Product information
UNITEST α2-Macroglobulin
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This kit is designed for the determination of α₂-macroglobulin (α₂-M) in human plasma. Dilute plasma is mixed with excess trypsin, and the α₂-M becomes complexed with the trypsin. The remaining, non-complexed trypsin is inhibited with soy bean trypsin inhibitor. The trypsin complexed with α₂-M is still able to cleave small substrates¹⁻² and releases p-nitroaniline (pNA) from a suitable chromogenic peptide substrate. The pNA concentration may be measured photometrically and is proportional to the α₂-M concentration.
Reagents
The kit reagents should be stored at 4°C until reconstituted.
1. Porcine Trypsin
Dissolve in 10ml of 1mmol/l hydrochloric acid. Stable for at least 4 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. Soybean Trypsin Inhibitor
Reconstitute with 10ml sterile distilled water. Stable for 8 hours at 4°C and 6 months at -20°C.
4. Buffer Concentrate
Dilute 1 part of buffer concentrate with 9 parts sterile distilled water.
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.
Required but not provided: 1mmol/l Hydrochloric Acid (for reagent preparation), acetic acid 50% (end point method).
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
The Standard Plasma is diluted with buffer as follows:
| STANDARD (% Activity) | PLASMA (µl) | BUFFER (µl) |
|---|---|---|
| 200 | 50 | 3950 |
| 150 | 25 | 2642 |
| 100 | 25 | 3975 |
| From the 100% standard prepare: | ||
| 75 | 300 | 100 |
| 50 | 200 | 200 |
| 25 | 100 | 300 |
| 0 | Use buffer alone |
Dilute 25µl of test plasma with 3975µl buffer.
Assay Method
Have the Thrombin 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 2 minutes, add: | |
| Soybean Trypsin Inhibitor | 200µl |
| Mix and incubate at 37°C for 2 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, add the reagents in reverse order and substitute 200µl of buffer for Trypsin Substrate. Subtract the A₄₀₅ values for the blanks from the test values before reading the α₂-M values from the standard curve.
Plot the results as A₄₀₅ against percentage α₂-M 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:
α₂-M (U/ml) = (% Activity x Potency of Standard*) / 100
*The potency of the Standard Plasma for α₂-M (lot UD-0137-0148) is 0.95 U/ml.
Performance Characteristics
The assay is linear up to 150%, and the intra-assay coefficient of variation is 4% at 1.00 U/ml.
Interpretation
Normal Range 0.70 - 1.50 U/ml.
α₂-M may be reduced in adult respiratory distress syndrome³ and septic shock⁴. Levels are higher in adult females than in adult males. Highest levels are found between birth and three years of age⁵.
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.
- Velasco F, Torres A, Guerrero A, Andres P, Guerrero R, Aljama P, Alvarez F. Behaviour of the contact phase of blood coagulation in the adult respiratory distress syndrome (ARDS). Thrombos Haemostas 1986; 55: 357-360.
- Martinez-Brotons F, Oncins JR, Mestres J, Amargos V, Reynaldo C. Plasma kallikrein-kinin system in patients with uncomplicated sepsis and septic shock - Comparison with cardiogenic shock. Thrombos Haemostas 1987; 58: 709-713.
- Ganrot PO & Schersten B. Serum alpha-2-macroglobulin concentration and its variation with age and sex. Clin Chim Acta 1967; 15: 113-120.
CATALOGUE NUMBER: 0041 PRODUCT: Unitest α₂-Macroglobulin Kit UNICORN DIAGNOSTICS Ltd, London, UK