Quantitation of Regulatory Activity for the Complement Alternative Pathway Using an Adaptation of the AP₅₀ in vitro Assay

Background

Complement is part of the innate immune system and acts to recognize and clear pathogens and stimulate adaptive immune responses. The three complement pathways, the classical (CP), lectin (LP) and alternative pathways (AP), constitute a series of cleavage and activation of complement proteins, generating enzymatically active protein complexes, such as convertase complexes, and bioactive split products, such as C3a. The three pathways converge on cleavage of the complement component C3 and one common downstream outcome is the generation of a membrane attack complex (MAC) and subsequent lysis of target cells (Ricklin et al., 2010). Functional activity of complement can be measured by assays such as the 50% complement hemolytic activity (CH₅₀ assay), where a stimulus (antibody coated red blood cells), is provided in vitro to the complement components in a biological sample. The subsequent lysis of the target red blood cells (RBC) is quantitated by release of hemoglobin and measured by an increase in optical density. If target complement components such as C3 are missing from the test biological sample, either genetically or due to complement consumption resulting from activation of complement in vivo, then CH₅₀ will be low. Similarly, the AP₅₀, specifically measures the activity of the AP where rabbit RBC spontaneously activate the human AP resulting in RBC lysis (Figure 1A). The presence of EGTA chelates calcium to prevent CP activation and the addition of Mg²⁺, restores this requirement for the AP, yielding buffer conditions specific to determine AP activity (Joiner et al., 1983). CH₅₀ and AP₅₀ assays are routinely used in many diagnostic laboratories to quantitate complement activity in human blood (Prohaszka et al., 2016). In the traditional application of the AP₅₀, a high AP activity is reflected by low availability of complement components due to complement ‘consumption’ and thus low lysis of rabbit RBC. The lysis of rabbit RBC is quantitated by detecting the release of free hemoglobin by an increase in optical density (Figure 1A). This traditional assay has been adapted here, to keep the starting complement components in the assay constant by use of one source of human serum and measure the ability of factors added in test substances to promote the cleavage of the complement components from human serum, under conditions specific for the AP (Figure 1B). In this case, with constant input of complement components, adding a sample containing a stimulator, such as factor D or B will result in increased RBC lysis, while adding a negative regulator, such as factor H (FH) will result in decreased RBC lysis. A previous study has applied a similar approach to measure complement convertase activity in human serum (Blom et al., 2014) and the method here has adapted this to measure the net stimulatory or inhibitory ability of cell culture supernatants for the AP (Cabezas et al., 2018).


Figure 1. Comparison of traditional (A) and adapted (B) AP activity assays. Method (A) uses variable levels of HuS to quantitate AP activity in the test HuS samples. Our adapted method (B) utilizes a constant HuS sample and quantitates the AP regulatory activity of added test samples. HuS = human serum; OD = optical density.