Samples were assayed for anti-malarial IgG antibody responses to P. falciparum and P. vivax merozoite surface protein (MSP-119) and apical membrane antigen 1 (AMA-1) recombinant protein [11 (link)] by enzyme-linked immunosorbent assay (ELISA) at the Amhara Region Health Research Laboratory Centre. Three-millimetre diameter circles were punched from DBS and reconstituted in PBS buffer as previously described [10 (link), 17 (link)]. Plates were coated with antigens at a concentration of 0.5 μg/mL overnight. Eluates from test samples were added at a dilution of 1:1000 and species-specific positive controls sera were added at serial dilutions on each plate to generate standard curves. After overnight incubation, horseradish-peroxidase-conjugated rabbit-anti-human IgG (DAKO, USA) secondary antibody was added followed by substrate Tetramethylbenzidine (TMB) (SurModics, USA) as color detection. Optical density (OD) values were read at 450 nm after 15 min incubation with the substrate.
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