1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N -hydroxysulfosuccinimide (Sulfo_NHS) were purchased from Pierce Biotechnology (Rockford, IL). 2-[N -morpholino] ethanesulfonic acid (MES), Tween-20, bovine serum albumin (BSA) sodium azide, biotinylated anti-human IgG, biotinylated anti-V5 antibody and phycoerythrin conjugated streptavidin were purchased from Sigma-Aldrich, USA.
The hyper-immune plasma pool was made up of plasma from ten individuals from a malaria endemic area of Liberia. Twenty samples from Danes who have never had malaria were used to make up the naïve pool. Sixty individual plasma samples, twenty each from people living in the three Tanzanian villages Mgome, Ubiri and Magamba with high, moderate and low malaria transmission , respectively were also analysed.
Protein expression was as described previously [9, 10]. Briefly, primer pairs designed to contain restriction enzyme sites (See additional file 1) were used to amplify Cysteine-rich inter-domain regions (CIDR) and Duffy binding-like (DBL) domains from 3D7 genomic DNA. The digested PCR products were cloned into the Baculovirus vector, pAcGP67-A (BD Bioscience), which was designed to contain the V5 epitope upstream of a histidine tag in the C-terminal end of the construct. The identity of the cloned fragments was verified by sequencing. Linearized Bakpak6 Baculovirus DNA (BD Biosciences Clontech) was co-transfected with pAcGP67-A into Sf9 insect cells for generation of recombinant virus particles and histidine-tagged proteins secreted into the supernatant of infected High-Five insect cell were purified on Co2+ metal-chelate agarose columns. Eluted products were dialysed overnight in PBS. The yield, integrity and purity of the recombinant proteins were estimated by analysis on SDS gel, comparing to BSA standards, and by western blotting using the anti-V5 antibody. All of the proteins coupled to the Luminex beads were estimated to be at or above 80% purity. The sizes of the different recombinant proteins ranged from 45 to 60 kDa.
Covalent coupling of recombinant PfEMP1 proteins to beads
Carboxylated Luminex beads were covalently coated with the different PfEMP1 protein domains through an interaction of their carboxyl groups and the amino groups on the proteins following the procedure suggested by the manufacturer. Beads (1.25 × 107 beads/ml) were brought to room temperature, vortexed for one minute and transferred to Eppendorf® tubes. The supernatant was removed after centrifugation for one minute at 16,000 × g. 1 ml of distilled water was added to the beads, vortexed to re-suspend followed by centrifugation for one minute at 16,000 × g. The beads were sonicated in a water bath sonicator into suspension and centrifuged for one minute at 16,000 × g. The supernatant was removed by a pipette and 1 ml of activation buffer (0.1 M NaH2PO4, pH 6.2) added to the pellet and vortexed to re-suspend. In separate tubes Sulfo_NHS and EDC were reconstituted to 50 mg/ml and 125 μl of each added to the beads, vortexed and incubated at room temperature for twenty minutes with inversions in the dark. The beads were centrifuged for one minute at 16,000 × g, re-suspended in 1 ml of 50 mM MES pH 5.0, centrifuged for one minute at 16,000 × g and the supernatant removed. The MES wash was repeated. The beads were re-suspended in 500 μl of MES. In separate tubes, the different protein samples (100 μg of each) were mixed with MES to a final volume of 500 μl and each was added to a separate bead population and incubated at room temperature for two hours in the dark with inversions. The beads were centrifuged for one minute at 16,000 × g and the supernatant removed. The beads were washed twice in 1 ml of PBS/TBN (0.02% Tween-20, 0.1% BSA and 0.05% sodium azide in PBS pH 7.4. The beads were re-suspended in 1 ml of PBS/TBN and stored at 4°C in the dark. To determine if coupling was effective, aliquots of the different bead sets were prepared for analysis as described below and analysed on the BioPlex100 system.
Analysis of coupled beads on the BioPlex100 system
The coated beads were diluted 1:333 in Assay Buffer E (ABE buffer: 0.1% BSA, 0.05% Tween-20, 0.05% sodium azide in PBS pH 7.4) and 50 μl aliquots of were dispensed into the wells of a 1.2 μm filter bottom 96-well microtiter plate (MSBVS 1210, Millipore, USA) pre-wetted with ABE buffer. The beads in 96-well plates were washed three times with ABE using a vacuum manifold (Millipore, USA). Frozen plasma samples were thawed at room temperature, mixed by vortexing, and spun at 16,000 × g for five minutes to remove particulates. Plasma samples were diluted 1:80 in ABE buffer and 50 μl aliquots of diluted sample was added to the beads and incubated in the dark on a shaking platform at 1100 rpm for thirty seconds followed by 300 rpm for thirty minutes. Excess antibody was removed using a vacuum manifold followed by three washes in ABE. 25 μl of biotinylated human IgG detection antibody diluted 1:500 in ABE was added to the beads, incubated in the dark with shaking at 1100 rpm for thirty seconds followed by 300 rpm for thirty minutes and washed three times in ABE. 50 μl of phycoerythrin-conjugated streptavidin diluted 1:500 in ABE was added to the beads and incubated in the dark with shaking at 1100 rpm for thirty seconds followed by 300 rpm for ten minutes. Excess phycoerythrin conjugated streptavidin was removed followed by three washes in ABE. The beads were then re-suspended in 125 μl of ABE and analysed on the BioPlex100 system. The reader was set to read a minimum of 100 beads with identical unique detection signal and the results were expressed as median fluorescent intensity (MFI). In order to determine whether the human IgG detection antibody bound non-specifically to the coated beads, the beads were analysed against naïve plasma sample from Danes who have not been exposed to malaria.
Multiplexing and lyophilization of beads
Equal volumes of the coated beads were pooled together and mixed by vortexing. This bead mix was divided in half and one half was stored at 4°C in the dark. Sucrose and Tween 20 were added to the other half to 3% and 0.05% respectively, mixed by vortexing and single-use aliquots were lyophilized (adVantage, Wizad™ 2.0, Virtis) in polypropylene vials, sealed under nitrogen gas and stored at -80°C. Immediately prior to use, lyophilized beads were reconstituted with distilled water and used for analysis as described above.
Enzyme-linked immunosorbent assay (ELISA)
Antibody reactivity levels to the recombinant PfEMP1 proteins were measured in an ELISA system as described by Jensen et al
]. The wells of Maxisorp microtitre plates (Nunc, Roskilde, Denmark) were coated by overnight incubation at 4°C with 100 μl of the recombinant PfEMP1 protein (5 μg/ml) diluted in 0.1 M glycine-HCl (pH 2.75). The plates were emptied and any residual binding sites were blocked by addition of 200 μl of blocking buffer (1% BSA, 0.5 M NaCl, 1% Triton X-100, in phosphate buffered saline (PBS) pH 7.2) per well followed by thirty minutes incubation at room temperature on a shaker. 100 μl of plasma sample diluted 1:80 in blocking buffer was added in duplicate wells and incubated for one hour at room temperature on a shaker. Following four washes in wash buffer (0.5 M NaCl, 1% Triton X-100 in PBS pH 7.4), the plates were incubated for thirty minutes at room temperature with 100 μl per well of peroxidase-conjugated goat anti-human IgG (Dako, Glostrup, Denmark) diluted 1:3000 in blocking buffer. The plates were washed four times in wash buffer and 100 μl of o
-phenylenediamine substrate (Dako) activated with H2
was added to each well. The plates were incubated in the dark at room temperature before adding 100 μl of 2.5 M H2
. Optical densities were measured at 492 nm (OD492
). All samples were analysed in duplicate. On each assay microtiter plate, a reference positive control plasma pool was included in addition to negative control wells without plasma (background levels). Results were calculated as arbitrary ELISA units (EU) to account for plate-to-plate variation as described by Jensen et al
These same samples were analysed by the bead-based assay and the results compared to those from ELISA.