Download Calculation of antibody concentration in leaf tissue

Supplementary Data S1
Transient expression by agroinfiltration
The agroinfiltration of N. tabacum was performed using a vacuum pump. A 10 mL starting
culture of the transformed agrobacteria was grown overnight to inoculate a 500 mL culture in
YEN-B medium. When OD600 reached 0.6 the culture was spun at 5000xg for 30 minutes
and agrobacteria were resuspended in an equal volume of infiltration buffer (10 mM MES, 10
mM MgCl2, 1.5 mM acetosyringone). The expression of a fully assembled TBG65 mAb, the
‘mini-RICs’ or the TB-RICs was achieved by mixing equal volumes of agrobacteria
suspensions harbouring the coding sequences for heavy and light chain antibody constructs.
The plants were submersed in the bacterial suspension inside a chamber connected to a
vacuum pump and depression was applied for 2-3 minutes. Air was then released very
slowly to allow penetration of the bacterial suspension into the leaf tissue. Following
infiltration, plants were incubated under normal growing conditions for 5 days before harvest.
The collected leaf tissue was frozen in liquid nitrogen and kept at -80oC until employed in the
purification procedure.
Calculation of antibody concentration in leaf tissue
The level of expression of TB-RICs in plants was calculated in a leaf sample by
homogenising 0.5 g leaf tissue in 1.0 ml PBS, followed by centrifugation. The concentration
of total soluble protein (TSP) in the supernatant was determined by the BCA protein assay
(Perbio, Cramlington, UK) using bovine serum albumin as the standard. The specific
antibody was then determined by a sandwich ELISA using an anti- (capture) and
peroxidase-conjugated anti- (detection) anti-IgG antibody (Caltag, Burlingame, CA, USA),
and hybridoma-derived TBG65 mAb as the standard. Specific protein concentration was
expressed as percentage of total soluble protein (TSP).
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Protein electrophoresis and Western blotting
Protein samples were mixed with 1/4 of their volume of 4x SDS-loading buffer (plus 100 mM
DTT for reducing conditions), boiled for 3 minutes and loaded onto a 4-12% gradient
polyacrylamide gel (Tris-Glycine; BioRad). Following completion of electrophoresis, the gels
were subjected to either Coomassie or silver staining, or Western blot analysis. For the
latter, the gel was electro-blotted onto nitrocellulose membrane using a semi-dry transfer
system (Hoefer™ TE70, Amersham Biosciences) according to manufacturer’s instructions.
Blots were blocked overnight with 5% (w/v) non-fat dried milk in PBS at 4oC and incubated at
room temperature for 2 h with appropriate primary and secondary antibodies diluted in PBSdried milk 5% (w/v). For detection of assembled TBG65 in plant extracts, only the secondary
antibody conjugated to peroxidase was used, probing either for the heavy chain (goat antimouse IgG-Fc, subclass 1 specific, HRP-conjugated antiserum, 1:1000 dilution; Jackson
Immuno Research) or for the light chain (goat anti-mouse light chain affinity purified HRPconjugated antiserum diluted 1:1000; Jackson Immuno Research). When detecting Acr
within TB-RICs, hybridoma TBG65 mAb was used at 1 g/ml, followed by a goat anti-mouse
IgG light chain horseradish peroxidase-conjugated antibody (1:1000; Jackson Immuno
Research). Since the samples were analysed under the reducing conditions, the goat
antibody recognised the light chain of the TBG65 hybridoma mAb bound to Acr within plant
TBG65-Ag85B-Acr, confirming therefore its expression. For detection of Ag85B, a rabbit
polyclonal anti-Ag85B antibody (1:1000) was used (a gift from Kris Huygen, Institute
Pasteur, Brussels), followed by a donkey anti-rabbit antibody conjugated to horseradish
peroxidase (1:1000; Jackson Immuno Research). After the secondary antibody incubation,
the blots were washed with PBS (4 times for 5 minutes) and developed using enzymatic
chemiluminescence
(ECL-Enhanced
Chemiluminescence,
Amersham
Biosciences)
according to manufacturer’s instructions.
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Antigen-specific and C1q ELISA
In order to evaluate the specific humoral responses induced in immunised mice, 96-well
plates (Nunc Maxisorp Immuno™ plates) were coated with 10 g/mL antigen solution (either
Acr or Ag85B) in PBS and incubated for 2 h at 37oC. The plates were then washed with
distilled water and blocked overnight with a solution of 2.5% (w/v) BSA in PBS at 4 oC.
Following a second washing step the samples were incubated for 2 h at 37oC. 3-fold serial
dilutions of sera in PBS-2.5% (w/v) BSA were added and the plates incubated at room
temperature for 2 h. For C1q complement ELISA, the 96-well plates were coated with 10
g/mL of human C1q (Calbiochem), a component of the complement system, and after
blocking of free sites with PBS-2.5% (w/v) BSA, 3-fold dilutions of TB-RICs (10 g/ml
starting concentration) were added and incubated at 37oC for 2 h. For both types of ELISA,
the plates were then washed 4 times with distilled water containing 0.1% (v/v) Tween-20,
rinsed twice with distilled water and incubated for 1 h with the detection antibody (goat antimouse IgG horseradish peroxidase-conjugated antibody (1:1000; Binding Site) in PBS-2.5%
(w/v) BSA. Following further washing as before, the peroxidase reaction was developed by
adding 100 L of TMB substrate to each well and then stopped by addition of 2 M sulphuric
acid. The absorbance was read on an ELISA plate reader (Sunrise Tecan) at 450 nm.
T cell proliferation and IFN- release assay
Spleens were aseptically extracted from mice and kept on ice. Tissues from mice within the
same group were pooled and processed together. The spleens were cut into small pieces
and squeezed through a 70 m cell strainer (BD Falcon™) in complete RPMI medium
supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin and 0.1 mg/mL
streptomycin. The released cells were spun for 5 min at 190 g in a ROTINA 48R centrifuge
(Hettich Zentrifugen) and pelleted. Supernatant was discarded and cells were resuspended
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in 3 mL of Red Blood Cell Lysing Buffer and incubated for 2 min at room temperature to
eliminate the red blood cells. Complete RPMI medium was added to the cell suspension and
the cells were recovered by centrifugation; this was repeated two more times before the cells
were counted and used in the T cell proliferation assay. 96-well round-bottom plates (BD
Falcon™) were seeded with 3x105 cells/well in complete RPMI medium. Concanavalin A (5
g/mL) was used as a non-specific stimulant, while Ag85B and Acr were both used at 10
g/mL. Cells stimulated with medium alone were used as a negative control for proliferation.
Cells were incubated at 37oC for 48 h and 100 L of culture supernatant was removed and
used for IFN- detection by ELISA (Mouse IFN- kit, R&D systems, USA). The cultures were
then pulsed with 1Ci/well of [3H]-thymidine (GE Healthcare) and incubated for a further 24 h
before harvest. Cells were harvested onto a glass fiber filter (Wallac) using a TOMTEC
harvester and fixed with a scintillator sheet (MeltiLex™ A, Perkin Elmer) prior to counts
reading (Wallac counter).
Intracellular cytokine staining
Splenocytes from immunised mice were stimulated with either Ag85B or Acr (both at 10
g/ml), PMA (1 g/ml), Ionomycin (1 g/ml) or medium only for 8 h at 37°C, 5 % CO2. For
the last 4 h of stimulation, GolgiPlug (BD Biosciences) was added. Cells were then washed
and resuspended in 10% FCS containing antibodies to CD3 (FITC), CD4 (APC-Cy7) and
CD8 (PerCP-Cy5.5), (purchased from Biolegend, UK). Cells were incubated for 20 min,
washed, and the pellets resuspended in Cytofix (BD Biosciences, UK), followed by an
additional 15 min incubation at RT, followed by overnight incubation at 4 °C. Cells were then
resuspended in Permwash (BD Biosciences, UK), and antibodies specific for IFN-γ (PE), IL2 (PE-Cy7) and TNF-α (APC) (all from BD Biosciences) added. After incubation for 1 h at 4
°C, cells were washed twice more in Permwash, resuspended and run on the BD
FACSCanto II (BD Biosciences, UK). Compensation was carried out using OneComp
eBeads (eBioscience, UK), and data were collected using BD FACSDiva software (BD
Biosciences, UK). Data files were analysed using FlowJo V10 (TreeStar Inc., USA).
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Lymphocytes were gated for CD3+ CD4+ staining, and analysed for IFN-γ, IL- 2 or TNF-α
expression. Graphical analysis was carried out in SPICE V 5.2 software (Mario Roederer,
Vaccine Research Center, NIAID, NIH, USA).
Cell surface staining and binding assay
106 Balb/c mouse bone marrow derived dendritic cells (BMDC, prepared by culture of bone
marrow cells for 7 days RPMI 1640 medium supplemented with 10 % bovine fetal serum and
20 ng/ml GM-CSF) were washed three times in PBS, and resuspended in 100 L of binding
buffer (PBS, 5% bovine serum albumin (BSA), 0.1% Na-azide). PE-conjugated
(phycoerythrin) antibodies (BD Biosciences), specific for cell surface markers, were added at
1 g/mL final concentration and cells incubated on ice for 1 h. An unrelated mouse IgG2bPE conjugate (BD Biosciences) was used as the negative isotype control. Cells were then
washed three times with binding buffer, and analysed for red fluorescence on a BectonDickinson flow cytometer. For TB-RICs binding assay, 10 g of RICs (or TBG65 antibody
alone) were added to 106 cells and following 1 h incubation on ice, cells were washed 3
times by addition of 2 mL of binding buffer, spun as above and resuspended in 100 L of
binding buffer. Secondary goat anti-mouse IgG-FITC (fluorescin-5-isothiocyanate) antibody
conjugate (Sigma Aldrich) was added at a final concentration of 1 g/mL and cells incubated
for one further hour on ice. Cells were then washed 3 times in binding buffer as before,
resuspended in 1 mL of binding buffer and analysed for green fluorescence in a BectonDickinson flow cytometer. Ten thousand cells were counted and results expressed as
percentage of cells positive for FITC staining.
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