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Serine (Or Cysteine) Peptidase Inhibitor, Clade C (Antithrombin), Member 1 (SERPINC1) ELISA Kit

Details for Product No. ABIN612656, Supplier: Log in to see
  • AI114908
  • At-3
  • At3
  • AT3
  • AT3D
  • THPH7
  • AT-III
  • serine (or cysteine) peptidase inhibitor, clade C (antithrombin), member 1
  • serpin peptidase inhibitor, clade C (antithrombin), member 1
  • Serpinc1
Kits with alternative reactivity to:
Methode Type
Competition ELISA
Minimum Detection Limit
0.0625 µg/mL
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Purpose The AssayMax AT III ELISA kit is designed for detection of human AT III in plasma and serum
Sample Type Plasma
Detection Method Colorimetric
Specificity Reference Value: The normal blood level of AT III is averaged 2, aeg/ml.
Components AT III Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against AT III. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes, which can be cut to fit the format of the individual assay. AT III Standard: AT III in a buffered protein base (4 aeg/ml, lyophilized). Biotinylated AT III: 1 vial, lyophilized. EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). 1 Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate, 100x): A 100-fold concentrate (80ael). 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 ATIII (SERPINC1 ELISA Kit Abstract)
Background The serine-protease-inhibitor Antithrombin III (AT III), the most important natural inhibitor of thrombin activity, has been shown to exert marked anti-inflammatory properties and proven to be efficacious in experimental models of sepsis, septic shock, and disseminated intravascular coagulation. It has often been recommended for the therapy of septic patients as it provides anticoagulant and anti-inflammatory actions. Antithrombin III (AT III) deficiency is a rare hereditary disease that predisposes to thromboembolic complications. AT III levels are positively correlated with plasma total cholesterol levels, plasma low-density lipoprotein cholesterol levels, plasma triglycerides and D-dimer levels.
Sample Volume 25 μL
Assay Time < 3 h
Plate Pre-coated,Strips (12 x 8)
Protocol This assay employs a quantitative competitive enzyme immunoassay technique that measures ATIII in less than 3 hours. A polyclonal antibody specific for AT III has been pre-coated onto a 96-well microplate with removable strips. AT III in standards and samples is competed with a biotinylated AT III sandwiched by the immobilized antibody and 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. AT III Standard: Reconstitute the 4 g of human AT III Standard with 1 ml of EIA Diluent to generate a stock solution of 4 g/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 (4 g/ml) 1:2 with EIA Diluent to produce 2, 1, 0.5, 0.25, 0.125, and 0.0625 g/ml. EIA Diluent serves as the zero standard (0 g/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [ATIII] ( g/ml) P1 1 part stock (4 g/ml) 4.00 P2 1 part P1 + 1 part EIA Diluent 2.00 P3 1 part P2 + 1 part EIA Diluent 1.00 P4 1 part P3 + 1 part EIA Diluent 0.500 P5 1 part P4 + 1 part EIA Diluent 0.250 P6 1 part P5 + 1 part EIA Diluent 0.125 P7 1 part P6 + 1 part EIA Diluent 0.0625 P8 EIA Diluent 0.0000 Biotinylated AT III (1x): Dilute Biotinylated AT III with 4 ml EIA Diluent to produce a working solution. Allow the biotin to sit for 10 minutes with gentle agitation prior to use. 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 plasma 1:300 with 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 serum 1:300 with EIA Diluent. The undiluted samples can be stored 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 25 µL of standard and/or sample per well and immediately add 25 µL of Biotinylated AT III to each well (on top of the standard or sample). Cover wells with a sealing tape and incubate for two hours at room temperature. 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 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 7 minutes or until 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 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 7.4 % respectively.
Restrictions For Research Use only
Handling Advice Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated- protein, 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 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. Opened EIA 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 be returned to the foil pouch with the desiccant packets, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
Supplier Images
ELISA image for Serine (Or Cysteine) Peptidase Inhibitor, Clade C (Antithrombin), Member 1 (SERPINC1) ELISA Kit (ABIN612656) Serine (Or Cysteine) Peptidase Inhibitor, Clade C (Antithrombin), Member 1 (SERPINC1) ELISA Kit
Background publications Erem, Hacihasanoğlu, Celik, Ovali, Ersöz, Ukinç, Deger, Telatar: "Coagulation and fibrinolysis parameters in type 2 diabetic patients with and without diabetic vascular complications." in: Medical principles and practice : international journal of the Kuwait University, Health Science Centre, Vol. 14, Issue 1, pp. 22-30, 2004 (PubMed).

Oelschläger, Römisch, Staubitz, Stauss, Leithäuser, Tillmanns, Hölschermann: "Antithrombin III inhibits nuclear factor kappaB activation in human monocytes and vascular endothelial cells." in: Blood, Vol. 99, Issue 11, pp. 4015-20, 2002 (PubMed).

Kulka, Tryba, Lange: "[Are there certified indications for the use of antithrombin III in intensive care]." in: Anästhesiologie, Intensivmedizin, Notfallmedizin, Schmerztherapie : AINS, Vol. 36, Issue 3, pp. 143-53, 2001 (PubMed).

Mitchison, Cramer: "Actin-based cell motility and cell locomotion." in: Cell, Vol. 84, Issue 3, pp. 371-9, 1996 (PubMed).

Hanstein, Lange, Schneider-Poetsch, Grolig, Wagner: "Detection of actin and localization of phytochrome in the green alga Mougeotia by monoclonal antibodies." in: Acta histochemica. Supplementband, Vol. 41, Issue 9, pp. 223-30, 1992 (PubMed).