ESPN 50th Annual Meeting

ESPN 2017

M.A. Michels 1 M. Okroj 2 A.M. Blom 3 S.A. van Kraaij 1 N.C. van de Kar 1 E.B. Volokhina 1 L.P. van den Heuvel 1

1- Radboud university medical center
2- Medical University of GdaƄsk
3- Lund University

The autoantibody C3 nephritic factor (C3NeF) plays a pathogenic role in C3 glomerulopathy (C3G) by stabilizing the key enzyme of complement activation, the C3 convertase. However, reliability of currently used assays to detect C3NeF is limited. Recently, we developed a method to measure convertase stability in whole human serum and we now optimized the method for simple detection of convertase-stabilizing factors such as C3NeF in large patient cohorts.

Material and methods:

Convertase stability was measured in a hemolytic assay using the C5-blocking agent eculizumab to separate the alternative pathway (AP) into two steps: formation of C3/C5 convertases by test sera in step 1 (a time-variable step) and formation of lytic membrane attack complexes in a standardized second step for readout. Samples of 15 controls, 33 patients with (suspected) C3G or closely related disorders, and family members with Factor B (FB) mutation (p.Lys323Glu) and atypical hemolytic uremic syndrome (aHUS) were analyzed.


Healthy controls were tested to define the normal convertase activity profile: maximal convertase activity was reached at t=10/15 min and activity returned to background levels from on t=30. When serum or purified Ig fraction containing C3NeF was added to control serum, convertase stability was increased at t=30 min (P<0.001). Thus, detectable convertase activity at t=30 min or later was chosen as a marker for presence of convertase-stabilizing factors such as C3NeF. In our cohort, 17 out of 33 (52%) patients showed increased convertase stability. Interestingly, prolonged convertase activity was also detected in an aHUS family and segregated with the FB mutation in affected and non-affected family members.


We present optimization of a simple, reliable, and cost- and time-effective assay for detecting convertase-stabilizing factors (C3NeF and some mutations) in patients with various complement-mediated renal diseases. This study may give insight in disease pathogenesis and treatment strategies in these patients.