Quantitation of Regulatory Activity for the Complement Alternative Pathway Using an Adaptation of the AP(50) in vitro Assay

Complement pathways function to identify and remove pathogens and infected cells. There are three complement pathways: the classical, lectin and alternative pathway (AP). While all pathways are activated following pathogen stimuli, the AP is constitutively active and tightly controlled by activators (e.g., Factor B, Factor D) and negative regulators (e.g., Factor H). Complement activity can be measured by well-established methods that are often used in a diagnostic setting to determine the CH50 (50% complement hemolytic activity) or AP(50), specifically to measure AP activity. The protocol here has adapted the traditional AP(50) method designed to measure AP activity in human sera, to measure the positive or negative AP regulatory activity within a given test sample. The assay relies on the ability of AP components in human serum to lyse rabbit erythrocytes under in vitro conditions specific for the AP with subsequent release of hemoglobin that is quantitated by measurement of optical density. Our method has added test substances, such as cell culture media with defined changes in individual complement components and determined the ability to either promote or inhibit AP activity in vitro. Thus, this protocol reflects the overall functional ability of a sample to effect AP activity and can be used in the research laboratory to determine AP regulatory activity in a complex biological sample, or to test the ability of drugs or novel biomolecules to regulate AP activity.