Anti-FIIa Assay: Procedure

Published on 2014/8/5 10:30:54

Anti-FIIa Assay: Solutions

Procedure

Take 18 microcentrifuge tubes (1.5 mL, with lids), label and arrange the tubes as B1, S1, S2, S3, S4, T1, T2, T3, T4, T1, T2, T3, T4, S1, S2, S3, S4, B2.

Add each reagent as Table 3.

Table 3. Adding Reagents
Sequence Solution Volume Incubation Temperature Reaction time
Note: After each addition of a reagent, the incubation mixture should be mixed without allowing bubbles to form.
1 Standard, sample
or Tris-NaCl buffer (pH 7.4)
50 uL 37 °C -
2 Antithrombin 50 uL 37 °C 1 minute
3 Thrombin 100 uL 37 °C 1 minute
4 Chromogenic substrate 250 uL 37 °C 4 minutes
5 Stop solution 375 uL - -

Calculation

Prepare a reference solution according to Table 3. Use S1 and don't add thrombin solution.

Measure the absorbance of each tube at 405 nm wavelength using a spectrophotometer.

Calculate the anti-FIIa potency with a parallel-line assay program.