alpha-Thrombin ELISA Kit

Catalog No. ABIN612790

Product Details alpha-Thrombin ELISA Kit

Target: alpha-Thrombin

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Minimum Detection Limit: < 300 pg/mL

Application: ELISA

Purpose: The AssayMax Human Thrombin ELISA kit is designed for detection of alpha Thrombin in human cell culture supernatants

Brand: AssayMax

Sample Type: Cell Culture Supernatant

Analytical Method: Quantitative

Specificity: This assay recognizes both natural and recombinant human Thrombin.

Cross-Reactivity (Details): This kit has about 70% cross reactivity to human prothrombin.

Components: Thrombin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody against alpha Thrombin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Thrombin Standard: Purified human Thrombin in a buffered protein base (640 ng, lyophilized). Biotinylated Thrombin Antibody (100x): A 100-fold biotinylated polyclonal antibody against Thrombin (80µl). EIA Diluent Concentrate (10x): A 10-fold 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). 1 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

Application Details

Sample Volume: 50 μL

Assay Time: 4 h

Plate: Pre-coated

Protocol: This assay employs a quantitative sandwich enzyme immunoassay technique that measures Thrombin in 4 hours. A monoclonal antibody specific for Thrombin has been pre-coated onto a microplate. Thrombin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for Thrombin, 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. EIA Diluent Concentrate (10x): Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 640 ng of human Thrombin Standard with 4 ml of EIA Diluent to generate 160 ng/ml of stock solution. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. The stock solution can be further dilute 1:8 with EIA Diluent to produce 20 ng/ml standard solution. Prepare triplicate standard points by serially diluting the Thrombin standard solution 1:2 with equal volume of EIA Diluent to produce 10, 5, 2.5, 1.25, 0.625 and 0.313 ng/ml. EIA Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [alpha Thrombin] (ng/ml) P1 Standard (20 ng/ml) 20.000 P2 1 part P1 + 1 part EIA Diluent 10.000 P3 1 part P2 + 1 part EIA Diluent 5.000 P4 1 part P3 + 1 part EIA Diluent 2.500 P5 1 part P4 + 1 part EIA Diluent 1.250 P6 1 part P5 + 1 part EIA Diluent 0.625 P7 1 part P6 + 1 part EIA Diluent 0.313 P8 EIA Diluent 0.000 2 Biotinylated Thrombin Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the 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 EIA Diluent. Any remaining solution should be frozen at -20°C.

Sample Collection: Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. The samples can be stored 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. 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 Biotinylated Thrombin Antibody to each well and incubate for one hour. Wash five times with 200 µL of Wash Buffer as 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 five times with 200 µL of Wash Buffer as above. Add 50 µL of Chromogen Substrate per well and incubate for approximately 10 minutes or till 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. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. Please note that some unstable black particles may be generated at high optical densities to reduce the readings after stopping the reaction for about 10 minutes.

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 four-parameter or log-log 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 5.0% and 7.0% respectively.

Restrictions: For Research Use only

Handling

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 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 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.

References for alpha-Thrombin ELISA Kit (ABIN612790)

Chen, Huang, Wang: "Bioinspired affinity DNA polymers on nanoparticles for drug sequestration and detoxification." in: Biomaterials, Vol. 35, Issue 36, pp. 9709-18, (2014) (PubMed).

Liao, Ho, Hou, Hsu, Tsai, Chen, Chen, Lee, Yu, Lee, Yang: "Heparin co-factor II enhances cell motility and promotes metastasis in non-small cell lung cancer." in: The Journal of pathology, Vol. 235, Issue 1, pp. 50-64, (2014) (PubMed).

Target Details for alpha-Thrombin

Target: alpha-Thrombin

Alternative Name: Thrombin (alpha-Thrombin Products)

Synonyms: PT ELISA Kit, RPRGL2 ELISA Kit, THPH1 ELISA Kit, coagulation factor II, thrombin ELISA Kit, F2 ELISA Kit

Background: Thrombin (activated Factor II [IIa]) is a coagulation protein that has many effects in the coagulation cascade. Thrombin is a serine protease (EC 3.4.21.5) that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions. Thrombin is in the form of alpha-thrombin tat is the immediate end product of prothrombin activation, two further thrombin products can be identified, beta- and gamma-thrombin. Theses are degraded form that may arise from autodigestion of a thrombin preparation.