Determination of PKA in albumin and immunoglobulin preparations with Chromogenic Substrate S-2302™
Measurement Principle
Prekallikrein (prekininogenase) is activated to kallikrein by prekallikrein activator (PKA). The kallikrein formed catalyses the splitting of p-nitroaniline (pNA) from the chromogenic substrate H-D-Pro- Phe-Arg-pNA (chromogenic substrate S-2302). The rate at which pNA is released is measured photometrically at 405 nm and can be followed on a recorder (initial rate method).
The correlation between the change in absorbance per minute (ΔA/min) and the prekallikrein activator concentration is linear between 0 and 51 IU/ml of prekallikrein activator.
The concentration of prekallikrein activator is calculated using an international standard.
PKA
Prekallikrein → Kallikrein
Kallikrein
H-D-Pro-Phe-Arg-pNA + H2O → H-D-Pro-Phe-Arg-OH + pNA
Reagents
- Chromogenic Substrate S-2302, 25 mg Art. No. S820340
Reconstitute the substrate S-2302 (MW: 611.6) with 6.8 ml of distilled water. Working solution: dilute one volume of the stock solution with nine volumes of the buffer (Reagent 2). The working solution is stable for 8 hours at 20-25°C. - Tris Buffer, pH 7.8 (25°C)
Tris 6.1 g (50 mmol/l)
NaCl 0.7 g (12 mmol/l)
Distilled water 800 ml
Adjust the pH to 7.8 at 25°C by adding approximately 38 ml of 1 mol/l HCl. Fill up to 1000 ml with distilled water. The buffer, if not contaminated, will remain stable for six months at 2-8°C. - Prekallikrein Activator
The 1 st International Standard 1984 (NIBSC, 82/530) contains 85 International Units per ampoule. Reconstitute with 1 ml of distilled water. Refer to PJ Kerry et al., Br J Haematol, 345-352, (1985) for more information on the standardization of PKA. - Prekallikrein fraction
A prekallikrein fraction is prepared according to the chromatography procedure described in the appendix. Check the quality of the prekallikrein according to paragraph J in the appendix before each test run. The prekallikrein solution is stable for at least one year at -70°C.
Sample
Albumin and immunoglobulin preparations.
Dilute the sample to a corresponding prekallikrein activator concentration of 10-40 IU/ml.
Standard curve
The 1st International Standard has a PKA concentration of 85 IU/ml and is diluted as indicated in the table below.
| PKA IU/ml | International Standard µl | Buffer µl |
|---|---|---|
| 0 | – | 1000 |
| 10.2 | 120 | 880 |
| 20.0 | 235 | 765 |
| 34.9 | 410 | 590 |
| 50.2 | 590 | 410 |
Method
| Initial rate method |
|---|
| Step A for sample and standard | Sample Tube No. 1 |
|---|---|
| Sample or standard | 25 µl |
| Prekallikrein | 100 µl |
| Mix and incubate at 37°C in capped tubes | 45 min |
| Step B for sample and standard | Sample Tube No. 2 |
| Substrate (37°C) | 1000 µl |
| Mixture from tube No.1 | 25 µl |
| Mix |
Transfer sample immediately to a 1 cm semi-microcuvette (preheated to 37°C) for measurement of the absorbance change for at least two minutes in a photometer at 405 nm and at 37°C. Immunoglobulin may occasionally contain significant kallikrein activities and thus a blank reading is necessary.
| Step A for immunoglobulin blank | Blank Tube No. 1 |
|---|---|
| Immunoglobulin | 25 µl |
| Buffer (37°C) | 100 µl |
| Mix | |
| Step B for immunoglobulin blank | Blank Tube No. 2 |
| Substrate (37°C) | 1000 µl |
| Mixture from tube No.1 | 25 µl |
| Mix |
Transfer sample immediately to a 1 cm semi-microcuvette (preheated to 37°C) for measurement of the absorbance change for at least two minutes in a photometer at 405 nm and at 37°C.
Calculation
Calculate ΔA/min. Perform the following calculation for the assay of prekallikrein activator in Immunoglobulin preparations:
ΔA/min sample – ΔA/min blank
Plot ΔA/min for the standards against their prekallikrein activator concentration. Calculate the prekallikrein activator concentration of the sample from the established standard curve.
Bibliography
- Snape TJ et al. The assay of prekallikrein activator in human blood products. Dev Biol Stand 44, 115-120 (1979).
- Kerry PJ et al. Standardisation of prekallikrein activator (PKA): the 1st International Standard for PKA. Br J Haematol 60, 345-352 (1985).
- Briseid K et al. Part of prekallikrein removed from human plasma together with IgG-immunoblot experiments and functional tests. Scand J Clin Lab Invest 59, 55-63 (1999).
- Briseid K et al. Removal of IgG from normal plasma and plasma from untreated patient active Crohn’s disease-effect on levels of contact factors. Scand J Clin Lan Invest 60, 237-45 (2000).