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

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Antigen
  • AT3
  • AT3D
  • ATIII
  • THPH7
  • SERPINC1
  • DKFZp470C1733
  • AI114908
  • At-3
  • At3
  • ANTITHROMBIN, AT-III
  • AT-III
  • serpin peptidase inhibitor, clade C (antithrombin), member 1
  • antithrombin-III
  • serine (or cysteine) peptidase inhibitor, clade C (antithrombin), member 1
  • SERPINC1
  • CpipJ_CPIJ000472
  • CpipJ_CPIJ013111
  • Serpinc1
Reactivity
Human
Alternatives
Kits with alternative reactivity to:
21
14
6
6
3
3
2
2
1
1
1
1
Method Type
Competition ELISA
Detection Range
0.063-4 μg/mL
Minimum Detection Limit
0.063 μg/mL
Application
ELISA
Options
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Purpose The AssayMaxTM Human Antithrombin III ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of ATIII in human plasma and serum samples. This assay employs a quantitative competitive enzyme immunoassay technique that measures human ATIII in approximately 3 hours. A polyclonal antibody specific for human ATIII has been pre-coated onto a 96- well microplate with removable strips. ATIII in standards and samples is competed with a biotinylated human ATIII protein sandwiched by the immobilized antibody and streptavidin-peroxidase (SP) 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 Plasma, Serum
Analytical Method Quantitative
Detection Method Colorimetric
Specificity Reference Value: The normal blood level of AT III is averaged 2, aeg/ml.
Components Human Antithrombin III Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human ATIII. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Antithrombin III Standard: Human ATIII in a buffered protein base (9.6 μg, lyophilized). Biotinylated Human Antithrombin III Protein (6x): 1 vial, lyophilized. EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). SP Conjugate (100x): A 100-fold concentrate (80 l). Chromogen Substrate (1x): A stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution (1x): A 0.5 N hydrochloric acid solution 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)
Alternative Name Antithrombin III (AT3) (SERPINC1 ELISA Kit Abstract)
Background The serine protease inhibitor antithrombin III (ATIII, serpin C1), the most important natural inhibitor of thrombin activity, has been shown to exert marked anti-inflammatory properties (1). ATIII levels are positively correlated with plasma total cholesterol levels, plasma low-density lipoprotein cholesterol levels, plasma triglycerides, and D-dimer levels (2).
Gene ID 462
UniProt P01008
Sample Volume 25 μL
Assay Time 3 h
Plate Pre-coated
Protocol
  • Step 1. Add 25 μL of Standard or Sample and 25 μL of Biotinylated Protein per well. Incubate 2 hours.
  • Step 2. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
  • Step 3. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 12 minutes.
  • Step 4. 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 10-fold with reagent grade water to produce a 1x solution. Store for up to 30 days at 2-8 °C. Human Antithrombin III Standard: Reconstitute the Human Antithrombin III Standard (9.6 g) with 2.4 mL of EIA Diluent to generate a 4 g/mL standard stock solution. Allow the vial to sit for 10 minutes with 4 gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution (4 g/mL) 2-fold with equal volume of EIA Diluent to produce 2, 1, 0.5, 0.25, 0.125, and 0.063 g/mL solutions. EIA Diluent serves as the zero standard (0 g/mL). Any remaining stock solution should be stored at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. Standard Point Dilution [ATIII] (μg/mL) P1 1 part Standard (4 μg/mL) 4.0 P2 1 part P1 + 1 part EIA Diluent 2.0 P3 1 part P2 + 1 part EIA Diluent 1.0 P4 1 part P3 + 1 part EIA Diluent 0.5 P5 1 part P4 + 1 part EIA Diluent 0.25 P6 1 part P5 + 1 part EIA Diluent 0.125 P7 1 part P6 + 1 part EIA Diluent 0.063 P8 EIA Diluent 0.0 Biotinylated Human Antithrombin III Protein (6x): Reconstitute the Biotinylated Human Antithrombin III Protein with 4 mL of EIA Diluent to produce a stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to dilution. From the stock solution, dilute 6-fold with EIA Diluent to produce a 1x working solution. Any remaining stock solution should be stored at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 20-fold with reagent grade water to produce a 1x solution. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with EIA Diluent to produce a 1x solution. The undiluted conjugate should be stored at -20 °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 collect plasma. A 300-fold sample dilution is suggested into EIA Diluent, however, user should determine optimal dilution factor depending on application needs. 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 an anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes and remove serum. A 300-fold sample dilution is suggested into EIA Diluent, however, user should determine optimal dilution factor depending on application needs. 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, 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 25 l of Human Antithrombin III Standard or sample to each well, and immediately add 25 l of Biotinylated Human ATIII Protein to each 5 well (on top of the standard or sample). Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. 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 SP Conjugate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape 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 to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 12 minutes or until the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. 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 low 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 5.1 % and 7.4 % respectively.
Restrictions For Research Use only
Handling Advice This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (diluent buffer, wash buffer, standard, 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 insert. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial before opening and using contents. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date. 2
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 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 and Biotinylated Protein at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.
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).