METHOD FOR DOSING THE CONTROL CAPACITY OF C1INH
The present invention relates to a method for dosing the control capacity of plasma serpin (SERine Protease INhibitor) C1 inhibitor (C1Inh) on the basis of a patient blood sample. The invention also relates to a kit specially designed for said dosing.
1 . A method for dosing the control capacity of the protein C1 plasma inhibitor (C1Inh) based on a sample of plasma from a patient, comprising the following steps:
a) a reaction mixture is prepared based on prekallikrein (pKK), high molecular weight kininogen (HK) and activated Hageman factor protease (FXIIa), the reaction mixture being adjusted in such a manner as to have a pH higher than 7;
b) the plasma sample of the patient is incubated with a serine protease inhibitor for a duration at least equal to 5 minutes, in such a manner as to obtain a plasma sample without spontaneous protease activity and in which said inhibitor is inactivated or becomes inactive with regard to the reaction mixture prepared at step a);
c) the plasma sample obtained at step b) is incubated with the reaction mixture prepared at step a) for a duration lower than or equal to 20 minutes;
d) a chromogenic or fluorogenic substrate of the kallikrein (KK), liable to release a chromophore or a fluorophore after hydrolysis by KK, is added to the plasma sample obtained at step c);
e) the possible release of the chromophore or fluorophore obtained at step d) is detected over time; and
f) the control capacity of C1Inh is determined based on the detection achieved at step e).
2 . The dosing method according to claim 1 , according to which the ratio (mol/mol) pKK/HK of the reaction mixture ranges between 1/10 and 10/1, preferably for example between 1/2 and 2/1.
3 . The dosing method according to claim 1 , according to which the reaction mixture exhibits a pH ranging between 7 and 8.5.
4 . The dosing method according to claim 1 , according to which the serine protease inhibitor is selected from among di-isopropyl fluorophosphate (DFP) or phenylmethylsulfonyl fluoride (PMSF).
5 . The dosing method according to claim 1 , according to which at step d) the peptide H-D-Pro-Phe-Arg-para-nitroanilide is used as chromogenic substrate of the KK and at step e) the possible presence of the chromophore pNA is detected by spectrophotometric reading at 405 nm.
6 . The dosing method according to claim 1 , according to which the volume of plasma required to perform the dosing is lower than 2 μl.
7 . A kit for implementing the method according to claim 1 , comprising:
prekallikrein (pKK), high molecular weight kininogen (HK) and activated Hageman factor protease (FXIIa),
solutions allowing obtaining a pH higher than 7,
a serine protease inhibitor, and
a chromogenic or fluorogenic substrate of KK.
8 . A method for determining the quantity of plasma C1Inh required for stopping the effects of an angioedema in a patient, comprising:
a) dosing the control capacity of the C1Inh protein based on a sample of plasma from the patient, according to claim 1 , and
b) determining the quantity of C1Inh to be administered to the patient.
9 . A method for determining the quantity of plasma C1Inh stimulator required for stopping the effects of an angioedema in a patient, comprising:
a) dosing the control capacity of the C1Inh protein based on a sample of plasma from the patient, according to claim 1 , and
b) determining the quantity of stimulator of C1Inh to be administered to the patient.
10 . A method for monitoring the control capacity of C1Inh in a patient over time consisting in dosing at two different times the control capacity of C1Inh based on a sample of plasma from this patient according to the method of claim 1 .