1 and neuronal injury." in: Nature reviews. Immunology, Vol. 5, Issue 8, pp. 629-40, 2005 (PubMed).

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