Antithrombin (ATIII) BioAssay(TM) ELISA Kit (Antibodies only)

Matched-Pair Antibody Set for ELISA of human Antithrombin antigen (ATIII). Sufficient reagents to perform 5 x 96 well plates.\n\nAntithrombin, also known as Antithrombin III (ATIII), is a member of the SERPIN family of proteinase inhibitors. It is the primary inhibitor of thrombin in plasma. It is produced in the liver and circulates in plasma at ~200ug/ml (~3.5uM). Antithrombin inhibits a broad spectrum of serine proteases including thrombin, activated forms of Factor X, Factor IX, Factor XI, Factor XII, as well as kallikrein, plasmin and urokinase. Enzyme inhibition by antithrombin occurs through proteolytic cleavage at Arg385-Ser386 and subsequent rapid formation of a stable, inactive 1/1 enzyme-antithrombin complex. Heparin has a profound accelerating effect on the inhibitory activity of antithrombin towards some enzymes. For example, the rate of inhibition of thrombin and activated Factor X is increased 1000-fold in the presence of optimal concentrations of heparin. Whereas heparin has relatively little effect on the inhibition rate of activated Factor XI, activated Factor XII and kallikrein. Antithrombin is a single chain molecule with a molecular weight of 59kD. Interaction with thrombin results in an SDS-stable thrombin-antithrombin complex of 96kD.\n\nPrinciple of Sandwich-style ELISA:\nAffinity-purified antibody to ATIII is coated onto the wells of a microtiter plate. The plates are washed. Plasma or other fluids containing ATIII are applied. The coated antibody will capture the ATIII in the sample. After washing the plate to remove unbound material, a second anti-ATIII antibody labeled with peroxidase is added to the plate to bind to the captured ATIII. After washing the plate to remove unbound labeled antibody, the peroxidase activity is expressed by incubation with o-phenylenediamine (OPD). After a fixed development time, the reaction is quenched with the addition of sulfuric acid. The color produced is quantified using a microplate reader. The color generated is proportional to the concentration of ATIII present in the sample.\n\nKit Components:\nA2298-19A: Capture Antibody, 1x500ul (Yellow cap). Polyclonal affinity purified anti-ATIII, 50% glycerol solution. Suitable for use as coating antibody. \nA2298-19B: Detection Antibody (HRP), 5x10ml. Pre-diluted polyclonal anti-ATIII. Labeled with peroxidase. Suitable for use as detection antibody.\n\nQuantity:\nSufficient reagents for 5x96 well plates.\n\nStorage and Stability:\nMay be stored at 4 degrees C. For long-term storage, aliquot and store at -20 degrees C. Aliquots are stable for 6 months. For maximum recovery of product, centrifuge the original vial prior to removing the cap.

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SPECIFICATIONS

Catalog Number

A2298-19

Size

1Kit

Applications

ELISA

Reactivities

Hum

References

1. Damus, P.S. and Rosenberg, R.D., Methods in Enzymology 45: 653-669 (1976). 2. Harpel, P.C., Blood Proteolytic Enzyme Inhibitors: Their Role in Modulating Blood Coagulation and Fibrinolytic Enzyme Pathways; in Hemostasis and Thrombosis, eds. R.W. Colman, J. Hirsh, V.J. Marder and E.W. Salzman, pp. 738-747, J.B. Lippincott Co., Philadelphia PA, USA, 1982. 3. Griffith, M.J., et al., JBC 260: 2218-2225 (1985). 4. Nix, B., Wild, D., in Immunoassays, A Practical Approach, editor J.P. Gosling, pp. 239-261, Oxford University Press, 2000. 5. NCCLS. Evaluation of the Linearity of Quantitative Analytical Methods; Proposed Guidline, Second Edition. NCCLS Document EP6-P2 (ISBN 1-56238-446-5, NCCLS, Wayne, Pennsylvania USA, 2001. 6. FDA Guidance for Industry. Bioanalytical Method Validation; May 2001, available on the internet: www.fda.gov/cder/guidance/index.htm