Wells. 4.10. Anti-DENV Complement-Fixing Pirenperone 5-HT Receptor antibody Assay Qualities The limit of detection (LOD), limit of quantitation (LLOQ), linearity, and precision are described within the supplemental material. LOD and LLOQ had been determined per Quinn et al. [64]. 4.11. Homology of Flavivirus Envelope Protein Many sequence alignment and phylogenetic tree evaluation from the envelope amino acid sequence of DENV1 (strain Western Pacific; GenBank accession No. AAN06981.1), DENV2 (strain New Guinea C; GenBank accession No. AAP73862.1), DENV3 (strain Sleman/78; GenBank accession No. AAT69740.1), or DENV4 (strain 1228; GenBank accession No. AER00190.1), Zika virus (strain PRVABC59; GenBank accession No. AMZ03556.1), yellow fever virus (strain Stamaril, Sanofi Pasteur, Lyon, France; GenBank accession No. QGN18661.1), West Nile virus (strain NY99; GenBank accession No. AAG02038.1), Japanese encephalitis virus (strain SA 14-14-2; GenBank accession No. ARE67893.1), and tick-borne encephalitis virus (strain K23; GenBank accession No. AAC62093.1) had been carried out using web interface for Clustal Omega and protein blast available at https: //www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 19 Could 2021) and https://blast.ncbi. nlm.nih.gov/Blast.cgi# (accessed on 19 Could 2021), respectively. The former was applied to construct a phylogenetic tree, while the latter, to calculate protein percent identity. 4.12. Statistical Evaluation Specificity of anti-DENV complement-fixing antibodies had been calculated determined by the percentage of samples with MNT50 10 (seronegative) that had been below the threshold titer of 3 EU/mL. On the other hand, sensitivity was calculated as the percentage of samples with MNT50 ten (seropositive) that have been equal to or above the threshold titer of three EU/mL. The receiver operating characteristic (ROC) curves have been made by plotting the accurate seropositive price (defined because the rate of complement-fixing antibodies with titers equal to or above the threshold titer of 3 EU/mL in seropositive samples (MNT50 10)) against the false seropositive price (defined as complement-fixing antibody titers equal or above the threshold titer of three EU/mL in samples thought of seronegative (MNT50 ten)) at numerous complement-fixing antibody thresholds from three EU/mL to roughly 1000 EU/mL. The area beneath the ROC was calculated as a single numerical measurement to describe the functionality of the complement-fixing antibody titers as a biomarker to classify dengue serostatus. Correlation evaluation amongst complement-fixing antibody titers and microneutralization titers or total binding IgG concentrations was carried out using log10 -transformed values utilizing JMP version 13.1.0 computer software (SAS BOC-L-phenylalanine-d8 supplier Institute Inc., San Diego, CA, USA). Samples have been color-coded depending on DENV serostatus (blue–seronegative; red–seropositive). Correlation analysis involving complement-fixing and C3d deposition antibody concentrations was carried out making use of log10 -transformed values making use of GraphPad Prism version eight.1.0 (GraphPad Computer software, Inc., San Diego, CA, USA).Int. J. Mol. Sci. 2021, 22,16 ofSupplementary Components: The following are offered on the net at mdpi/article/10 .3390/ijms222112004/s1. Author Contributions: Conceptualization, E.J.M.N. and H.J.D.; methodology, E.J.M.N., R.B., E.K.; formal analysis, E.J.M.N., B.N., A.P., R.B.; E.K. and H.J.D.; investigation, E.J.M.N., B.N., A.P., R.B., E.K. and H.J.D.; information curation, E.J.M.N., B.N., A.P.; writing–original draft preparation, E.J.M.N., B.N., A.P., R.B., E.K. and.