Blood elution and multiplex bead assays
A 3 mm DBS was punched from each card using a Harris Uni-Core Puncher. Each 3 mm DBS was eluted in 125 µL Elution Buffer (PBS, 0.05% sodium azide, 0.3% Tween-20, filtered with a 0.2 µm filter) overnight at 4 °C in a polystyrene round bottom 96-well plate (Costar®, Corning). It was assumed each 3 mm DBS contained 1.25 µL of serum (50% hematocrit); therefore, to perform a 1:100 dilution of the serum, the blood spot was eluted in a final volume of 125 µL. The DBS elutions were diluted 1:4 by adding 75 µL of the elution into 225 µL Buffer B (PBS, 0.5% polyvinyl alcohol, 0.8% polyvinylpyrrolidone, 0.5% casein, 0.3% Tween-20, 0.02% sodium azide, filtered with a 0.2 µm filter and containing 3 µg/mL crude Escherichia coli extract) for a final serum concentration of 1:400 used for the IgG detection assay. For the antigen detection assay, a 6 mm DBS punch was taken (10 µL whole blood), and whole blood eluted in the same manner as described above to a final dilution of 1:20 whole blood used for the assay.
Assays for
Plasmodium antigen detection were performed as described previously [
19]. Two unique bead regions (Bio-Plex COOH bead, BioRad, Hercules, CA) 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) and
P. falciparum PfHRP2 (20 μg/12.5 × 10
6 beads, mouse IgG anti-HRP2, Abcam). For the assay, a mix of the two anti-
Plasmodium coupled bead regions was made in 5 mL Buffer A (PBS, 0.5% bovine serum albumin (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 at 1:20 dilution of whole blood were incubated with 50 µL of the bead mix in 0.2 µm filter bottom plates (Millipore) for 90 min under gentle shaking and subsequently washed three times with 100 µL wash buffer (PBS, 0.05% Tween20: PBS-T). Beads were incubated for 45 min with a 50 µL mix of detection antibodies: anti-pAldo (1:1000×, rabbit anti-aldolase, Abcam), and anti-HRP2 (1:500× of 1:1 mix of mouse IgG:IgM anti-HRP2, Abcam). All detection antibodies were previously biotinylated by Thermo Scientific EZ-Link Micro Sulfo-NHS-Biotinylation Kit (ThermoFisher Scientific) according to the manufacturer’s protocol. Plates were washed three times, and subsequently incubated with streptavidin-R phycoerythrin (1:200×, Invitrogen, Carlsbad, CA). Plates were washed three times, and after a final 30 min wash step with reagent diluent, beads were washed once and resuspended in 100 μL PBS and read on a Bio-Plex 200 instrument (BioRad, Hercules, CA
) by generating the median fluorescence intensity (MFI) signal for a minimum of 35 beads in each unique region, and then the mean fluorescence intensity of the MFIs between duplicates. Positive and negative controls were included on each plate to ensure appropriate validity of data. 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.
Six
Plasmodium antigens were used for IgG antibody detection: the species-specific merozoite surface protein 19kD (MSP1) fragments for
P. falciparum,
P. vivax, and
P. malariae (as described previously [
8]), and three other
P. falciparum antigens: liver stage antigen 1 (LSA1, [
20]), circumsporozoite protein (CSP, NANPx5 repeat, [
20]), and glutamate-rich protein R0 fragment (GLURP-R0, [
21]). The three
Plasmodium MSP1 19 kD antigens were produced recombinantly and purified as described previously [
8], and the three other
P. falciparum antigens were produced as peptides. The
Schistosoma japonicum glutathione-
S-transferase antigen was produced recombinantly and served as a generic protein to assess IgG non-specific binding. All antigens were coupled to magnetic beads (Luminex Corporation, Austin, TX) in the same manner as prior studies [
8,
22]. Briefly, beads were pulse vortexed, transferred to a microcentrifuge tube and centrifuged for 1.5 min at 13,000
g. Supernatant was removed and beads were washed with 0.1 M sodium phosphate, pH 6.2 (NaP). Beads were activated by suspending in NaP with 5 mg/mL of EDC (1-ethyl-3-[3′-dimethylaminopropyl]carbodiimide hydrochloride) and 5 mg/mL sulfo-NHS (sulfo
N-hydroxysulfosuccinimide) and incubating with rotation for 20 min at room temperature (RT) protected from light. After a wash with coupling buffer (50 mM 2-(4-morpholino)-ethane sulfonic acid, 0.85% NaCl at pH 5.0), antigens were coupled to beads in presence of coupling buffer for 2 h at an antigen concentration of 20 µg/mL for all antigens except for CSP and GLURP-R0 at 30 µg/mL and GST at 15 µg/mL. Beads were washed once with PBS, and suspended in PBS with 1% BSA with incubation for 30 min at RT by rotation. Beads were then resuspended in storage buffer (PBS, 1% BSA, 0.02% NaN
3, 0.05% Tween-20, protease inhibitors) and stored at 4 °C.
For the IgG assay, all incubation steps were performed at RT in 50 µL reaction volumes protected from light as previously described [
23]. Incubation steps were followed by washes with 200 µL PBS-T performed by attaching plates to a handheld magnet (Luminex Corp., Austin, TX) for 1 min then inverting to evacuate supernatant. A master mix of beads was made by combining 125,000 beads per region in 5.5 mL of Buffer A. Bead master mix was aliquoted into a black flat-bottom 96-well plate (BioRad) for a total of 1250 beads per well per region. Beads were then incubated with eluted samples for 90 min and washed three times, followed by 45 min with secondary antibodies diluted in Buffer A (50 ng/well biotinylated mouse anti-human IgG and 20 ng/well biotinylated mouse anti-human IgG4; Southern Biotech, Birmingham, AL). After three washes, wells were incubated with 250 ng/well of streptavidin-R phycoerythrin, incubated for 30 min, washed three times, and incubated for 30 min with Buffer A alone. After one wash, beads were resuspended in 100 µL PBS and then stored overnight at 4 °C protected from light and read the following day. Before reading, plates were shaken at room temperature for at least 20 min, and the old PBS was removed. Fresh 100 µL PBS was added and plates were read on a MAGPIX machine (Luminex Corp) with Luminex xPonent
® software. Median fluorescence intensity (MFI) signal was generated for a minimum of 35 beads/region, and background MFI from wells incubated with Buffer B was subtracted from each sample to give a final value of MFI-bg.
Statistical analysis
To determine an assay signal (MFI-bg signal) which would indicate positivity to antigen or antibody, a panel of 86 US resident blood samples were run by both the multiplex antigen assay (at 1:20 whole blood dilution) and multiplex antibody assay (at 1:200 whole blood dilution, equivalent to 1:400 serum) to obtain the mean and standard deviation for a “malaria non-exposed” population. For each antigen or antibody, the mean + 3 standard deviations (s.d). MFI-bg value for this non-exposed population was used at the positivity threshold. For antigen detection, the MFI-bg thresholds were: pAldo (325) and HRP2 (100), which correspond to a limit of quantification of approximately 128 pg/mL for pAldolase and 8.8 pg/mL for HRP2. For the IgG antibody detection assay, the MFI-bg thresholds were: PfMSP1 (75), PvMSP1 (146), PmMSP1 (207), LSA1 (99), CSP (120), GLURP-R0 (62). Positivity of a sample to any of the three P. falciparum-specific antigens (LSA1, CSP, GLURP-R0) considered that child positive to “Pf short-term” antibodies.
To obtain unweighted odds ratios (ORs) for IgG carriage to Plasmodium antigens, data was fit to logistic regression model in SAS v9.4 (Cary, NC) using PROC LOGISTIC with the MODEL statement. Wald OR estimates were obtained for camp of residence, age category, or duration at camp of residence. Duration categories were selected based on the seasonality of malaria for this region. Statistical significance was considered if 95% confidence interval did not include 1.0.