Affinipure goat anti human iga igg igm h l

ELISAs were performed by capturing biotinylated bait proteins into individual wells of streptavidin-coated 384-well plates (Greiner Bio-one). Plates were washed for 30 min with 50 μL PBS-T (0.2% Tween) and blocked with PBS-2% BSA for a minimum of 3 h. 20 μL of a bait protein diluted in PBS-2% BSA at a concentration previously determined as the amount required to saturate the biotin binding capacity of the well were added in triplicate and incubated for at least 16 h at 4 °C. Antisera were centrifuged at 13,000 rpm for a minimum of 1 h at 4 °C, diluted in PBS-2% BSA and incubated with rotation for at least 16 h at 4 °C before adding to the antigen-coated plates for 1 h. Serum dilutions used were: native Tanzanians 1:5000, native Malaysians 1:1000, imported malaria 1:500–1:1000, European travellers 1:100–1:500, and Malawian pooled sera resuspended to 20 mg mL⁻¹ and used 1:1000. Plates were washed 3× in PBS-T before incubating with 1:10,000 dilution of peroxidase-conjugated AffiniPure goat anti-human IgA + IgG + IgM (H + L) (Jackson ImmunoResearch) in PBS-2%BSA for 1 h. Plates were washed in PBS-T and the HRP substrate ABTS (KPL) was added and absorption at 405 nm determined using an automated plate reader (FluoStar Optima, BMG Labtech).