SPECTROZYME® FIXa, chromogenic substrate for FIXa assays

Description
Highly sensitive chromogenic substrate for factor IXa. The sensitivity of this substrate is significantly enhanced in the presence of alcohols, especially ethylene glycol.

Chemistry
Formula: CH₃SO₂-(D)-CHG-Gly-Arg-pNA·AcOH
Chemical name: Methylsulfonyl-D-cyclohexylglycyl- glycyl-arginine-paranitroanilide monoacetate salt
MW: 628.7 Dalton
Solubility: up to 20 mM in water
Biochemical Characteristics:
k_cat = 4.4/sec                                     K_M = 1.3 mM
k_cat/K_M= 3.38 l/mM·s (determined in the presence of 33% ethylene glycol)

Intendet use
Determination of Factor IXa in concentrates or other blood products according to Ref 1.
Conditions: The test temperature can be selected but should be kept constant during the assay. All reagents should be kept at the test temperature prior to use. Do not work with chilled reagents directly from the refrigerator. For the kinetic version 37°C may be used, especially when a thermo-stated cell holder is available.
Automation: The assay can be either performed on a spectrophotometer or microtiter plate reader at 405 nm. Kinetic or endpoint versions are possible. An adaptation on fully automated chemistry analyzers at 405 nm may be possible but has not been tested.
Buffer: 50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM CaCl₂, 40 % (vol/ vol) ethylene glycol.
Notes: The sensitivity of this substrate for factor IXa is significantly increased in the presence of 33% ethylene glycol.
All volumes of the described pipetting scheme may be adapted for assay in regular cuvettes. An example is given below.
Interference by turbidity or from colored samples in the endpoint assay can lead to falsely elevated results. This can be prevented by running a sample blank as follows:
Pipette a sample blank in the following sequence: Acetic Acid/„Stop reagent“ - Buffer sample - Substrate

Microtiter plate format
0.200 ml buffer
0.025 ml Spectrozyme^® FIXa (10 mM)
0.020 ml Sample (factor IXaβ, 2 μM)
⇒ Determination of optical density at 405 nm
0.025 ml Acetic acid (50 %) to stop the reaction after 5-10 minutes
 
Spectrophotometer format
0.800 ml buffer
0.100 ml Spectrozyme  FIXa (10 mM)
0.080 ml Sample (factor IXaβ, 2 μM)
⇒ Determination of optical density at 405 nm
0.100 ml Acetic acid (50 %) to stop the reaction after 5-10 minutes

Calculate the activity of factor IXa according to:
F IXa activity = (OD sample – OD sample blank)

Presentation
Amber glass vial containing 10 μmoles of lyophilized substrate.

Reconstitution
Reconstitute with filtered deionized water to create a 10 mM solution. Shake gently before use.

Storage
May be used by the expiry date given on the label when stored unopened, protected from moisture, in the dark, 2-8°C. Avoid contamination of the reagents by microorganisms.

Category: Research use only

Type: Substrate

Product Availability: Worldwide

Manufacturer: ImmBioMed GmbH & Co KG, Germany

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