Nunc opaque maxisorp 96 well plates

The ADRB assay was performed as previously described (Llewellyn et al., 2015 (link), Murungi et al., 2016 (link)). Briefly, PEMS were thawed and coated overnight onto individual wells of Nunc opaque MaxiSorp 96-well plates (Thermo Scientific, USA) at 18.5 × 10⁵/ml. Following three washes with 1 X PBS, the plates were blocked and incubated with plasma (1:50) for 1 h at 37 °C. The plates were washed and 50 μl of PMNs (1 × 10⁷ PMNs/ml) added, followed by 50 μl of isoluminol (0.04 mg/ml). Chemiluminescence was measured for 1 s every 2 min over an hour. Control wells containing a pool of UK adult plasma and a pool of plasma from Kilifi adults were included in each plate. Readings were expressed relative to those obtained from the pool of plasma from Kilifi adults.

100 μL PEMS at 18.5 × 10⁵ schizonts/mL was adsorbed onto Nunc opaque Maxi-sorp 96-well plates (Thermo Scientific) at room temperature (RT) overnight. Plates were then washed three times with PBS and blocked for 1 h with Casein block solution (Pierce, UK) before a second set of 3x washes. 100 μL serum diluted 1:50 in PBS (unless stated otherwise) was then added and incubated for 1 h at 37 ^°C. Within 2 min of a final wash of the assay plate in PBS, 50 μL isoluminol (Sigma Aldrich, UK) (0.04 mg/mL) and 50 μL isolated human PMNs at 1 × 10⁷ PMNs/mL were added to each well and luminescence was read (in relative light units [RLU]) every 2 min for 1 h using a Varioskan Flash luminometer. For Fig. 1D, serum was heat inactivated by heating to 56 ^°C for 30 min. A positive control sample of pooled hyper-immune Kenyan serum was tested on each plate at a dilution of 1:50 and used to index samples as described in Results.