Multiplexed High-Throughput Serological Assay for Human Enteroviruses

Figures

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  Figure A1. Western blot analysis for antigens. Lanes 1-7 are VP1 proteins for CVA4, CVB1, PV1 EV-D68, RV-A89, RV-B14, RV-C3, lane 8 is BSA, and lane 9 is 2A. Panel (A) shows the Ponceau-staining of the nitrocellulose membrane after transfer, (B) 3A6 anti-CVB1 monoclonal antibody, (C) anti-his antibody and (D) Dako 5D8/1.

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  Figure 1. Multiplex assay development. Representative VP1 protein sequences from all human infecting enterovirus and rhinovirus species were equipped with an N-terminal GST-tag and a C-terminal 6xhis-tag for purification after bacterial expression. The purified recombinant VP1 proteins along with 2A and 3C proteases from CVB3 and a BSA control were biotinylated and each antigen was coupled to a unique U-PLEX linker. During coating each U-PLEX linker coupled antigen would target a specific site at the bottom of the well. After coating, the assay is run like a regular indirect ELISA, except for having a secondary antibody with electrochemiluminescence tag, and a suitable substrate and an MSD plate reader.

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  Figure 2. Antigen quality control. (A) The detection of biotinylation using neutral chimeric avidin and anti-avidin antibody. (B) Antigen detection in Western blot using pooled human serum (Nanogam).

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  Figure 3. Different sample groups show variable raw signal ranges in antibody responses for EV and RV antigens in the multiplex assay. (A) Adult serum antibody responses grouped by IgM status. (B) Antibody responses grouped by the age groups of samples. * p < 0.05 in Wilcoxon rank-sum test after correcting for multiple testing (see Table 2).

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  Figure 4. Scatterplots and correlations of antibody responses between different enterovirus antigens. The lower diagonal has scatterplots showing how pairs of antibody responses are correlated in (A) adults and (B) children and in the upper diagonal shows the corresponding Spearman correlation coefficients. In panel A, the diagonal shows the distribution of antibody responses grouped by IgM status: blue represents IgM positive group, green the IgM negative group and red the Tampere biobank samples collected from healthy adults, for which we do not know the IgM status, and which are representing the population background.

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  Figure 5. Antigen specific min-max normalized antibody responses and correlations of antibody responses between different enterovirus antigens. Antigen specific min-max normalized antibody responses (see Equation (2)) in the multiplex assay illustrated as radar plots for individual (A) adults and (D) children. Radii of the sectors represent normalized signal intensities. Age grouped Spearman correlations for antibody responses are illustrated as matrices for adults (B) and children (D). Min-max normalization was performed separately for adults and children, due to differences in signal levels in the two groups. Comparing (A) to (D) and (B) to (C), the differences between antibody responses in adults and children become apparent.