Measuring Complement Activation by Malaria Antigens

96-well plates (Maxisorp, Nunc) were coated with purified merozoites at 5 × 10⁶ merozoites per well or recombinant merozoite surface antigens at 1–2 μg/ml^{24 (link)}. Briefly, plates were blocked with 1% casein, then incubated with serum samples (1/100–1/500) for 1 h, then washed with PBS-Tween 0.01%, followed by incubation for 30 min with purified C1q (10 μg/ml, Millipore) or normal serum (NS), heat-inactivated serum (HIS) from malaria naive donors, or C1q-depleted serum (Millipore) at 10% final concentration as a source of complement. After washing plates, C1q fixation was detected by incubating with rabbit anti-C1q antibodies (in-house) and then with anti-rabbit-IgG-HRP (Millipore), incubated for 1 h each. ABTS (Life Technologies) was added, incubated for 30 mins and absorbance quantified using a plate reader. All steps were conducted at room temperature (~21 °C). For studies of membrane attack complex (MAC) formation, normal serum was used as a complement source, and (rabbit-) anti-C5b/C9 antibodies (Millipore), and anti-rabbit-IgG-HRP (Millipore) were used for detection. IgG subclass ELISAs were done as previously described^{29 (link),46 (link),54 (link)} using mouse anti-human IgG subclass monoclonal antibodies (Thermo Fischer Scientific) and anti-mouse-IgG HRP (Millipore) detection antibodies. (Detailed protocols are available from the authors on request).