Three different routes may drive the complement system, an important constituent of innate immunity: the classical, lectin, and alternative pathways. Whereas the first two pathways are initiated by certain stimuli such as antibodies bound to the cell surface or the presence of pathogen-specific sugar moieties, the alternative pathway is constantly active at a low level and its further propagation depends on the lack of restriction by endogenous complement inhibitors [
1]. The alternative or classical/lectin pathway complement convertases are the elements of the cascade, which are particularly controlled by complement inhibitors. Otherwise, convertases produce opsonins, anaphylatoxins, and initiate the formation of the membrane attack complex aimed to lyse target cells [
2]. Under physiological conditions, the maximal activity of convertases is reached relatively quickly after pathway initiation (seconds or minutes, depending on complement availability, as shown in [
3]) and after this time their activity decreases due to spontaneous and inhibitor-driven decay as well as proteolysis of C3b and C4b components by factor I [
4]. Diverse modes of complement activation determine the risk of autoinflammatory diseases resulting from misguided or uncontrolled complement attack. A loss of regulation of the alternative pathway is sufficient for pathologic events whereas in the case of the classical and lectin pathways, an analogous condition must be accompanied with a pathway-specific stimulus present on self cells. Indeed, mutations in alternative pathway components are well-known etiologic/risk factors for diseases such as C3 glomerulopathies (C3G) [
5] or atypical hemolytic uremic syndrome (aHUS), which manifest already at a very young age [
6]. Another condition, which leads to the unrestricted activity of alternative convertases, is the presence of autoantibodies termed C3NeF. C3NeF binds neoepitopes formed upon the assembly of the convertase and stabilizes it, resulting in consumption of C3 (hypocomplementemia) and amplification of downstream events in the complement cascade. Occurrence of C3NeF is reported in almost all patients with dense deposit disease and in half of the patients with C3GN [
7]. Interestingly, this autoantibody was also found in an individual showing no disease symptoms [
8] and its presence in some C3GN patients did not cause hypocomplementemia [
9], but, on the other hand, the same study reported C3NeF-positive C3GN in the absence of other known disease-causing agents [
9]. In contrast to the deregulation of alternative pathway convertases by either mutations in pivotal pathway components or autoantibodies, similar phenomena for the classical pathway convertases are poorly described, with not a single report about a gain-of-function mutation and only a few reports about C4NeF (analogous to C3NeF). Incidence of C4NeF was briefly reviewed in [
10] and reported in postinfectious glomerulonephritis [
11,
12], membranoproliferative glomerulonephritis [
13], systemic lupus erythematous [
14], and recently in a patient with sepsis caused by
Neisseria meningitidis [
15]. There are two reports on C4NeF occurrence in large cohorts with renal diseases, which show C4NeF in 19 out of 100 patients [
13] and 19 out of 197 patients [
16], respectively. Interestingly, the percentage of patients double-positive for C3NeF and C4NeF was 52 [
13] and 10 % [
16] depending on cohort, showing that these two kinds of activities may appear independently of each other. Also, there are reports showing that C4NeF may stabilize not only C3 classical convertase but also C5 classical convertase [
15,
17]. There is no routine diagnostic procedure for C4NeF determination. Available experimental methods are based on multistep hemolytic assays performed on sheep erythrocytes coated with purified components of classical convertases (EAC142 or EAC1423) [
13,
15] or precipitation of stabilized fluid-phase C4b2a complexes followed by detection by sandwich ELISA [
18]. An obvious limitation of detection systems based on purified complement components is elimination of interactions with other components from autologous serum, which are normally present under physiological conditions and may influence convertase formation and stability. On the other hand, detection of stabilized, fluid-phase classical convertase precipitated from patient serum does not give any information about enzymatic activity. We have designed a new method for assessment of convertase activity directly in patient’s serum or plasma, which makes use of C5 blockers: OmCI or eculizumab [
19]. Thereafter, we showed that our approach enables proper detection of clinical samples with altered function of alternative convertases caused by either autoantibodies (C3NeF, anti-factor H) or mutations in complement proteins (C3, factor B) [
19]. Herein, we report the usage of this method for screening for abnormally prolonged activity of classical convertases and, by doing so, identification of C4NeF activity in a patient with C3 glomerulonephritis of previously unknown etiology. Further analysis revealed that the specific activity responsible for the phenotype was conferred in the Ig fraction isolated from plasma.