Anti-FIIa Assay: Solutions

Published on 2014/8/5 10:25:11

This method is designed to determine the anti-FIIa activity of low molecular weight heparins.

By using this manual assay and our reagents (α-thrombin, antithrombin and S-2238), we can get precise and accurate results.

Solutions

Tris-NaCl Buffer (pH 7.4)

Dissolve 4.385 g of NaCl, 3.04 g of Tris in 400 mL of water. Adjust with 1 mol/L HCl to pH 7.4. Dilute with water to 500 mL. Store it at 4 °C.

Tris-EDTA Buffer (pH 8.4)

Dissolve 5.12 g of NaCl, 3.04 g of Tris, 1.40 g of Na2EDTA in 450 mL of water. Adjust with 1 mol/L HCl to pH 7.4. Dilute with water to 500 mL. Store it at 4 °C.

Thrombin Solution

Dissolve a vial of thrombin (50 IU) with 10 mL of Tris-NaCl buffer (pH 7.4), obtaining a 10 IU/mL thrombin solution.

Antithrombin Solution

Dissolve a vial of antithrombin (10 IU) with 10 mL of water, obtaining a 1 IU/mL antithrombin stock solution.

Before use, dilute 5 mL of 1 IU/mL antithrombin stock solution with 5 mL of Tris-NaCl buffer (pH 7.4), obtaining a 0.5 IU/mL antithrombin solution.

Chromogenic Substrate Solution

Dissolve a vial of chromogenic substrate (category number: AG00-0101) with 5 mL of water, obtaining a 3 mM chromogenic substrate stock solution.

Before use, dilute 5 mL of chromogenic substrate stock solution with 25 mL of Tris-EDTA buffer (pH 8.4), obtaining a 0.5 mM of chromogenic substrate solution.

Stopping Solution

Prepare a 30% (v/v) acetic acid solution.

Standard Solutions

According to the anti-FIIa potency, dissolve an ample of low-molecular-weight heparin RS with an appropriate amount of Tris-NaCl buffer (pH 7.4), obtaining a 1 IU/mL of standard stock solution.

Dilute 0.1 mL of 1 IU/mL standard stock solution with 0.9 mL of Tris-EDTA buffer (pH 8.4), obtaining a 0.1 IU/mL standard solution.

Dilute 0.1 IU/mL standard solution with Tris-EDTA buffer (pH 8.4), obtaining standard dilute solutions as Table 1.

Table 1. Standard Solutions for Anti-FIIa Assay
Label S1 S2 S3 S4
Concentration (IU/mL) 0.06 0.045 0.034 0.025

Sample Solutions

Accurately weigh 0.100 g of low-molecular-weight heparin. According to the estimated anti-FIIa potency, dissolve it with an appropriate amount of Tris-NaCl buffer (pH 7.4), obtaining a 1 IU/mL of sample stock solution.

Dilute 1 mL of 1 IU/mL sample stock solution with 9 mL of Tris-EDTA buffer (pH 8.4), obtaining a 0.1 IU/mL standard solution.

Dilute 0.1 IU/mL sample solution with Tris-EDTA buffer (pH 8.4), obtaining sample dilute solutions as Table 2.

Table 2. Sample Solutions for Anti-FIIa Assay
Label T1 T2 T3 T4
Concentration (IU/mL) 0.06 0.045 0.034 0.025

Anti-FIIa Assay: Procedure