Bacterial strains and media
E. coli strain Top10 (TOP10 Chemically Competent E. coli Cells, Invitrogen) was used for the amplification of plasmids. Bacteria were grown in LB medium containing 100 μg/ml ampicillin.
Construction of plasmids and transformation
A double-stranded oligonucleotide encoding the secretion signal sequence of bee-venom melittin was ligated to NheI digested pcDNA3.1 (Invitrogen) resulting in plasmid pcDNA3.1_BVM. Mouse glycophorin-A mRNA was obtained from mouse bone marrow cells and cDNA generated by two-step reverse-transcription PCR using primers designed to amplify specifically the cDNA of the transmembrane encoding region of glycophorin-A. This sequence was then ligated to NotI digested pcDNA3.1_BVM resulting in plasmid pcDNA3.1_BVM_GP_His. The plasmid was constructed such that unique NheI and NotI sites were preserved in the multiple cloning site. Then plasmid pUC57 containing codon optimised synthetic genes encoding P. falciparum genes PF14_0325, PFD1130W or PFF0620C respectively that lack sequences for the secretion signal peptide and for the GPI-attachment signal peptide (Genscript), were ligated into NotI/NheI digested pcDNA3.1_BVM_GP_His. Thereafter a double-stranded oligonucleotide encoding a FLAG tag was ligated into the NotI site resulting in expression plasmids pcDNA3.1_BVM_PF14_0325_FLAG_GP_His, pcDNA3.1_BVM_PFD1130W_FLAG_GP_His and pcDNA3.1_BVM_PFF0620C_FLAG_GP_His.
Culture of eukaryotic cells
The human embryonic kidney cell line 293 HEK was obtained from the American Type Culture Collection (CRL-1573, ATCC). 293 HEK cells were cultured in DMEM supplemented with 10% foetal calf serum, glutamine and penicillin/streptomycin at 37°C in a humidified incubator.
Establishment of HEK 293 cell lines stably expressing PF14_0325, PFD1130W or PFF0620C
293 HEK cells were transfected with pcDNA3.1_BVM_PF14_0325_FLAG_GP_His, pcDNA3.1_BVM_PFD1130W_FLAG_GP_His and pcDNA3.1_BVM_PFF0620C_FLAG_GP_His respectively using JetPEI™(PolyPlus) transfection reagent. One day prior to transfection, a total of 5 × 105 293 HEK cells were seeded in 35-mm dishes. Transfection was performed following the manufacturer's protocol. 3 μg of expression plasmid and 6 μl transfection solution was used. Antibiotic selection was started 48 h after transfection. The selection medium containing 500 ug/ml of Geneticin (Gibco) was exchanged every 3-4 days.
Generation of anti-FLAG tag and anti-His tag mAb
The mAbs His-6/9 and FLAG-27 were raised in Naval Medical Research Institute (NMRI) mice injected intraperitoneally with 20 μg of the respective peptides CGGHHHHH and CGGDYKDDDDL conjugated to KLH (Imject® Maleimide Activated mcKLH, Pierce) and emulsified in aluminum hydroxide gel (Alhydrogel-2%, Brenntag Biosector) containing CPG-OGN according to Davis et al 1998 [
27]. The animals received up to four booster injections each at 3-week intervals with the same antigen preparation. As soon as the animals showed a specific immune response to the immunogen, the best responders were boosted and after 3 days, the spleens were removed and the isolated cells fused to PAI myeloma cells, a variant of the P3-x63-AG8 myeloma [
1,
28].
Immunofluorescence staining of methanol-fixed HEK cells
HEK cells were collected using enzyme free dissociation buffer (Cell dissociation buffer enzyme-free Hanks'-based, Gibco), washed with PBS and spotted on multiwall glass slides (multitest slide, 12-well, 7 mm, ICN Biomedicals Inc.). When air-dried, cells were fixed in methanol for 10 min. Immunostaining was performed by incubating the wells with 30 μl of an appropriate mAb diluted in PBS or hybridoma culture supernatant for 20 min in a humid chamber at 37°C. After rinsing twice and washing for 15 min in PBS, 30 μl of 125 μg/ml FITC-conjugated goat anti-mouse IgG antibodies (RAM/IgG(H+L)/FITC, Nordic Immunological Laboratories) diluted in PBS were added to the wells and incubated for 20 min in a humid chamber at 37°C. Finally, slides were rinsed twice and washed for 15 min in PBS, mounted with mounting solution (50% PBS 50% glycerol) and covered with a coverslip. Stainings were assessed by fluorescence microscopy on a Leica CTR500 fluorescence microscope and a Leica DFC300 FX digital camera.
Immunofluorescence staining of living HEK cells
For immunofluorescence staining of live HEK cells chamber slides (4-well chamber-slide, Lab-Tek™, Nunc™) were used. Wells were coated with 100 mg/l poly-D-lysine in H2O in a humid box at room temperature over night. After washing the wells three times with sterile H2O, 40'000 cells were seeded per well. Three days later the immunostaining was performed by incubating the wells with 500 μl of an appropriate mAb diluted in serum-free culture medium for 30 min on ice. After washing two times with serum-free culture medium 500 μl of 100 μg/ml FITC-conjugated goat anti-mouse IgG antibodies (RAM/IgG(H+L)/FITC, Nordic Immunological Laboratories) diluted in serum-free culture medium were added to the wells and incubated for 30 min on ice. Finally, the wells were rinsed twice with serum-free culture medium and once with D'PBS (Dulbecco's Phosphate-Buffered Saline containing calcium, Gibco). The slides were mounted with mounting solution containing DAPI (ProLong® Gold antifade reagent with DAPI, Invitrogen) and covered with a coverslip. Stainings were assessed as described above.
Western Blotting analysis
HEK cells were collected using enzyme free dissociation buffer (Cell dissociation buffer enzyme-free Hanks'-based, Gibco), and washed two times with PBS. To prepare cell lysate, 106 cells were lysed with 0.1 ml of lysis buffer (1% NP40, 10% glycerol, 2 mM EDTA, 137 mM NaCl, 20 mM TrisHCl, pH8, Protease Inhibitors) for 10 min on ice. The lysate was cleared by centrifugation at 20'000 g for 5 min.
Blood stage parasite lysates were prepared essentially as described previously by saponin lysis of
P. falciparum 3D7-infected erythrocytes [
29]. In brief, cultured parasites were washed once with PBS. Pelleted infected red blood cells were lysed by mixing with 20 volumes of 0.015% (w/v) saponin in PBS and incubated on ice for 20 min. Finally, the pelleted parasites were resuspended in 3 volumes of PBS and stored at -80°C until further use.
For SDS-PAGE cell- or parasite lysate was resolved on precast 4-12% gradient gels (NuPAGE® Novex 4-12% Bis-Tris Gel, Invitrogen) with MES running buffer according to the manufacturer's directions. The proteins were electrophoretically transferred to nitrocellulose membrane using a dry-blotting system (iBlot, Invitrogen). After blocking the membrane over night in blocking buffer (5% Milk in PBS) at 4°C, specific proteins were detected with appropriate dilutions of mAbs in blocking buffer for 1 h at room temperature. The membrane was then washed four times for 5 minutes in blocking buffer and incubated with horseradish peroxidase conjugated anti-mouse IgG mAb (GAM/IgG (γ-chain)/HRP) diluted 1:10'000 in blocking buffer at room temperature for 1 h. After washing again, blots were developed using ECL Western blotting detection reagents (ECL Western Blotting Substrate, Pierce) to visualise bands.
FACS and Flow cytometric analysis of HEK cells
For sorting stably transfected cells into high-expressing cell pools, cells were dissociated with enzyme-free dissociation buffer (Cell dissociation buffer enzyme-free Hanks'-based, Gibco), washed with blocking buffer (PBS containing 3% BSA). The cells were then incubated with 200 μl of 100 μg/ml FLAG-27 mAb diluted in blocking buffer for 15 min on ice. The cells were then washed with blocking buffer and incubated with 200 μl of 100 μg/ml FITC-conjugated goat anti-mouse IgG antibodies (RAM/IgG(H+L)/FITC, Nordic Immunological Laboratories) diluted in blocking buffer for 15 min on ice. After a final wash the labelled cells were analysed and sorted using a Becton Dickinson FACSAria running Diva software. All analyses were performed using appropriate scatter gates to exclude cellular debris and aggregates. Gating settings were set to collect highly labelled cells. Post-sorting, the cells were collected in culture medium with 20% FCS and plated in 35 mm wells
For monitoring surface expression of PF14_0325, PFD1130W or PFF0620C on transfected cell lines, cells were stained as described above. FACS analysis was performed on a FACScan (Becton Dickinson) using CellQuest software (Becton Dickinson). 20'000 thousand events were collected for each sample. Untransfected cells served as negative control. For analysing seroconversion of immunised mice, transfected cell lines were stained using a 1:600 serum dilution in blocking buffer. Cells were staining and analysed as described above.
Immunisation of mice
All procedures involving living animals were performed in accordance with the Rules and Regulations for the Protection of Animal Rights (Tierschutzverordnung) of the Swiss Bundesamt für Veterinärwesen. Naval Medical Research Institute (NMRI) mice were immunised by intravenous injections of 106 stably transfected HEK cells. Cells were thawed, washed and resuspended in 0.9% NaCl. Injections were accomplished on three consecutive days and after two weeks again on three consecutive days. Two to three weeks after the boost, blood was collected and the serum was tested for the presence of anti-PF14_0325, anti-PFD1130W or anti-PFF0620C antibodies, respectively by IFA and flow cytometry using stably transfected 293 HEK cells. Mice immunised with PF14_0325-HEK cells were boosted a second time after 4 weeks.
Fusion and cell-based selection
Animals with serum strongly reactive with expressing cells were selected for fusion. These received a final injection of 106 cells two and one day before the fusion. Mice were sacrificed and the spleen was removed. Spleen cells were harvested by trituration under sterile conditions and fused with the myeloma cell partner (PAI mouse myeloma cells, derived from P3-x63-AG8) using polyethylene glycol 1500 (Roche Diagnostics). The fusion mix was plated into multiwell plates and hybridomas were selected by growing in HAT medium supplemented with culture supernatant of mouse macrophages P388. Wells were screened for specific IgG production between 2-3 weeks post-fusion by ELISA and IFA as described below. Cells from wells positive in initial screens were cloned by limiting dilution to obtain monoclonal populations.
IgG ELISA screen
Maxisorp™plates (Nunc) were coated overnight at 4°C in a humid box with 100 μl of 5 μg/ml goat anti-mouse IgG (γ-chain specific) mAb (Sigma) diluted in PBS. After two washings with PBS containing 0.05% Tween-20, wells were blocked with blocking buffer (50 mM Tris, 140 mM NaCl, 5 mM EDTA, 0.05% NONidet P40, 0.25% gelatine, 1% BSA) for 1 h at 37°C and afterwards washed two times. 50 μl hybridoma supernatants were added to the wells and incubated for 1 h at 37°C. After washing 4 times, plates were incubated with 50 μl horseradish peroxidase-conjugated goat anti-mouse IgG (γ-chain specific) (Sigma) diluted 1:1000 in blocking buffer for 1 h at room-temperature in a humid box in the dark. After washing 4 times, TMB peroxidase substrate solution was added and the colour change monitored.
Antibody production and characterisation
Identification of antibody subclasses was performed using a Mouse Monoclonal Antibody Isotyping Kit (ISO2, Sigma). For large-scale mAb production hybridoma cell lines were cultured in 500 ml roller-bottles (Corning). MAbs were purified by affinity chromatography using protein A or protein G Sepharose.
P. falciparum blood stage culture
P. falciparum strain 3d7 was cultured essentially as described previously [
29]. The culture medium was supplemented with 0.5% AlbuMAX (Gibco) as a substitute for human serum [
30]. Cultures were synchronised by sorbitol treatment [
31]. Erythrocytes for passages were obtained from the Swiss Red Cross (Switzerland).
Immunofluorescence staining of P. falciparum
Erythrocytes from in vitro cultures of
P. falciparum strain 3d7 were fixed in paraformaldehyde-glutaraldehyde as described previously [
3]. Cells were blocked by incubation in 3% BSA in PBS for 1 h. Immunostaining was performed by incubation with an appropriate mAb dilution in blocking solution for 1 h. After three washes with PBS, 5 μg/ml cyanine dye (Cy3)-conjugated affinity-pure F(ab')2 fragment goat anti-mouse IgG (Fc-specific) antibodies (Jackson Immuno Research Laboratories) diluted in blocking solution was added and incubated for 1 h at RT. Finally cells were washed three times with PBS, mounted with mounting medium containing DAPI (ProLong® Gold antifade reagent with DAPI, Invitrogen) and covered with a coverslip. Antibody stainings were assessed as described above.
For immunfluorescence staining of sporozoites, air-dried unfixed
P. falciparum (strain NF54) salivary gland sporozoites attached to microscope glass slides were incubated with mAbs and detected with cyanine dye (Cy3)-conjugated affinity-pure F(ab')2 fragment goat anti-mouse IgG (Fc-specific) antibodies (Jackson Immuno Research Laboratories) as previously described [
32].
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