Materials collected from Plasmodium falciparum HB3 in vitro culture
All procedures were followed in accordance with CDC malaria parasite culture protocols and performed in the biosafety cabinets using aseptic technique. The HB3 strain was selected as it is an established culture strain that has the gene for pfhrp3 deleted and therefore does not express HRP3 antigen , allowing for the purification of HRP2 without risking contamination with the paralogue HRP3. P. falciparum HB3 parasites used in this study had previously been cryopreserved in a 57% glycerolyte solution. Vials were thawed for 1-2 minutes in a water bath maintained at 37°C with gentle shaking while ensuring that the cap remained above the water level. To prevent lysis of the red blood cells, the 1-2mL volume of frozen parasite stock was transferred to a 50 mL conical tube for processing with a series of sterile salt solutions of decreasing concentration starting at 12% NaCl. A volume of 0.2 mL of 12% NaCl per mL of thawed culture stock was added drop by drop to the conical tube while placed on a low-rpm vortex. Following the addition of the first salt solution, the culture sample was incubated for 3 minutes without shaking, then 10 mL of 1.6% NaCl was added per mL of culture as before. The parasite-salt solution was then centrifuged at 500 ×g for 10 minutes and the supernatant was removed by aspiration to leave 0.5 mL. To each culture stock, 10 mL of a 0.9% NaCl + 0.2% dextrose solution was added per mL of initial culture stock as before and the sample was centrifuged. The supernatant was then removed to leave 0.5 mL volume containing a packed pellet of parasites for inoculation.
The parasite pellet was then transferred to a T25 culture flask containing 4.5 mL of complete medium consisting of RPMI 1640 (Gibco, ThermoFisher Scientific, Waltham, MA) with 10% human O+ serum, prewarmed to 37°C, by first resuspending the pellet in the 50 mL conical tube using 2 mL of warmed media from the flask. Washed human O+ erythrocytes were added to the flask to obtain a total RBC volume ≤ 0.5 mL and mixed by recapping and gently shaking. The culture flask was then gassed with a mixture of 5% CO2 + 5% O2 and 90% N2 for 30-45 seconds and then tightly capped and placed on an orbital rotator in a 37°C incubator without CO2. Parasite cultures were maintained daily by changing out the medium with complete RPMI prewarmed to 37°C. The old culture media was collected each day and labeled with the parasitemia and stored at -20°C for further use. Following the addition of new media, the flask was gassed with the CO2+O2+N2 mixture as before and placed back on an orbital rotator inside the 37°C incubator. Parasitemia values were obtained daily from thin blood smear readings using Giemsa stain. To prepare a thin blood smear from the culture flask, a 200 µl aliquot of the culture was transferred to a 1.5 mL Eppendorf tube and centrifuged. Most of the supernatant was then removed, leaving only a volume of supernatant equal to that of the pellet for the thin smear. The culture flask was subcultured every third day, or when the parasitemia was greater than 3%, to an increasing flask size, starting at a T25 flask and going to a T75 flask then a T150 flask. The larger flask size allowed for a larger total volume of the resulting sample. Upon each subculture, new cultures were started with at least 1% parasitemia at 10% hematocrit. The flasks containing the subcultured sample were then placed onto an orbital rotator inside the incubator overnight on a daily basis to increase the overall number of single-infected iRBCs. This process allowed the parasitemia to increase exponentially within a few days, especially when there were many schizonts present. The supernatant was harvested daily by transferring the content of each culture flask into a sterile centrifuge tube, centrifuged at 500 ×g for 10 minutes to separate the supernatant from the pellets. The supernatant was then pipetted into another sterile centrifuge tube, labeled with the strain name, date, and parasitemia, and stored at -20°C for later use. Cultures were harvested when parasitemia was between 9-12%, usually around the fourth day. If the parasitemia of the cultures was less than 9% after the fourth day since subculture, the culture was subcultured to a new flask or flasks, at a starting parasitemia of 1% and at 10% hematocrit.
Purification of native HRP2 antigen from culture supernatant, iRBCs, and whole parasite lysate
All centrifugations were carried out using a Sorvall RC6 Plus Superspeed centrifuge (Thermo Fisher Scientific, Waltham, MA), using a Sorvall GS3 rotor for centrifugation at 1,200 and 1,500 rpm and a Sorvall SS-34 rotor for centrifugation at 17,000 rpm. Supernatant pools were centrifuged at 1,500 rpm for 10 minutes before being filtered through a 0.45 µM filter (Corning, Corning, NY), and an equal volume of 4°C 1X Phosphate buffer with 50 mM NaH2PO4, and 300 mM NaCl at pH 8.0 was added to the pooled supernatant. Imidazole (Sigma, St. Louis, MO) was added to each supernatant pool to obtain a final concentration of 10 mM imidazole and allowed to incubate at 4°C with continuous stirring at 1,500 rpm for 2 hours. The mixture was centrifuged at 1,200 rpm for 5 minutes and nHRP2 was purified using affinity chromatography through Ni-NTA slurry using the batch purification method described by the manufacturer (Qiagen, Hilden, Germany). Briefly, 1 mL of Ni-NTA agarose was used for every 250 mL of supernatant and mixed via gentle shaking at room temperature for 1 hour. Because the Ni-NTA column methodology relies on the immobilization of histidine-rich targets by nickel (Ni), the use of the HB3 culture strain which has HRP2 but lacks the histidine-rich HRP3 protein is essential to ensure the purification of only HRP2 and not HRP3.
The supernatant-Ni-NTA mixture was loaded into the chromatography column and the flow-through was collected. The Ni-NTA slurry in the column was washed with 20 mM Imidazole, and protein was subsequently eluted using washes with 500 mM Imidazole and collected. Each fraction, including flow-through, washes, and elutes was tested for purity and molecular weight estimation of the antigen present using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting followed by assessment via bead-based HRP2 detection assay as described below. In order to estimate the efficiency of nHRP2 binding to the Ni-NTA column, loss of antigen during the washing steps, and absolute elution of antigen from the column, each fraction during the column purification was assessed for levels of nHRP2. Two independent column purification experiments (Exp1 and Exp2) were performed under slightly different wash and elution conditions to purify nHRP2 obtained from the HB3 culture supernatant (Table 1), iRBCs (Table 2), and parasite pellet (Table 3).
For storage, elutes containing purified nHRP2 were pooled, dialyzed, and concentrated using an Ultra-15 centrifugal filter (Millipore, Burlington, MA). The final concentration of antigen in solution was determined by comparison with a standard curve of 0.5 – 20 µg/mL using a Micro BCA Protein Assay Kit (Thermo Scientific, catalog# 23225) . The final purified protein was stored at -80 0C until further use.
For purification of nHRP2 from iRBC, the iRBC fractions were thawed on ice and pooled. PBS with 0.1% saponin (Sigma) was added at five times the volume of the RBCs volume and incubated for one hour on ice with gentle shaking. This suspension was centrifuged for 30 minutes at 17,000 rpm at 4°C. Infected RBC lysate was used to purify nHRP2 using the same protocol as described for the supernatant.
For purification of nHRP2 from whole parasite lysate, extraction buffer containing 50 mM Tris-HCl (pH 7.5) + 5 µl Triton-X-100 + 100 mL of 150 mM NaCl and 10 mM Sodium Phosphate+ 5 µl Traysol (Sigma) at pH 7.4 and protease inhibitor (Roche, Penzberg, Germany) was prepared as previously described . The iRBCs were then pelleted by centrifuging at 17,000 rpm for 30 minutes at 4°C. The pellet was then washed three times with cold PBS to remove hemoglobulin. Extraction buffer was added to the parasite pellet at 5 times the volume of the pellet and incubated at 4°C on ice for 30 minutes. The suspension was kept on ice and sonicated a total of five times for 30 seconds with five-minute intervals between each round of sonication until the mixture appeared homogeneous. After sonication, the lysate was centrifuged at 17,000 rpm at 40C for 45 minutes. nHRP2 was purified from the supernatant of the parasite lysate following the procedure described above.
SDS-PAGE and Western Blotting
The washes, flow-throughs, and elutes obtained from the Ni-NTA column during nHRP2 purification were diluted at a 1:1 ratio with reducing SDS sample buffer containing Tris/Glycine/SDS prepared from a 10X stock solution (BioRad, Hercules, CA) with 0.05% 2-mercaptoethanol to prepare the sample under reducing conditions. The samples were then loaded onto an SDS gradient gel (5-20%, BioRad) with Precision plus protein dual-color standard (BioRad) used as the molecular weight marker for all western blots. Gels were electrophoresed at room temperature for 1.5 to 2 hours at 100V in a Mini Protean II Electrophoresis Cell (BioRad).
After electrophoresis, the gels were soaked for 10 minutes in transfer buffer containing 25 mM Tris, 192 mM glycine at pH 8.3 (BioRad), and proteins were transferred to Trans-Blot nitrocellulose paper (GE Healthcare Life Sciences, Chicago, IL) at 100 milliamps constant current at 4°C for 1 hour. Following transfer, the membrane was rinsed with 1X PBS (pH 7.2) and stained with Ponceau S stain. The membrane was then washed three times with PBS 1X before blocking with 5% (wt/vol) non-fat dry milk.
For the western blot, a mouse anti- P. falciparum HRP2 monoclonal antibody ((MPFG-55A, ICL Labs, Portland, OR) was used as the primary antibody at dilution of 1:500 in PBS 1X with 0.5% BSA and 0.05% Tween20. The blots were then washed three times with 50 mL of PBST (PBS with 0.05% Tween20) and incubated with the secondary antibody, goat anti-mouse IgG H+L alkaline phosphatase (ThermoFisher Scientific) at a dilution of 1:2,000 in PBS 1X with 0.5% BSA and 0.05% Tween20 for 45 minutes. Following incubation with the secondary antibody, the blots were washed three times with 50 mL of PBST as before and subsequently washed with PBS 1X for five minutes. Bands were visualized using Western Blue stabilized substrate for alkaline phosphate according to the manufacturer’s protocol (Promega, Madison, WI).
Enzyme-linked immunosorbent assay (ELISA)
A 96-well ELISA plate (Immulon 2B, ThermoFisher Scientific) was coated with nHRP2 protein at 1 µg/mL in carbonate buffer containing 0.04 M sodium bicarbonate and 0.006 M sodium carbonate (pH 9.6) and incubated overnight at 4°C. The following day, the plate was blocked with 3% non-fat milk in PBS for 1 hour at 37 0C. Serial dilutions of the mouse anti-P. falciparum HRP2 monoclonal antibody (MPFG-55A) were added to the plate in triplicate at dilutions of 1:100 to 1:204,800 in PBS with 0.5% bovine serum albumin and 0.05% Tween20 and allowed to incubate for 1 hour at 37°C. Following incubation with the primary antibody, the plate was washed three times with PBST and incubated with goat anti-mouse antibody conjugated to horseradish peroxidase (HRPO) from (Abcam, Cambridge, United Kingdom) at 1:500 (equivalent to 2 µg/mL) for 45 minutes at 37°C. Following the final three washes with PBST, bound antibodies were detected using TMB substrate (KPL/SeraCare, Milford, MA) and absorbance values were read at 450 nm with a Molecular Devices SpectraMAX spectrophotometer (Sunnyvale, CA).
Bead-based HRP2 detection assay
The bead-based multiplex assay for malaria antigen detection was performed as described previously . Magnetic microbeads (xMAP, Luminex Corp., Austin, TX) were covalently bound to HRP2 capture antibodies (MPFG-55A, ICL Labs, Portland, OR) by the Luminex antibody coupling kit according to manufacturer’s instructions at a concentration of 20 µg/mL. Detection antibodies were also prepared in advance by biotinylation using the EZ-link Micro Sulfo-NHS-Biotinylation Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. The final prepared dilution of detection antibodies was 1.0 mg/mL for anti-HRP2 (1:1 antibody mixture of MPFG-55A and MPFM-55A, ICL Labs). Conjugated beads and prepared detection antibodies were stored at 4o until use in the immunoassay.
All assay reagents were diluted in buffer containing PBS pH 7.2, 0.05% Tween20, 0.5% BSA, 0.02% sodium azide. For all wash steps, the assay plate was affixed to a handheld magnet (LuminexCorp, Austin, TX), and gently tapped for two minutes to allow bead magnetization before the evacuation of liquid and washing with 100 µL PBS, 0.05% Tween20. The capture beads were combined in dilution buffer and pipetted onto a BioPlex Pro 96-well assay plate (BioRad) at a quantity of approximately 800 beads/region. Plates were washed twice, and 50 µL of controls or samples were pipetted into appropriate wells. Following a 90-minute gentle shaking at room temperature protected from light, plates were washed three times. Detection antibodies were prepared in dilution buffer at a concentration of 1:500 and 50 µL added to each well for a 45-minute incubation. After three washes, 50µL of streptavidin-phycoerythrin at 1:200 (Invitrogen – ThermoFisher Scientific) was added for a 30-minute incubation. Plates were washed three times, and 50 µL dilution buffer was added to each well for a 30-minute incubation. Plates were washed once and beads resuspended in 100 µL PBS. After brief shaking, plates were read on a MAGPIX machine (LuminexCorp) with a target of 50 beads per region. The median fluorescence intensity (MFI) value is generated for all beads collected for each region by assay well and subtracting the assay signal from wells with dilution buffer blank provides an MFI-background (MFI-bg) value used for analyses. To extrapolate from assay signal to antigen concentration, Type B recombinant HRP2 (rHRP2) was kindly provided by MicroCoat (Starnberger See, Germany) and standard curves were prepared to create a regression equation.
Assessment of reactivity of RDTs to purified nHRP2 from HB3 culture strain
The malaria RDTs selected for this study were Carestart™ Malaria Pf (HRP2) Ag RDT (Access Bio, Inc., RMOM-02571, Lot MO19F68) and Carestart™ Malaria Pf (HRP2/pLDH) Ag RDT (Access Bio, Inc., RMPM-02571, Lot MP19F61). Six 10-fold serial dilutions (in parasite-negative blood) starting at a dilution of 2060 ng/mL were prepared for the native HRP2 and recombinant HRP2 (ICL Labs). Each dilution was then tested on RDTs following manufacturer instructions by pipetting 5 µl sample and two drops of manufacturer-provided buffer to each RDT cassette. Band intensity read with a score of 0-4 was estimated for all tests. A score of 0 indicates no band was present and while scores 1 to 4 indicate positive tests with increasing band intensity. In addition to assessing RDT reactivity to the purified nHRP2, dilutions of P. falciparum culture strains 3D7 and US06F Nigeria XII were prepared from cultures with known parasite densities. Eight serial dilutions were prepared for 3D7 and Nigeria XII culture samples ranging between 1,000-6.25 parasites/µl. Sample concentrations used for RDT readings are displayed in ng/mL for nHRP2 and recombinant HRP2 while parasites per µl (p/µl) concentrations are displayed for 3D7 and Nigeria XII samples.