Collection of blood samples and laboratory assessments
Blood was obtained at visit 2,4 and 17 (prior to C1-INH dose
administration) and at 18 (follow-up visit). C1-INH function was
measured with a chromogenic assay (Sanquin, the Netherlands). C4 levels
and total IgE were determined at visit 1. In women <50 years
of age, pregnancy was excluded via a urine dipstick βHCG test. CRP,
d-dimer and leukocyte count were determined with routine assays. C1-INH,
FXII, PK and HK levels were analysed using immunoblot. For this, EDTA
plasma was diluted 40 times in four times reducing sample buffer (15.5%
glycerol, 96.8 mM Tris-HCL, 3.1% SDS, and 0.003% bromophenol blue, 25
mM DTT), boiled for 10 minutes, and 5 µL per sample was loaded and ran
on a 4-12% Bis-Tris gel at 165V for 60 minutes and transferred onto
Immobilon-FL membranes at 125V for 55 minutes. For detection, polyclonal
goat anti human IgG antibodies (anti-human FXII Cl20055AP, anti-human PK
Cl20090A, anti-human HK Cl20027AP, anti-human C1-INH CL200323AP,
Cedarlane, Burlington, Canada) and Alexa Fluor 680 donkey anti-sheep IgG
(lot#1878516, Dako, Glostrup, Denmark) were used.
Levels of cHK in EDTA plasma, an indirect marker for bradykinin release,
were determined with ELISA as described above.22 The
upper normal limit was assessed using values of ~50
healthy individuals for cHK and ~20 healthy controls for
C1-INH complexes.