Product information
UNITEST ?Factor XIIa Inhibitors
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This kit is designed for the determination of βFactor XIIa (βFXIIa) Inhibitors in human plasma. Purified βFXIIa is added to dilute plasma and a proportion of the enzyme complexes to its plasma inhibitors. The residual βFXIIa activity is then measured using a chromogenic peptide substrate. The concentration of pNA cleaved from the substrate is measured photometrically and is inversely proportional to the concentration of βFXIIa inhibitors.
Reagents
The kit contents should be stored at 4°C until reconstituted.
1. βFactor XIIa
Dissolve in 10ml sterile distilled water. Stable at 4°C for up to 6 hours, store at -20°C.
2. Factor XII Substrate
10µmol/vial 2AcOH.H-D-CHT-Gly-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 below -20°C.
3. βFXIIai Buffer Concentrate
Dilute the vial contents (10ml) with 90ml distilled water. This gives a buffer of 0.05M Tris-HCl pH7.9.
4. Kallikrein Inhibitor
Dissolve the contents of 1 vial in 10ml distilled water. Stable for 1 month at -20°C. Dilute 5ml of kallikrein inhibitor with 95ml buffer. Stable for 8 hours at 4°C.
5. Standard Plasma
Add 1ml distilled water, leave for 5 minutes at room temperature and then mix gently until completely dissolved. Stable for 8 hours at 4°C.
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 2 hours of blood collection and should be assayed immediately or stored frozen at -20°C.
Preparation of the Standard Curve
Dilute the Standard Plasma in buffer containing Kallikrein Inhibitor as follows:
| Standard (% Activity) | Plasma (µl) | Buffer (µl) |
|---|---|---|
| 150 | 25 | 1475 |
| 100 | 25 | 1975 |
| From the 100% Standard Prepare: | ||
| 75 | 300 | 100 |
| 50 | 200 | 200 |
| 25 | 100 | 300 |
| 0 | Use buffer plus Kallikrein Inhibitor alone |
Dilute 25µl of each test plasma with 1975µl of buffer plus Kallikrein Inhibitor.
Assay Method
Warm the substrate to 37°C. Into siliconised semimicro cuvettes or plastic tubes pipette:
| Step | Volume |
|---|---|
| Plasma dilution or Buffer plus Kallikrein Inhibitor | 200µl |
| Incubate for 4 minutes at 37°C, add: | |
| βFXIIa | 200µl |
| Mix and incubate for 15 minutes at 37°C, add: | |
| Factor XII Substrate | 200µl |
| Record the change in optical density at 405nm (rate assay), or incubate for exactly 30 minutes at 37°C, add: | |
| Acetic Acid 50% (end-point assay) | 200µl |
Read the absorbance at 405nm (end-point assay).
Calculation
For the end-point assay, prepare blanks by substituting 400ul buffer for the βFXIIa and the Factor XII Substrate. Subtract the blank values from the test values. Plot the results as Log A₄₀₅ against percentage βFXIIa Inhibitor 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:
βFXIIa Inhibitors (U/ml) = (% Activity x Potency of Standard*) / 100
*The potency of the standard plasma for βFXIIa Inhibitors (lot UD-0137-0466) is 1.25 U/ml.
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.
CATALOGUE NUMBER: 0052 PRODUCT: Unitest Beta FXIIai UNICORN DIAGNOSTICS Ltd, London, UK.