Product information
UNITEST α1-Antitrypsin
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This kit is designed for the determination of α₁-antitrypsin (α₁-AT) in human plasma. Plasma is diluted in buffer containing methylamine, which inactivates α₂-macroglobulin. Excess trypsin is added and during an incubation period, it complexes with α₁-AT. The remaining free trypsin is measured by its ability to cleave a chromogenic peptide substrate. The release of p-nitroaniline (pNA) from the substrate is measured at 405nm, and the log absorbance is inversely proportional to the plasma α₁-AT concentration¹⁻².
Reagents
The kit reagents should be stored at 4°C until reconstituted.
1. Porcine Trypsin
Dissolve in 10ml of 1mmol/l HCl, then further dilute 1 volume with 39 volumes of 1mmol/l HCl. Stable for at least 8 hours at 4°C and 6 months at -20°C.
2. Trypsin Substrate
Reconstitute in 10ml sterile distilled water. Stable for 8 hours at 4°C and 6 months at -20°C, if free from contamination.
3. Buffer Concentrate
Dilute 1 part of buffer concentrate with 9 parts sterile distilled water.
4. Standard Plasma
Dissolve in 1ml distilled water. Stable for 8 hours at 4°C.
Required but not provided: 1mmol/l Hydrochloric Acid (for reagent preparation), acetic acid 50% (end-point assay).
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 25µl Standard Plasma with 975µl buffer and then further dilute as follows:
| STANDARD % | Dil PLASMA | BUFFER |
|---|---|---|
| 200 | 100µl | 1900µl |
| 150 | 75µl | 1925µl |
| 100 | 50µl | 1950µl |
| From the 100% standard prepare: | ||
| 75 | 300µl | 100µl |
| 50 | 200µl | 200µl |
| 25 | 100µl | 300µl |
| 0 | Use buffer alone |
Dilute 25µl of test plasma with 975µl buffer and then further dilute 50µl with 1950µl of buffer.
Assay Method
Have the Trypsin Substrate at 37°C. Into plastic tubes, siliconised glass tubes or siliconised microcuvettes, pipette:
| Step | Volume |
|---|---|
| Buffer or plasma dilutions | 200µl |
| Incubate at 37°C for 2 minutes, add: | |
| Porcine Trypsin | 200µl |
| Mix and incubate at 37°C for 5 minutes, add: | |
| Trypsin Substrate | 200µl |
| Mix and record the change in optical density at 405nm (Rate assay), or Incubate for exactly 2 minutes at 37°C, add | |
| Acetic acid (50%) | 200µl |
Read A₄₀₅ (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 α₁-AT values from the standard curve.
Plot the results as log A₄₀₅ against percentage α₁-AT 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), assuming that the standard plasma has 1U/ml α₁-AT.
Performance Characteristics
The assay is linear up to 150%, with a sensitivtiy limit of 10%. The intra-assay coefficient of variation = 5% at 1.00U/ml.
Interpretation
Normal Range 0.70 - 1.50 U/ml.
α₁-antitrypsin accounts for 90% of the inhibitory capacity in plasma for neutrophil elastase³, and decreased levels may be found in patients with multi-systems organ failure and sepsis. It is also an important inhibitor of FXIa⁴ and activated protein C⁵.
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.
References
- Friberger P. Chromogenic peptide substrates. Their use for the assay of factors in the fibrinolytic and plasma kallikrein-kinin systems. Scand J Clin Lab Invest 1982; 42: Suppl 162: pp 84-85.
- Gallimore MJ, Aurell L, Friberger P, Gustavsson S. Chromogenic peptide substrate assays for determining functional activities of alpha-2-macroglobulin and alpha-1-antitrypsin using a new trypsin substrate. Thrombos Haemostas 1983; 50: 230.
- Ohlsson K & Olsson A-S. Immunoreactive granulocyte elastase in human serum. Hoppe-Seyler's Z Physiol Chem 1978; 359: 1531.
- Heck LW & Kaplan AP. Substrates of Hageman factor: I. Isolation and characterization of human factor XI (PTA) and inhibition of the activated enzyme by alpha-1-antitrypsin. J Exp Med 1974; 140: 1615.
- Heeb MJ & Griffin JH. Physiologic inhibition of human activated protein C by α₁-antitrypsin. J Biol Chem 1988; 263: 11613-11616.
CATALOGUE NUMBER: 0076 PRODUCT: α₁-Antitrypsin Kit UNICORN DIAGNOSTICS Ltd, London, UK