Documente Academic
Documente Profesional
Documente Cultură
2016; 11: 19
Muhammad Haddad, Chadi Soukkarieh, Houssam Eddin Khalaf, Abdul Qader Abbady*
that make it more hydrophilic, promote their flexibility and and antibodies-online). Moreover, nanobodies are usually
solubility [7,8]. The recombinant form of VHH, also known detected by mean of recombinant tags, added at one of
as Nanobody, possesses many unique features such as their ends like 6His, c-Myc, glutathione S-transferase
small size and the ability to recognize epitopes which are (GST) or hemagglutinin (HA) [18]. Most of these tags have
otherwise incompatible with classical antibodies [9]. In commercialized antibodies which differ certainly in their
addition, amino acid substitutions in VHHs can explain optimal working conditions and concentrations. Here,
the high stability of their corresponding nanobodies in we have characterized a purified polyclonal rabbit anti-
expressing Escherichia coli, where they can be produced camel antibody which represents an interesting tool for
economically as soluble and non-aggregating proteins detecting all camel IgG subclasses as well as their derived
[10,11]. Nanobodies, because of their single domain nanobodies.
nature, offer several advantages for biotechnological and
medical applications.
Nanobodies are procured by cloning their genetic
2 Materials and Methods
repertoire from B cells circulating in the blood of an
immunized camel, constructing a cDNA library and 2.1 Purification of Rabbit polyclonal
panning by phage display [10,12]. Several published antibodies
reports described the involvement of HCAbs in camelid
immunity, especially in the response to pathogenic Polyclonal rabbit antibodies (rIgGs) were purified
antigens [4,13,14]. Hence, after camel immunization with from 2 ml of commercial rabbit serum by affinity
the antigen of interest, and during antibody purification, chromatography on a 5 ml HiTrap protein A column (GE
three fractions containing IgGs of distinct molecular weight Life Science) according to the manufacturers instructions.
can be isolated from the dromedary serum by differential Binding was performed in 0.02 M sodium phosphate (pH
adsorption on protein-A and protein-G columns [15]. The 7), and rabbit IgG was eluted with 0.1 M citric acid (pH
so-called IgG-1 subclass contains the conventional 3). Eluted IgG was collected and immediately neutralized
antibodies comprising two heavy and two light chains. The to physiological (pH 9), with 1 M Tris-base buffer. After
IgG-2 and IgG-3 subclasses contain HCAbs composed of purification, rIgG was dialyzed and then concentrated
heavy chains that are approximately 10 and 12 kDa smaller on a Vivaspin concentrator with a molecular mass cutoff
than the heavy chain of conventional antibodies [4]. The of 50 kDa (Vivascience) to 1 mg/ml and stored at -20C in
percentage of HCAbs and conventional IgG in the sera of phosphate buffer containing 50% glycerol.
camelids is variable; in camels it might reach 5080%,
whereas in South American camelid species, it totals up
to 1025% [16]. The participation of HCAb subclasses in 2.2 ELISA
the antigen-inducing immune response, as manifested in
antigen recognition in ELISA, is an important indication An indirect ELISA format was employed for the analysis
of a good camel immunizing process, since nanobodies of commercial immunized rabbit sera and the detection
are derived from the variable domain of these subclasses. of camel antibody subclasses. Maxisorp 96-well plates
Such information is indispensable before constructing an (Nunc) were coated overnight at 4C with conventional
expensive and laborious nanobody immune library. IgG-1, heavy chain camel antibodies IgG-2 and IgG-3
A limiting factor for investigations into camel immunity (0.2-1g/well), and nanobody mix (0.25 g/well) diluted in
has been a shortage of well-characterized, isotype- carbonate buffer. After coating, ELISA plates were washed
specific reagents. One of the key components in ELISA and 3 times with washing buffer TBST (20 mM Tris-base,
in various detection systems during this procedure is the 150 mM NaCl, 0.05% Tween-20, pH 7.5). Residual protein
specific antibody used against camel IgGs to evaluate the binding sites in the wells were blocked for 1 h at 37C with
raised immune response and the participation of HCAbs. 5 blocking buffer (3% skimmed milk and 1% BSA) in
The production of several monoclonal antibodies specific TBS-T. After the removal of blocking buffer, the indicated
to different subclasses of llama IgGs have been described, dilutions of purified rabbit anti-camel rIgG or anti-llama
but failed in detecting camel IgGs [17]. Furthermore, (kindly provided by Prof. Serge Muyldermans, Vrije
testing retrieved nanobodies from the library against Universiteit Brussel), were prepared in 1 blocking buffer
their antigens requires specific antibodies. Generally, and added in the wells for 1 hour at RT. After 3 washes,
camel IgGs are detected by anti-camel anti-serum detection of rabbit polyclonals was performed by 1 h
produced mainly from rabbit (Bethyl Laboratories, Inc. incubation at RT with goat anti-rabbit conjugated to HRP
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Purification of polyclonal IgG specific for Camelids antibodies and their recombinant nanobodies 3
(Bethyl Laboratories Inc.) at 1:3000 in 1 blocking buffer. was carried out to 0.25 g of different camel subclasses
After 5 additional washes, bound conjugate was detected antibodies (IgG1, IgG2, IgG3) and pure nanobodies mixture
with 3,3,5,5tetramethylbenzidine (TMB) substrate was then blotted onto 0.45 m nitrocellulose membranes
(Sigma), the reaction was stopped after 10 minutes with (BioRad) using 1 blotting buffer (25 mM Tris-base, 200 mM
the addition 1 M H2SO4. The spectroscopic absorbance of glycine, 0.1% SDS and 20% methanol). After incubation
the enzymatic reaction was measured in an automated in blocking buffer, membranes were incubated with the
plate reader at a wavelength of 450 nm. indicated dilutions of the rabbit anti-camel (1:1000) for 1h
at RT. After several washes with TBS-T, blots were finally
incubated with goat anti-rabbit AP conjugated antibodies
2.3 Fractionation of IgG subclasses (Bethyl Laboratories Inc.) at dilution 1:2000 for 1 h at RT.
Bands revelation was achieved by adding chromogen
An automated protocol to separate different IgG substrate (0.05% NBT and 0.025% BCIP; Sigma) in AP
subclasses from the 5 ml camel plasma was setup using buffer (100 mM Tris-base, 100 mM NaCl, 5 mM MgCl2, pH
the AKTAprime (GE Healthcare) fast protein liquid 9.5).
chromatography system (FPLC). This was performed
by differential adsorption on Hitrap protein-A and
Hitrap protein-G columns (GE Healthcare) as described 2.5 Nanobody detection by competitive
previously [4,19]. Briefly, 5 ml camel serum was diluted ELISA
with an equal volume of PBS and loaded at a flow rate
of 5 ml/min on the protein-G column (5 ml) equilibrated Apparent detection affinity was determined by competitive
with PBS. The flow-through was collected as this part indirect ELISA testing. In brief, nanobody mixture (0.2g/
contained the IgG-2 subclass of antibodies that do not well) was prepared in carbonate buffer and coated in
adsorb on protein-G. After a column-volume washing with 96-well ELISA plate at 4C overnight. After blocking and
PBS, IgG-3 was eluted with buffer A (150 mM NaCl, 0.58% washing, nanobody titrations (1 to 100 ng/ml), previously
acetic acid, pH 3.5), IgG-1 was then eluted with buffer B incubated with rabbit anti-camel rIgG (1:1000) for 1 h at
(100 mM glycineHCl, pH 2.7). The column was washed RT, were added (100 l) to the wells and incubated for an
intensively with PBS and the flow through was reloaded additional 1 h at RT. Plates were washed and bound rabbit
on the column to remove the residual IgG-1 and IgG-3 of anti-camel antibodies were detected with a goat anti-
the flow through (as all IgGs from camel bind to protein rabbit-HRP conjugate antibody (1:3000). The absorption
A). The flow through was captured and loaded on PBS at 450 nm was measured 15 min after adding the enzyme
equilibrated protein A column, IgG-2 was eluted from the substrate TMB for peroxidase conjugates.
column using buffer A. Monitoring the UV absorption at
280 nm facilitated fractionation and sample collection.
Once eluted, all IgG fractions were neutralized with 2.6 Dot blotting
1.0 M Tris pH 9.0, dialyzed with PBS, quantified, diluted
to 1 mg/ml, aliquoted and stored at 20C. The integrity Different antigens of recombinant human growth
and the purity of IgG fractions were verified by loading hormone (rhGH) from previous work [12] were
a sample of 2 g protein (denatured and reduced) onto a immobilized on nitrocellulose membrane by spotting
15% polyacrylamide SDS gel, followed by coomassie blue 2 l (1 g) of the samples at the center of the grid, and
staining. the membrane was left to dry. Blocking was conducted
with 5 blocking buffer in TBS-T, then spots were treated
with or without NbGH01 nanobody (1/500) [12] then with
2.4 Immunoblotting and coomassie blue different antibodies: R-anti-camel rIgG (1:1000), R-anti-
staining of SDS-PAGE 6His (Bethyl Laboratories Inc., 1:5000), R-anti-GFP
(homemade, 1:3000) [20], and R-anti-GH (homemade,
SDS-PAGE was performed using Bio-Rad mini-Protean 1:3000) [21]. After three washes with TBS-T, primary
II system following the manufacturers instructions. antibodies were detected with secondary conjugated AP
Gels were prepared using stacking gel 5% and running goat anti-rabbit (1:3000). Spots revelation was achieved
gel 15%. After electrophoresis, the gel was stained with by adding chromogen substrate NBT/BCIP in AP buffer as
coomassie blue for 2 h followed by destaining in 5% acetic described previously.
acid and 10% methanol. For immuno-blotting, separation
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4 M. Haddad, et al.
Fig. 2. Evaluation of the reactivity of rabbit IgG against camel immunoglobulins and nanobodies (A) An indirect ELISA using the indicated
serial dilutions (v:v) of rIgG in the absence (No Ag) or the presence of immobilized antigens (0.2 g/well). (B) Sensitivity of rIgG (1:1000 v:v)
was tested in the presence of serial dilutions (from 0 to 1000 ng/ml) of the immobilized antigens; camel total immunoglobulins (IgGt) or a
mixture of different nanobodies (Nb).
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Purification of polyclonal IgG specific for Camelids antibodies and their recombinant nanobodies 5
decreasing concentrations (ng/ml) of immobilized cIgGt column is necessary to assure the total purification of these
or nanobodies by indirect ELISA (Fig. 2B). Using this two cIgGs from the serum flow-through (Fig. 3A, middle
method, rIgG was able to detect low concentrations of panel), before further purification of cIgG-2 on protein-A
cIgGt (linear range from 3 to 100 ng/ml) and nanobodies column from this flow-through (Fig. 3A, right panel). The
at a concentration ten times greater. integrity and purity of these fractions were visualized by
SDS-PAGE (15%) followed by staining with coomassie blue
(Fig. 3B). Expectedly, cIgG-1 as conventional antibody
3.3 Purification of camel IgG subclasses for showed two distinct bands: one of the heavy chains
testing against rIgG (~55kDa) and a smaller one of the light chains (~25kDa),
whereas, HCAbs (IgG-2 and 3) showed smaller single
In order to evaluate the reactivity of rIgG for the different bands related to their heavy chains which lack the CH1
subclasses of camel cIgG, rIgG were prepared from 5 ml domain. The band of cIgG-2 (~50 kDa) was notably bigger
blood serum of an immunized camel with rhGH. The cIgG than that of cIgG-3 (~45kDa) because of the long hinge
subclasses (IgG-1, 2 and 3) were fractionated from the region characterizing this subclass of antibodies (Fig 3B).
serum sample by differential adsorption on protein-G and SDS-PAGE-loaded nanobody mixture appeared as a single
protein-A columns (Fig. 3A). IgG-1 and IgG-3 can be purified small band of ~15 kDa (Fig. 3B).
directly from camel serum by adsorption on protein-G
column and through two steps of elution (Fig. 3A, left
panel). An intermediate purification step on protein-G 3.4 Reactivity of rIgG against camel subclas-
ses and nanobodies
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6 M. Haddad, et al.
3.6 Detection of antigen-bound cIgG and incubated in the presence of rIgG (Fig. 5A). Interestingly,
nanobodies using rabbit anti-camel rIgG rabbit anti-camel rIgG succeeded in similarly recognizing
the antigen-bound cIgG in the positive crude serum (after
Finally, indirect ELISA was performed to test the capacity immunization) and in all pure subclasses, recognizing at
of anti-camel rIgG to efficiently detect different subclasses the same time the bound specific nanobody and not the
of cIgG and the nanobodies when they are bound to control. Impressively, the background signals in control
their specific antigen. For this aim, the microplate was or empty wells were very weak using pure rIgG for the
coated with rhGH (0.25 g/ml) before adding diluted detection of cIgG as compared with rabbit anti-camel
(1/10000) crude serum samples from before (S0) and after crude serum (data not shown).
eight weeks (S8) of camel immunization with the same
recombinant protein. Also, diluted (1/500) cIgG subclasses
(IgG-1, 2 and 3) as well as two different nanobodies (1/500)- 3.7 Testing the background of nanobody
-a GH specific (Nb+) and negative control nanobody detection using dot blotting
(Nb-)--were all added to the precoated wells and then
To confirm the utility of the anti-camel rIgG in the
detection of nanobody bound to its specific recombinant
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Purification of polyclonal IgG specific for Camelids antibodies and their recombinant nanobodies 7
antigen comparing with a commercial anti-6His in this immune response, since these last are the true
antibody, a dot blot experiment was carried out. For this representatives of nanobodies in camel blood. A simple
purpose, different recombinant antigens were spotted on ELISA on the immobilized antigen could provide the
a nitrocellulose membrane: rhGH, His-GH, GFP-GH, GFP, answers for these questions using several dilutions of total
GFP-TEV, with PBS used as a negative control (Fig. 5 B). camel serum or its IgG subclasses purified by differential
After blocking, spots were incubated with an anti-GH affinity chromatography. One key element in such ELISA
nanobody (NbGH01), and bound nanobodies were is the anti-camel detecting antibody, which must be very
detected either by anti-6His antibody or the purified anti- reactive and specific towards all camel IgG subclasses. We
camel rIgG. In another case, spots were directly treated showed in this work that rabbit anti-camel antibody from
with these antibodies in the absence of nanobodies. As Bethyl Laboratories Inc. might represent an interesting
expected, the two methods for nanobody detection were choice. Unfortunately, this antibody is commercialized
able to detect bound NbGH01 to GH in its different forms. as total rabbit serum, which imposes a first step of IgG
However, one confusing spot appeared using GFP-TEV as purification. For the purification of rabbit IgG from serum
antigen when anti-6His antibody was used for detection. we used protein-A sepharose affinity chromatography
This spot was not related to the nanobody as it was also since protein-A column is the best candidate to isolate
observed in the absence of nanobody. It is explained by the monoclonal and polyclonal IgG from ascites, serum,
capacity of this antibody to detect c-terminal 6His tags in tissue culture and bioreactor supernatants. Protein A
the recombinant proteins (like the nanobody itself), and is a recombinant cell wall protein from the bacterium
GFP-TEV is the only immobilized antigen that possess the Staphylococcus aureus with an important binding affinity
His tag at the c-terminal. for the constant domains (CH) in the Fc fragment of
immunoglobulins from different species [26,27]. Also,
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8 M. Haddad, et al.
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Purification of polyclonal IgG specific for Camelids antibodies and their recombinant nanobodies 9
[22] Flajnik M.F., Deschacht N., Muyldermans S., A case of [26] Goudswaard J., van der Donk J.A., Noordzij A., van Dam R.H.,
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[25] Muyldermans S., Nanobodies: natural single-domain
antibodies, Annu. Rev. Biochem., 2013, 82, 775-797
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