Background

Complement comprises more than 30 plasma and membrane-bound proteins that interact to induce a series of inflammatory responses to defend against infection, prime innate immunity and modulate adaptive responses. The complement cascade is activated by three pathways: classical (CP), lectin and alternative (Morgan et al., 2015; Zelek et al., 2019a). The CP was the first to be discovered when investigating the bacterolytic properties of serum in the 19^th century. More recent studies have shown that the complement cascade is functionally and structurally conserved in mammals, with the majority of components working across species (Slodkowicz et al., 2020; Moleón et al., 2006). CP is triggered by antigen-bound antibodies on surfaces; C1 binds and initiates a cascade with sequential generation of the CP C3 convertase C4b2a and C5 convertase C4b2a3b. The C5 convertase cleaves C5, releasing the C5a anaphylatoxin and initiating the terminal pathway through C5b; this binds C6 and C7, and the trimolecular C5b-7 complex attaches to the target surface. C8 and multiple copies of C9 are recruited to build a transmembrane pore (Podack et al., 1984; Serna et al., 2016; Menny et al., 2018). The complement system evolved to protect from pathogens; however, over-activation of complement drives inflammation in many diseases, including renal, autoimmune and neurological diseases (Zelek et al., 2019a; Carpanini et al., 2019). Emerging evidence of roles of complement activation in diverse diseases has triggered an explosion of interest in developing new complement targeting drugs and generated a need for testing in animal models. It is therefore important to have simple and robust assays measuring complement activity in rodents and other model species. Although the traditional CH50 assay, developed more than 70 years ago (Rice et al., 1952; Mayer et al., 1967), is well respected and used in research and clinical settings to measure human complement activity, its application for measuring mouse complement is limited because the “classical” targets, antibody-sensitised sheep erythrocytes (ShEA), are poor activators of mouse complement necessitating the use of high amounts of mouse serum required (typically, 25-50% concentration in assay). The utility of the assay for mouse can be improved by inclusion of an additional sensitisation step, incubating the ShEA with mouse anti-rabbit IgG, but serum requirement remains high (Zelek et al., 2019b) (Figure 1).

Here I provide modified haemolysis assay protocols for measuring the activity of individual components of mouse complement that require ~10-fold less mouse serum by combination with human depleted sera (Figure 1). Because of the high functional conservation in complement proteins between man and rodent, the test mouse component functionally restores haemolysis in the depleted human serum, providing a titratable assay.

Figure 1. Schematic of mouse classical pathway (CP) CH50 haemolytic assays. Top panel: diagram of modified CP CH50 haemolytic assay utilising double sensitised ShEA and high amounts of mouse serum (typically 25% male, 50% female). Bottom panel: diagram of the mouse component-specific CP CH50 haemolytic assay utilising 10% human serum depleted of the component of interest and different amounts of mouse serum as source of the component. MAC, membrane attack complex; ShEA, sheep erythrocytes sensitised with amboceptor.