Product information
UNITEST Factor XIa Inhibitors
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This kit is designed for the determination of inhibitors of factor XIa (FXIa) in human plasma. Plasma is diluted in buffer containing an inactivator of α₂-macroglobulin. Excess FXIa is added and during an incubation period it complexes with plasma inhibitors. The remaining free FXIa is measured by its ability to cleave a chromogenic peptide substrate and liberate p-nitroaniline (pNA). This can be measured photometrically, and the absorbance is inversely proportional to the plasma inhibition of FXIa.
REAGENTS
The reagents should be stored at 4°C until reconstituted.
1. Factor XIa Substrate
10µmol/vial 2AcOH.H-D-Lys(Cbo)-Pro-Arg-pNA, plus mannitol. Dissolve in 10ml sterile distilled water. Stable for at least 6 months at 4°C if kept free from contamination. It may also be stored in aliquots at -20°C.
2. Human FXIa
Reconstitute in 10ml sterile distilled water. Stable for 6 hours at 4°C and 6 months at -20°C.
3. Human Albumin
Dissolve in 10ml distilled water. Stable for 8 hours at 4°C and 6 months at -20°C.
4. Buffer Concentrate
Dilute 10ml of buffer concentrate with 85ml sterile distilled water, and add 5ml Human Albumin. This gives a buffer of 0.05M Tris-HCl, 0.4M NaCl, 0.5% albumin, pH 8.0, containing an inhibitor of α₂-macroglobulin. store at 4°C.
5. Standard Plasma
Add 1.0ml distilled water, leave for 5 minutes at room temperature and then mix gently until completely dissolved. Stable for 8 hours at 4°C.
Reagents required, but not provided: 50% acetic acid.
BLOOD COLLECTION AND PREPARATION OF PLASMA
Blood (9ml) is mixed with 0.106M Tri-sodium citrate (1ml) and centrifuged at 2000g for 15 minutes at room temperature. The plasma samples should be removed with plastic pipettes within two hours of blood collection and should be assayed immediately or stored frozen at -20°C.
PREPARATION OF THE STANDARD CURVE
Dilute 100µl Standard Plasma with 900µl buffer and then further dilute as follows:
| STANDARD % | PLASMA | BUFFER |
|---|---|---|
| 150 | 25µl | 1475µl |
| 100 | 25µl | 1975µl |
| From the 100% Standard prepare: | ||
| 75 | 300µl | 100µl |
| 50 | 200µl | 200µl |
| 25 | 100µl | 300µl |
| 0 | Use buffer alone |
Dilute 100µl of each test plasma with 900µl buffer, then further dilute 25µl of this with 1975µl buffer.
ASSAY METHOD
Have the FXIa Substrate at 37°C.
Into siliconised semi-micro cuvettes, siliconised glass or plastic tubes pipette:
| Step | Volume |
|---|---|
| Plasma dilution or buffer | 200µl |
| Incubate at 37°C for 2 minutes, add: | |
| FXIa | 200µl |
| Mix and incubate at 37°C for 15 minutes, add: | |
| FXIa Substrate | 200µl |
| Mix and record the change in optical density per minute at 405nm (rate assay), or incubate for exactly 30 minutes at 37°C, add: | |
| Acetic acid (50%) | 200µl |
| Mix and read optical density at 405nm (end point assay). |
CALCULATION
With the end point assay, if the test plasmas have high bilirubin levels, are lipaemic or have visible haemolysis, blanks must be performed. For the blanks take 200µl volumes of diluted plasma, add 400µl buffer and 200µl acetic acid and mix. The A₄₀₅ values for the blanks are subtracted from the test values before reading the FXIa inhibition values from the standard curve.
Plot the results as A₄₀₅ against percentage FXIa inhibition for the standard plasma dilutions and read the values for the test plasmas from the standard curve. The values can be expressed either as a percentage or in units per ml (U/ml) by applying the formula:
FXIa Inhibitors (U/ml) = (% Activity x Potency of Standard*) / 100
* The potency of the standard plasma for FXIa Inhibitors (lot 0137-0316) is 1.25 U/ml.
PERFORMANCE CHARACTERISTICS
The assay is linear up to 150%, with a sensitivity limit of 5%. The intra-assay coefficient of variation is 5% at 1.00 U/ml.
INTERPRETATION
Normal Range 0.70 - 1.50 U/ml (70-150%)
The major plasma inhibitor of FXIa is α₁-antitrypsin¹, although AT-III² and protein C inhibitor³ (PCI-1) contribute.
HAZARD WARNING
All materials of human origin were tested and found negative for the presence of HBsAG and anti-HIV antibody. However, as with all preparations of human origin, these products cannot be assumed to be free from infectious agents and suitable precautions should be taken in their use and disposal.
NOTE
The recommended standard and test sample dilutions may vary between different batches of this kit owing to differences in the specific activity of some batches of reagents.
REFERENCES
- Heck LW & Kaplan AP. Substrates of Hageman factor. I. Isolation and characterization of human factor XI (PTA) and inhibition of the activated enzyme by α₁-antitrypsin. J Exp Med 1974; 140: 1615.
- Scott CF, Schapira M, James HL, Cohen AB, Colman RW. The inactivation of factor XIa by plasma protease inhibitors. J Clin Invest 1982; 69: 844.
- Meijers JCM, Kanters DHA, Vlooswijk RAA, van Erp HE, Hessing M, Bouma BN. Inactivation of human plasma kallikrein and factor XIa by protein C inhibitor. Biochemistry1988; 27: 4231-4237.
CATALOGUE NUMBER: 0091 PRODUCT: Unitest Factor XIa Inhibitors Kit
UNICORN DIAGNOSTICS Ltd, London, UK
2.11.93