The presence and quantification of antigens was performed with similar methodology as described previously using the bead-based Luminex
® platform (Luminex Corp., Austin, TX) [
23]. Three unique bead regions (Bio-Plex COOH bead, BioRad, Hercules, CA; 171506XXX) were individually coated by the EDC/Sulfo-NHS intermediate reaction with separate antibodies specific for each antigen to be captured:
Plasmodium aldolase (12.5 μg/12.5 × 10
6 beads, rabbit IgG anti-aldolase, Abcam, Cambridge, UK; ab207494),
Plasmodium LDH (12.5 μg/12.5 × 10
6 beads, mouse IgG anti-LDH, BBI Solutions, Cardiff, UK; BM355-Z8F7), and
P. falciparum PfHRP2 (20 μg/12.5 × 10
6 beads, mouse IgG anti-HRP2, Abcam; ab9206). For the assay, a mix of the three coupled bead regions was made in 5 mL Buffer A (PBS, 0.5% BSA, 0.05% Tween20, 0.02% NaN
3) so that 1500 of each bead region would be added per well in the assay plate. Samples were incubated with 50 μL of the bead mix in filter bottom plates (Millipore; MABVN1250) for 90 min under gentle shaking and subsequently washed three times with 100 μL wash buffer (PBS, 0.05% Tween20). Beads were incubated for 45 min with a 50 μL mix of detection antibodies: anti-pAldo (1:1000×, rabbit anti-aldolase, Abcam; ab207494), anti-pLDH [1:500× of 2:1:1 mixture (BBI Solutions BM355-P4A2: BioRad Pv-pLDH HCA156: BioRad Pf-pLDH HCA158)], and anti-HRP2 (1:500×, mouse IgG anti-HRP2, Abcam, ab9203). All detection antibodies were previously biotinylated by Thermo Scientific EZ-Link Micro Sulfo-NHS-Biotinylation Kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Plates were washed three times, and wells subsequently incubated with 50 μL streptavidin–phycoerythrin (1:200×, Invitrogen, Carlsbad, CA) for 30 min. Plates were washed three times, and after a final 30 min wash step with reagent diluent, beads were washed once and re-suspended in 100 μL PBS and read on a Bio-Plex 200 instrument (BioRad, Hercules, CA
) by generating the median fluorescence intensity (MFI) signal for 50 beads in each unique region, and then the mean fluorescence intensity of the MFIs among duplicates. The final measure, denoted as MFI-bg, was reported by subtracting MFI values from beads on each plate only exposed to sample diluent during the sample incubation step. As antibodies used in this assay against HRP2 would also recognize the same epitopes present on HRP3, any positive signal for the bead assay using these antibodies is denoted as detection of HRP2/3 since the true signal would not be able to be differentiated between these two antigens from a sample.
To determine an assay signal (MFI-bg signal) which would indicate sample positivity to malaria antigens, a panel of 86 U.S. resident blood samples were run by both the multiplex antigen assay (at 1:20 dilution) in order to obtain the mean and standard deviation for a “malaria non-exposed” population. For each antigen, the mean + 3 s.d. MFI-bg value for this non-exposed population was used at the positivity threshold. To extrapolate an assay signal to an antigen concentration, standard curves of known recombinant antigens were run in order to generate equations to derive a concentration from a signal intensity of the bead assay [
23]. Recombinant pLDH and HRP2 antigens were provided by Microcoat Biotechnologie GmbH (Bernried, Germany), and lyophilized preparations were rehydrated according to the manufacturer’s instructions. The
Plasmodium vivax-specific isoform of aldolase was produced at the CDC as described previously [
23].