Fluorescein isothiocyanate (FITC), SigmaCell Type 20 and Whatman
CF11 were obtained from Sigma. Secondary fibres where kindly supplied
by Portucel Viana. All chemicals were of the highest purity available in the market.
Amorphous Cellulose Preparation
Amorphous cellulose was prepared by treating Whatman CF11 fibres
with phosphoric acid. Briefly, 0.17 g of Whatman CF11 were slowly mixed
with 10 mL of cold (4°C) phosphoric acid (85%), and left in contact for
5 minutes. Then, 600 mL of cold water were added and the suspension was
filtered through a test sieve (mesh width 71 μm,
according to DIN 4188). Finally, the fibres were extensively washed
first in tap water and afterwards with distilled water. The obtained
material was lyophilized and stored.
The CBDs were prepared according to the following methodology: the Celluclast® commercial
enzymatic preparation (Novozymes A/S, Denmark) was digested with Papain
(1:1200, protein basis). The CBDs were separated by ultrafiltration
through a 10 kDa membrane (Pellicon 2 TFF System from Millipore, USA)
and concentrated by precipitation with ammonium sulphate (Merck,
Darmstadt, Germany). After dialysis, the protein was injected on a
Sepharose Fast-Flow gel (Amersham Pharmacia Biotech AB, Sweden), and
the non-adsorbed protein was collected and lyophilized. The purity and
identity (CBD from T. reesei CBH I) of this protein has been demonstrated by N-terminal sequencing and MALDI-TOF .
The conjugation of CBD with the labelling probe was carried out by mixing 20 μg
of FITC per mg of CBD (2 mg protein/mL in 0.1 M HEPES buffer, pH 9.0).
This solution was incubated overnight in the dark, at room temperature,
with magnetic stirring. To eliminate the unbound FITC, the labelled CBD
mixture was filtered through a BIO-GEL P-4 (BIO-RAD, Hercules, USA)
column, previously equilibrated with 50 mM sodium acetate buffer
(Panreac, Barcelona, Spain).
Adsorption assays of FITC-labelled CBD were carried out at 4°C. The
conjugates were allowed to adsorb on cellulose fibres (20 g/L, in 50 mM
sodium acetate buffer, pH 5.0), with continuous magnetic stirring, in
the dark, for 2 hours. The supernatant with unbound CBD was removed by
centrifugation at 3219 RCF for 10 minutes (Heraeus Megafuge 1.0R). The
fibres were washed with acetate buffer to remove the non-adsorbed
Fluorescence microscopy observations were performed in a Zeiss
Axioskop microscope (Zeiss, Oberkochen, Germany) equipped with a Zeiss
AxioCam HRc attached camera (Zeiss, Oberkochen, Germany) and using the
AxioVision 3.1 software (Zeiss, Oberkochen, Germany). All images were
acquired at 1300 × 1030 pixels and 24 bits colour depths (8 bits per
channel). The FITC-CBD quantification was performed as described
Confocal observation was performed in an Olympus (Tokyo, Japan)
Fluoview 1000 in Laser Scanning mode equipped with a 60× UPLSAPO lens,
with a numerical aperture of 1.35 and a pinhole size of 105 μm.
CBD-specific antibodies were produced in a rabbit (Oryctolagus cuniculus)
maintained under standard conditions of housing with unrestricted
access to food and water; these conditions followed European Union
Directive no. 86/609/CEE. Briefly, the rabbit was immunized
intradermically (i.d.) with a 1:1 suspension of Phosphate Buffered
saline (PBS)/Complete Freund's adjuvant containing 500 μg CBD and boosted two weeks later i.d. with a 1:1 suspension of PBS/Incomplete Freund's adjuvant containing 500 μg
CBD. Blood was collected three weeks after the second immunization for
the preparation of immune serum. Purification of IgG antibodies from
this serum sample was performed as follows: the serum sample was
equilibrated in a binding buffer (20 mM sodium phosphate, pH 7.0) by
overnight dialysis and 3 ml of this preparation was applied to a
Protein G HP affinity column (HiTrap, Amersham Biosciences, UK). Bound
antibodies were eluted with Glycine -HCl buffer, pH 2.7 and recovered
in 50 μl of 1 M Tris-HCl pH 9.0 per ml of
eluent, according to the manufacturer's instructions. Recovered IgG
antibodies were further equilibrated in PBS in a VIVAPORE concentrator
with a 7.5 kDa cutoff membrane (Vivascience, Hanover, Germany) and
stored at -80°C in frozen aliquots. The anti-CBD antibody titre of this
preparation was determined by ELISA. Specific anti-Sap2 or anti CaS
antibodies in mice sera collected by retrorbital bleeding were
quantified by ELISA. Polystyrene microtitre plates (Nunc, Roskilde,
Denmark) were coated with 20 μg/ml of CBD
and incubated o.n. at 4°C. Wells were then saturated for 1 h at room
temperature with 1% BSA in PBS. Serial dilutions of the serum samples
were then plated and incubated for 2 h at room temperature. After
washing, bound antibodies were detected by adding alkaline
phosphatase-coupled monoclonal goat anti-rabbit-IgG antibody (Southern
Biotechnology Associates, Birmingham, ALA, USA) for 30 min at room
temperature. Substrate solution containing p-nitrophenyl phosphate
(Sigma, St. Louis, USA) was then added after washing and the reaction
was stopped by adding 0.1 M EDTA pH 8.0. The absorbance was measured at
405 nm. The ELISA antibody titres were expressed as the reciprocal of
the highest dilution giving an absorbance of 0.1 above that of the
control (no serum added). The titre of anti CBD antibodies in the
purified IgG preparation was of 4014. No antibodies with this
specificity were detected in the control sera from non-immunized
Immunolabelling in Transmission Electron Microscopy
The CBD-treated Whatman CF11 fibres were fixed in a freshly prepared
mixture of 0.2% glutaraldehyde (v/v), 2% paraformaldehyde (w/v) in 0.05
M phosphate buffer (pH 7–7.2). Successive periods of vacuum (5 to 10
min) and air inlet were carried out, up to two hours. Afterwards, the
fibres were washed 3 × 10 minutes with 0.05 M phosphate buffer. The
samples were then dehydrated through a graded series of ethanol and
embedded in London Resin White (hard mixture) polymerized for 24 h at
Immunolabelling was done on ultrathin transverse sections (500 Å) floating on plastic rings .
The sections were floated on several dilutions of the antiserum in 10
mM Tris buffered saline (500 mM NaCl) to determine the optimal ratio of
labelling and background .
The secondary marker was Protein A-gold (pA G5, BioCell). The gold
particles were further enhanced using a silver enhancing Amersham kit.
Finally, the thin-sections were transferred on copper-grids,
post-stained with 2.5% aqueous uranyl acetate and examined with a
Philips CM 200 Cryo-TEM at an accelerating voltage of 80 kV.
To guarantee semi-quantitative comparative labelling, experiments
were carried out in parallel on treated and non-treated samples of CBD.
Therefore, the exposure to the antibody was identical.