Peroxidase conjugated goat anti human igg igm

Total P. falciparum+ IgG and IgM in blood plasma were measured as previously described [21 (link)]. Briefly, microtiter plates were coated with schizont extract, blocked with 5 % skimmed milk (Sigma) for the IgG-assay and super block dry blend (Thermo Scientific) for the IgM-assay. Plates were incubated with peroxidase-conjugated goat anti-human IgG/IgM (Sigma) and bound antibody quantified using TMB (3,3′,5,5′-Tetramethylbenzidine) substrate (Promega). Optical density (OD) was read at 450 nm.

Pf+ total IgG and IgM in blood plasma were measured by enzyme linked immune-sorbent assay (ELISA) as described [58 (link)]. Briefly, microtiter plate wells were coated with 1 µg of schizont extract per well (overnight at 4 °C), and blocked with 5% skimmed milk (Sigma) for IgG and super block dry blend (Thermo Scientific) for IgM for 2 h at room temperature. Plasma specimens were diluted 1:200 with plasma dilution buffer (2.5% milk powder in phosphate buffered saline with tween 20 plus 0.02% sodium azide). Diluted plasma samples were added to the wells in quadruplets and incubated in the microtiter plates at room temperature for 1 h. The microtiter wells were washed 4 times between the incubation (coating, blocking, first and secondary antibody) steps. The wells were then incubated (45 min) with diluted (1:20,000) peroxidase-conjugated goat anti-human IgG/IgM (Sigma) and rewashed. Bound secondary antibody was quantified by adding TMB (3, 3′, 5, 5′-Tetramethylbenzidine) substrate (Promega). Optical density (OD) was read at 450 nm with a reference at 620 nm. Plasma samples of Swedish individuals unexposed to P. falciparum infections were used as negative controls. All specimens were analysed twice and the means of the ELISA OD used in the analysis.

Total anti-P. falciparum IgG and IgM in blood plasma were measured as described [39 (link)]. Briefly, microtiter plate wells were coated with schizont extract and blocked with 5% skimmed milk (Sigma) for IgG and super block dry blend (Thermo Scientific) for IgM. Diluted plasma samples were added to the wells in the microtiter plates and incubated at room temperature for 1 hour. Plates were washed 4 times between the incubation steps and subsequently incubated with peroxidase-conjugated goat anti-human IgG/IgM (Sigma) and rewashed. Bound secondary antibody was quantified using TMB (3,3′,5,5′-Tetramethylbenzidine) substrate (Promega). Optical density (OD) was read at 450 nm. All samples were analyzed twice and the means of the OD used in the analysis.