For validation and standardization of the multiplex assay of antibodies to P. falciparum antigens, a plasma pool made of 30 plasma samples from adults living in a Ugandan area of seasonal malaria transmission (positive control pool samples)
 and seven plasma samples from North American individuals never exposed to malaria (negative control samples) was used. For comparison of antibody testing by CBA and ELISA to multiple P. falciparum antigens, samples from 30 individuals from a malaria endemic area of western Kenya were used
. Kenyan samples were obtained from both children and adults, to enable testing of a broad range of antibody values. Written informed consent was obtained from the study participants or, in the case of minors, from their parent or guardian. Ethical approval for the study was obtained from Kenya Medical Research Institute National Ethical Review Committee and the Institutional Review Boards of Makerere University and University of Minnesota.
Plasmodium falciparum recombinant and peptide antigens
The ten recombinant P. falciparum antigens used for testing were apical membrane antigen-1 (AMA-1, full length ectodomain, 3D7 and FVO strains), erythrocyte-binding antigen (EBA-175, non-glycosylated region II), glutamate rich protein (GLURP, conserved non-repeat N-terminal region, amino acids 25–514, R0; and repeat C-terminal region, amino acids 705–1178, R2, 3D7 strain), merozoite surface protein-1 (MSP-119, E-KNG variant; MSP-142, 3D7, FUP and FVO strains), and merozoite surface protein-3 (MSP-3, C-terminus, FVO strain) were used for testing. Recombinant AMA-1 was expressed in Escherichia coli and provided by David Lanar, Walter Reed Army Institute for Research. Recombinant MSP-142 and MSP-3 were expressed in Escherichia coli, and recombinant EBA-175 was expressed in Pichia pastoris, and provided by David Narum, National Institutes of Health. Recombinant GLURP was expressed in Escherichia coli and provided by Michael Theisen, Statens Seruminstitut, Copenhagen, Denmark. Recombinant MSP-119 expressed in Saccharomyces cerivisiae, was provided by the Malaria Research and Reference Reagent Resource Center (Manassas, VA), and originally deposited there by David Kaslow. Blank samples consisted of the plasma diluent alone.
Coupling of recombinant antigens to microspheres for the cytometric bead assay (CBA)
Microspheres were purchased from Luminex Corporation (Austin, TX). The bead stock was resuspended by gentle inversion for 1 min. An aliquot of 612, 500 beads was removed and centrifuged at 16, 000 g (Labnet Wood bridge, NJ) for 3 min. After discarding the supernatant, 100 μl of distilled water was added and centrifuged at 16, 000 g for 3 min. Beads were resuspended in 80 μl of activation buffer, 100 mM monobasic sodium phosphate (Sigma, S3139); pH 6.2, by vortexing (Scientific Industries Bohemia, NY) and sonication, 20 sec each. To activate the beads for cross-linking to proteins, 10 μl of 50 mg/mlN- hydroxysulfosuccinimide sodium salt (Sigma, 56485) was added to the beads and mixed by vortexing for 10 sec at moderate speed. Next, 10 μl of 50 mg/ml N-[3-dimethylaminopropyl] – N`-ethylcarbodiimidehydrochloride (Sigma, E1769) was added and the beads mixed again by vortexing for 10 sec at moderate speed. All incubations of beads were performed in the dark (covered with foil). The bead mixture was rotated on a rotary shaker (Labnet Edison, NJ) at room temperature for 20 min and vortexed for 10 sec at 10 min and at 20 min, both at moderate vortexing speed. Beads were pelleted by centrifuging at 16, 000 g for 5 min and washed twice with 250 μl of 100 mM morpholineethane sulfonic acid (MES), (Sigma, M2933), pH 6.0 buffer. Finally, beads were centrifuged at 16, 000 g for 5 min to pellet. To coat the beads with antigens, pelleted beads were resuspended with the relevant antigen and the volume adjusted to 500 μl per reaction by addition of coupling buffer (100 mM MES pH 6.0). Beads were conjugated to 0.5, 1, 2 and 5, 10 and 1,000 μg of different antigens. The antigen and activated beads mixture was incubated on a rotary shaker for 2 h at room temperature in the dark to allow bead coupling to occur. After being coated with proteins beads were centrifuged at 16, 000 g for 3 min and washed twice with 250 μl of PBS-TBN (PBS, 0.1% BSA, 0.02% Tween, 0.05% sodium azide) and resuspended in 200 μl of PBS-TBN. To determine the percentage recovery after the coupling procedure, coupled beads were counted on a haemocytometer (Hausser Scientific Horsham, PA).
Testing for IgG antibodies to P. falciparum antigens by CBA
The volume of working solution (50 μl/ well) was calculated together with the number of beads that would result in 1,000 beads/region/well or 5,000 beads/region/well. Bead stocks were then combined in a 15 ml amber conical tube and diluted with PBNT (0.1% BSA, 0.05% Tween 20, 0.05% sodium azide in PBS) to result in 100 microspheres/μl or 20 microspheres/μl. 96 well-millipore microtiter plates (MABVN 1250, Millipore corporation, Billerica, MA) were pre-wetted with 100 μl of PBNT/well and aspirated using a millipore vacuum manifold and 50 μl of working bead solution was transferred to it. Plasma samples were thawed at room temperature, mixed and centrifuged at 16,000 g for 3 min. Plasma was diluted through a series of concentrations: 1:100, 1:200, 1:400, 1:1000, 1:2000 and 1:4000. Plasma samples were diluted in either buffer A (1xPBS, 0.1% BSA, 0.05% Tween 20, and 0.05% sodium azide) or buffer B (1xPBS, 1% BSA, 0.05% Tween 20, 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone). Buffer A is the standard buffer used by other studies of multiplex CBA for P. falciparum antigens
, while buffer B has been used for multiplex antibody testing to other antigens and found to decrease background reactivity
Fifty μl of diluted plasma was added to each of the well of the microtiter plate. The plasma was mixed with the beads three times by pipetting up and down. The plates were incubated in the dark on a shaking microplate shaker (IKA®MTS, Wilmington, NC) at 600 rpm for 30 sec, followed by 300 rpm for 30 min. Plates were aspirated using a millipore vacuum manifold and washed twice with 100 μl/well of PBNT, and beads were resuspended in 50 μl PBNT by pipetting. 50 μl of diluted 1:1,000 goat antihuman IgG (gamma- chain specific, F(ab`)2 fragment-R-phycoerythrin (Sigma, P-8047 St. Louis, MO) in PBNT was added to each well, and incubated in the dark with shaking at 600 rpm for 30 sec, followed by 300 rpm for 30 min. Plates were aspirated using a millipore vacuum manifold and washed twice with 100 μl/well PBNT. The beads were resuspended in 100 μl PBNT by mixing and analysed on bioplex machine (Hercules, CA). The reader was set to read a minimum of 100 beads with of unique fluorescent signature/region and the results expressed as median fluorescence intensity (MFI).
Testing for IgG antibodies to P. falciparum antigens by ELISA
To validate the multiplex assay, similar plasma samples were tested for IgG antibodies to the same P. falciparum antigens by enzyme-linked immunosorbent assay (ELISA). Recombinant antigens were dissolved in 0.01 M PBS to concentrations: 0.1 μg/ml for (AMA-1 3D7, AMA-1 FVO, EBA-175 and GLURP-R2), 0.2 μg/ml for (MSP-142 FVO, MSP-142 3D7, and MSP-142 FUP), 0.5 μg/ml for (GLURP-R0, MSP-119 and MSP-3 FVO). These antigen concentrations were found to be optimal in previous studies for AMA-1, EBA-175, MSP-142, GLURP-R0
[16, 18], MSP-119 and MSP-3
. Fifty microlitres of antigen solution was added to Immulon-4 plates (Dynex Technologies,Chantilly, VA). Following overnight incubation at 4°C, washing with PBS-0.05% Tween 20, and blocking in 5% (wt/vol) nonfat powdered milk in PBS, duplicate 50 μl samples of serum diluted through a series of concentrations ranging from 1:50, 1:100, 1:200, 1:500, 1:1000, and 1:2000 in 5% powdered milk were added to wells and incubated for 2 h at room temperature. After washing with PBS −0.05% Tween 20, 50 μl of alkaline phosphatase-conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:1,000 in 5% powdered milk was added and incubated for 1 h. After extensive washing with PBS-0.05% Tween 20, p-nitrophenylphosphate was added in accordance with the manufacturer’s instructions (Sigma, S0942 St. Louis, MO). The optical density (OD) was measured at 405 nm (Molecular Devices, Sunnyvale, CA).
The degree of association between MFI or OD values was assessed using Pearson’s correlation (r). Comparison of MFI values with Buffer A vs. Buffer B was done using Student’s t-test. All statistical tests were 2-sided and a P value of less than or equal to 0.05 was considered to be statistically significant for all comparisons. Analyses were conducted with Stata software version 10.0 (Stata Corporation, College Station, TX).