CHROMOGENIC SUBSTRATES | METHODS | HIRUDIN
Determination of hirudin levels in plasma with the chromogenic substrate S-2366
Hirudin is a protein originating from the medical leech (1,2). It is the most potent thrombin inhibitor with a dissociation constant below 10 -12 mol/l. Due to the production of hirudin through recombinant technology, this protein is now readily available in abundant amounts and hence it has become an interesting candidate as a new antithrombotic agent (3-6). The therapeutic range of hirudin is roughly 0.5 2.5 µg/ml. Hirudin may be determined by ELISA methods, by clotting methods (APTT, thrombin time or Ecarin clotting time (ECT)) or by chromogenic methods (7,8). The latter is insensitive to oral anticoagulants (9) and is suitable for automation. The ECT method is the most competitive of the clotting methods being essentially insensitive towards heparin. However it shows some sensitivity to changes in plasma concentrations of prothrombin and fibrinogen (9, 10).
The chromogenic methods described here may be used for the monitoring of hirudin levels in plasma samples. The same principle may be used for the potency assessment of hirudin preparations. In this case the potency of hirudin is expressed in thrombin inhibitory units (TIU) (11) and the international standard for thrombin must be used.
Thrombin is added in excess to the sample. Thrombin activity is neutralised in proportion to the amount of hirudin contained in the sample, and the remaining amount hydrolyses the chromogenic substrate S-2366.
The pNA released upon hydrolysis of the substrate is then monitored photometrically at 405 nm.
|Thrombin (excess) +Hirudin||Thrombin
|S-2366||Thrombin||pNA + Peptide|
Blood (9 volumes) is mixed with 0.1 mol/l sodium citrate (1 vol) and centrifuged at 2000 x g for 20 minutes at 20-25°C. Separate plasma carefully from the blood cells.
Standard and Sample Dilution
Low range (0 2 µg/ml)
High range (0 10 µg/ml)
Samples and Standards
|Dilution||Low dose range||High dose range|
|Sample / Standard||50 µl||25 µl|
|Buffer||800 µl||2000 µl|
Microplate Assay Procedure
|Samples/Standard||50 µl||50 µl|
|Incubate at 37°C||3-4 min||-|
|Thrombin (20-25°C)||50 µl||-|
|Incubate at 37°C||2 min||-|
|Incubate at 37°C||2 min||-|
|Acetic acid, 20%||50 µl||50 µl|
Read the absorbance at 405 nm, using a reference
wavelength of 490 nm. The color is stable for at least 4 hours. Subtract the
absorbance for the blank from the absorbance of the corresponding standards and
test plasma sample. Plot the corrected absorbances for the standards against
hirudin concentrations in a lin-lin graph and draw the standard curve from
The concentration of hirudin in the test sample is calculated from the standard curve.
|Fig. 1. Low range standard curve with the microplate method. Recombinant hirudin was used.|
|Fig. 2. High range standard curve with the microplate method. Recombinant hirudin was used.|
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