There were low antibody responses to these antigens while there was a higher signal to the GST alone. pools and samples. The plots represent the levels of antibodies measured in serial dilutions of the positive pools (1:3 starting at 1:50 for IgG, IgG1C4 and IgM; and 1:2 starting at 1:10 for IgE), and the RTS,S samples (1:10 starting at 1:500 for IgG, 1:100 for IgM, 1:50 for IgG1C4; and 1:2 starting at 1:10 for IgE). Data on IgG3 and IgE levels measured in RTS,S vaccinees were?not available. Isolated dots represent the levels measured in the technical blanks. 12936_2018_2369_MOESM2_ESM.pdf (158K) GUID:?5ED15D4A-E942-4AB7-8F02-4D13D4A0773A Additional file 3. Comparison of the IgG and IgG1C4 predicted curves between the WHO reference reagent and the WHO-CSP pool incubating at 4?C ON. IgG and IgG1C4 predicted curves from a non-linear equation were measured against a 23-multiplex panel. Isolated dots represent the levels measured in the technical blanks. 12936_2018_2369_MOESM3_ESM.pdf (63K) GUID:?D73B192E-6769-40ED-A012-26692B039D22 Additional file 4. IgG and IgG1C4 50% effective concentrations (EC50) to RTS,S-specific antigens measured in the WHO reference reagent and the WHO-CSP pool, and EC50 ratios between pools. The functions used to fit the standard curves were 4PL (SSl4) or exponential (SSexp) equations. 12936_2018_2369_MOESM4_ESM.pdf (52K) GUID:?BA64611A-D2BE-43C1-B650-9475620B679D Additional file 5. Correlations between GST vs. antigens included in the RTS,S vaccine, and GST vs. non-RTS,S antigens in plasmas from RTS,S-vaccinated children. Scatterplots with levels of IgG (log10MFI) to GST alone in the X-axis and to GST-fused proteins (orange) or proteins not fused to Adipor2 GST (green) in the Y-axis. Linear regression lines with 95% confidence intervals (in grey) and BIO-5192 Spearman correlation coefficients (r2) for each antigen. Correlations between IgGs to RTS,S proteins and GST were high but similar between GST-fused (CSP NANP & C-terminus) and non GST-fused proteins (CSP full length and HBsAg). Antibody levels against the GST-fused CSPs (Y-axis value) were higher than to the GST alone (X-axis value). IgG levels to GST fusion proteins representing non-RTS,S antigens (e.g. EBA-175, MSP-2) were not correlated with IgG levels to GST alone. There were low antibody responses to these antigens while there was a higher signal to the GST alone. Overall, the patterns of correlations were similar between GST-fused and non-GST fused proteins. Responses to GST and to GST fusion proteins appeared to be independent. 12936_2018_2369_MOESM5_ESM.pdf (25K) GUID:?742008F5-2D6F-4A66-B59A-C699C5B41889 Additional file 6. Levels of IgG and IgG2-4 to 15 antigens measured in the WHO reference reagent compared to negative control and blanks under three different incubation conditions. Curve plots of the antigen-specific antibody levels measured in serial dilutions of the WHO reference reagent, negative control and blanks at three different incubation conditions: 37?C 2?h (37?C 2?h), 4?C overnight (4?C BIO-5192 ON) and room temperature for 1?h (RT 1?h). neg means negative control. 12936_2018_2369_MOESM6_ESM.pdf (188K) GUID:?78EEAE98-337D-4D0A-B6B4-EB1BDA8494EB Additional file 7. Levels of IgM and IgE measured to the 40-multiplex panel in the WHO reference reagent and IgM pool compared to negative control and blanks under two different incubation conditions. Incubation conditions compared are: 4?C (4?C ON) vs 2?h at 37?C (37?C 2?h). A) Predicted 5PL curves of IgM levels in the IgM pool. B) Predicted 5PL curves of IgM levels in the WHO reference reagent. C) IgE levels in the WHO reference reagent. 12936_2018_2369_MOESM7_ESM.pdf (83K) GUID:?A96E452D-537A-4E41-B06B-ED1257D4E1C0 Data Availability StatementData obtained in this study and more details are available from the corresponding author on reasonable request. Abstract Background The quantitative suspension array technology (qSAT) is a useful platform for malaria immune marker discovery. However, a major challenge for large sero-epidemiological and malaria vaccine studies is the comparability across laboratories, which requires the access to standardized control reagents for assay optimization, to monitor performance and improve reproducibility. Here, the antibody reactivities of the newly available WHO reference reagent for anti-malaria human plasma (10/198) and of additional customized positive controls were examined with seven in-house qSAT multiplex assays measuring IgG, IgG1C4 subclasses, IgM and IgE against a panel of 40 antigens. The different positive controls were BIO-5192 tested at different incubation times and temperatures (4?C overnight, 37?C 2?h, room temperature 1?h) to select the optimal conditions. Results Overall, the WHO reference reagent had low IgG2, IgG4, IgM and IgE, and also low anti-CSP antibody levels, thus this reagent BIO-5192 was enriched with plasmas from RTS,S-vaccinated volunteers to be used as standard for CSP-based vaccine studies. For the IgM assay, another customized plasma pool prepared with samples from malaria primo-infected adults with adequate IgM levels proved to be more adequate as a positive control. The range and magnitude of IgG and IgG1C4 responses were highest when the WHO reference reagent was incubated with antigen-coupled beads at 4?C overnight. IgG levels measured in the negative control did not vary.