Details for Product No. ABIN612721, Supplier: Log in to see
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Method Type
Sandwich ELISA
Minimum Detection Limit
1 ng/mL
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Supplier Product No.
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Purpose The AssayMax Human Kininogen ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human Kininogen in urine and cell culture supernatant
Brand AssayMax
Sample Type Cell Culture Supernatant
Analytical Method Quantitative
Detection Method Colorimetric
Components Human Kininogen Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human Kininogen. 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 Kininogen Standard: Human Kininogen in a buffered protein base (200 ng, lyophilized). Biotinylated Kininogen Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against Kininogen (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). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). 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 Kininogen (HMW)
Background High molecular-weight kininogen (HK) is a plasma protein coagulation cofactor serving for the activation of zymogens prekallikrein, Factor xII and Factor xI and is also a substrate of each of their proteolytic forms. It circulates as a complex with these zymogens and links the plasma coagulation, fibrinolysis, complement activation, and blood pressure control. HK is produced by the liver and weighs 120 kDa with 626 amino acids. Its plasma concentration ranges from 55 to 90 µg/ml and (1 - 5). HK exhibits anticoagulant properties and is a strong inhibitor of cysteine proteases. Upon cleavage by kallikrein, the released active peptide bradykinin mediates NO release, vasodilation, hypotension and pain. The remaining cleaved HK (HKa) exhibits antiadhesive and antiangiogenic activity, enhancing cell-associated fibrinolysis and releasing cytokines and chemokines to enhance inflammation. Patients with HK deficiency exhibit abnormal surface-mediated activation of fibrinolysis (6 - 7).
Sample Volume 50 μL
Assay Time < 4 h
Plate Pre-coated
Protocol This assay employs a quantitative sandwich enzyme immunoassay technique that measures human Kininogen in less than 4 hours. A polyclonal antibody specific for human Kininogen has been pre-coated onto a 96-well microplate with removable strips. Kininogen in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for Kininogen, 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. Standard Curve: Reconstitute the 200 ng of Kininogen Standard with 2 ml of MIx Diluent to generate a solution of 100 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the standard solution (100 ng/ml) 1:2 with equal volume of MIx Diluent to produce 50, 25, 12.5, 6.25, 3.13 and 1.56 ng/ml solutions. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [Kininogen] (ng/ml) P1 Standard (100 ng/ml) 100.00 P2 1 part P1 + 1 part MIx Diluent 50.00 P3 1 part P2 + 1 part MIx Diluent 25.00 P4 1 part P3 + 1 part MIx Diluent 12.50 P5 1 part P4 + 1 part MIx Diluent 6.25 P6 1 part P5 +1 part MIx Diluent 3.13 P7 1 part P6 +1 part MIx Diluent 1.56 P8 MIx Diluent 0.00 Biotin Kininogen 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 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 MIx Diluent. Any remaining solution should be frozen at -20°C.

Sample Collection Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes and assay. Dilute samples 1:200 into MIx Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Saliva: Rinse your mouth at least twice with cool water. Then chew sugarless gum for 1-2 minutes, swallowing saliva as usual. You may continue to chew the gum during saliva collection but please do not spit it into the collection tube. If, for some reason, you are not able to chew the gum, saliva collection is still possible but may take longer. Dilute samples 1:20 into MIx Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Milk: Centrifuge samples at 600 x g for 10 minutes and assay. Dilute samples 1:200 into MIx Diluent. Store samples 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 samples at -20°C or below. Avoid repeated freeze-thaw cycles. 2
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 Kininogen 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. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to completely remove liquid at each step. Add 50 µL of Biotinylated Kininogen Antibody to each well and incubate for one hour. Wash five times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to complete remove liquid at each step. 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 five times with 200 µL of Wash Buffer. Add 50 µL of Chromogen Substrate per well and incubate for about 10 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. Please note that after the reaction is stopped for about 10 minutes, some black particles may be generated at high concentration point, 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 4.8% and 7.3% respectively.
Restrictions For Research Use only
Handling Advice 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.
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Product cited in: Vijayashankar, Arumugam, Sridharan: "Urine proteome analysis to evaluate protein biomarkers in children with autism." in: Clinica chimica acta; international journal of clinical chemistry, 2015 (PubMed).

Background publications Coffman, Parsonage, DAgostino, Torti, Torti: "Regulatory effects of ferritin on angiogenesis." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, Issue 2, pp. 570-5, 2009 (PubMed).

Hassan, Sainz, Khan, Bradford, Isordia-Salas, Kashem, Sartor, Colman: "Antithrombotic activity of kininogen is mediated by inhibitory effects of domain 3 during arterial injury in vivo." in: American journal of physiology. Heart and circulatory physiology, Vol. 292, Issue 6, pp. H2959-65, 2007 (PubMed).

Reddigari, Kuna, Miragliotta, Shibayama, Nishikawa, Kaplan: "Human high molecular weight kininogen binds to human umbilical vein endothelial cells via its heavy and light chains." in: Blood, Vol. 81, Issue 5, pp. 1306-11, 1993 (PubMed).

Kellermann, Lottspeich, Henschen, Müller-Esterl: "Completion of the primary structure of human high-molecular-mass kininogen. The amino acid sequence of the entire heavy chain and evidence for its evolution by gene triplication." in: European journal of biochemistry / FEBS, Vol. 154, Issue 2, pp. 471-8, 1986 (PubMed).

Kitamura, Kitagawa, Fukushima, Takagaki, Miyata, Nakanishi: "Structural organization of the human kininogen gene and a model for its evolution." in: The Journal of biological chemistry, Vol. 260, Issue 14, pp. 8610-7, 1985 (PubMed).

Maier, Austen, Spragg: "Kinetic analysis of the interaction of human tissue kallikrein with single-chain human high and low molecular weight kininogens." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 80, Issue 13, pp. 3928-32, 1983 (PubMed).

Kerbiriou, Griffin: "Human high molecular weight kininogen. Studies of structure-function relationships and of proteolysis of the molecule occurring during contact activation of plasma." in: The Journal of biological chemistry, Vol. 254, Issue 23, pp. 12020-7, 1980 (PubMed).

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