The carboxylated beads were sourced from Bio-Rad (Bio-Plex COOH Beads, 1ml, 1.25x10 7 beads/ml 120 and Bio-Plex Pro Magnetic COOH Beads, 1ml, 1.25x10 7 bead/ml) and stored at 2-4°C. Optimisation of 121 coupling procedures for non-magnetic and magnetic beads were done separately, due to the larger size 122 of the magnetic beads generally requiring more protein (see Results). To be able to measure all plasma 123 samples at the same dilution, we optimized all protein concentrations by generating a log-linear 124 standard curve with a positive control plasma pool from immune PNG donors (high responders to 125 Plasmodium antigens).
Coupling of P. vivax proteins to non-magnetic beads was performed as previously described [10] .
Briefly, the optimised antigen concentration (Tables 1 and 2 ) was coupled to 2.5x10 6 pre-activated 129 microspheres, in 100 mM monobasic sodium phosphate buffer pH 6.0, using 50mg/ml sulfo-NHS and 130 50 mg/ml of EDC to cross-link the proteins to the beads. The activated beads were washed and stored 131 in PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Na-azide, pH 7.4 at 4°C until use. For the coupling to 132 magnetic beads, a magnet rack was used for pelleting the beads, instead of the centrifugation step for 133 non-magnetic beads. We qualitatively assessed the stability of the coupled beads by visual comparison 134 of the MFI of the standard curve over a nine-month period. Table 1 for a complete list of proteins and the optimised amount coupled to 141 non-magnetic and magnetic beads. 
To enable these parametric correlations, data were log-transformed prior to the analysis to better fit the 186 normal distribution. 
It was again observed that there was a strong correlation between results obtained using the non- 
During the same week assays were performed to measure total IgG antibodies against these P. vivax (Table 3 , scatter plots in Figure S1 ). The same correlation analysis was then performed on 272 data converted in R using the standard curves (to account for any plate-plate variation). Strong 273 correlation coefficients were observed for all 12 proteins, including PVX_094255 (r 2 values >0.51, 274 p<0.0001) ( Table 3 , scatter plots in Figure S2 ). For the majority of proteins, the correlation was stronger 275 after conversion (Table 3) . This is expected given the conversion, based on the standard curve 276 generated with a plasma pool from immune PNG donors, is used to account for any plate-plate 277 variation.
These results indicate that data generated using this multiplexed assay are highly reproducible in a vivax. Importantly, we also assessed the stability of the coupled beads by running the standard curve 284 10 times over a period of 9 months (intensely for 2 months) ( Figure S3 ). For most proteins the coupled 285 beads were highly stable (11/16 tested over 9-months), with the MFI dropping for three proteins and 286 increasing for two proteins. This is supported by previous research that has indicated the stability of 287 protein-coupled beads [13] , noting that the stability may vary by antigen [15]. 
The aim of this study was to demonstrate that multiplexing assays performed using magnetic beads or 295 non-magnetic beads are highly comparable, independent of the beads and platform used to analyze the 296 assays. We compared here a total of 19 P. vivax proteins that were coupled to both magnetic beads 297 and non-magnetic beads. The protein concentration used for the couplings was individually determined 298 by optimisation for each protein for the chosen bead type (Table 1) 349 Figure S3 : Stability of protein-coupled magnetic beads over 9-months. The original coupled beads 350 were tested at every week for 2 months after coupling, then again at 9 months post-coupling. The MFI
of the standard curves are presented (S1 = 1/50, then 2-fold serial dilution 