Open Life Sci.

2016; 11: 19

Research Article Open Access

Muhammad Haddad, Chadi Soukkarieh, Houssam Eddin Khalaf, Abdul Qader Abbady*

Purification of polyclonal IgG specific for

Camelids antibodies and their recombinant
nanobodies
DOI 10.1515/biol-2016-0001
Received July 22, 2015; accepted December 21, 2015
List of Abbreviations
Abstract: Camelids heavy-chain antibody (HCAb) consists FPLC: fast protein liquid chromatography
of only two heavy chains and lacks the two light chains HCAb: heavy-chain antibody
together with the CH1 domain usually found in conventional HRP: horseradish peroxidase
immunoglobulins. A recombinant single antigen-binding TMB: 3, 3,5 ,5tetramethylbenzidine
entity, named VHH (or Nanobody) was generated
by reengineering the variable domains from HCAb.
This study focuses on the detection of camelids
1 Introduction
immunoglobulins as well as their derivative nanobodies
Antibodies, either polyclonal or monoclonal, from
using a universal anti-camel antibody produced in rabbit
animal or human origin are essential for a very broad
(rIgG). Starting from a crude rabbit serum, a standard stock
range of applications, from use as a research tool for
of rIgG (1 mg/ml) was prepared after purification by affinity
target detection or purification, to aiding in medical
chromatography using protein-A column. As expected,
diagnosis and therapy. The demand for an affordable
rIgG was able to detect camel antibodies in ELISA and
and renewable source of antibodies is continuously
immunoblotting, and its reactivity was equal against all
increasing [1]. The overall antibody structure consists
different camel IgG subclasses, which were purified from
of two identical heavy (H-chain) and two identical light
serum by differential affinity chromatography on protein-G
(L-chain) polypeptides, which is highly conserved in
and -A. Interestingly, rIgG also recognized nanobodies
mammals [2]. Whole antibodies, with a molecular weight
since they were originally part of camel HCAbs, providing
of about 150 kDa, sometimes lead to practical drawbacks,
an alternative method to detect the corpus of these
such as a slow production, high cost, or weak tissue
recombinant proteins rather than targeting their artificial
penetration. In addition, for a range of applications,
tags. These data suggest that the anti-camel rIgG described
such as radioimmunotherapy or in vivo imaging, the
here could be efficiently applied at different stages of
Fc-mediated cellular effects or prolonged half-life in blood
nanobody technology, including the quantitation of the
are usually serious bottlenecks. Consequently, smaller
issued nanobodies and their detection when bound to
recombinant antibody fragments have become top listed
target antigens.
as an emerging new class of drugs [3].
Reduced antibodies in structure, which are naturally
Keywords: Camel, nanobody, heavy chain antibody,
devoid of light chains, were found by chance in sera of
polyclonal antibody, affinity chromatography.
species from the family Camelidae and evidently contribute
to the immune response of these animals [4]. Hence, the
variable domain in these heavy chain antibodies (HCAbs)
*Corresponding author: Abdul Qader Abbady, Department of
Molecular Biology and Biotechnology, Atomic Energy Commission of
is represented in a single antigen-binding entity (VHH)
Syria (AECS), Damascus, Syria, E-mail: ascientific@aec.org.sy which is joined directly to the hinge region, lacking
Muhammad Haddad, Chadi Soukkarieh, Department of Animal the first constant domain (CH1) which is necessary for
Biology, Faculty of Sciences, Damascus University, Syria anchoring the light chain in conventional antibodies [5,6].
Houssam Eddin Khalaf, Department of Molecular Biology and Bio- In HCAbs, extended hinge regions, deletion of the CH1
technology, Atomic Energy Commission of Syria (AECS), Damascus,
domain, and amino acid substitutions in the V domain
Syria

2016 Muhammad Haddad et al., published by De Gruyter Open.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
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2 M. Haddad, et al.
that make it more hydrophilic, promote their flexibility and
solubility [7,8]. The recombinant form of VHH, also known
as Nanobody, possesses many unique features such as
small size and the ability to recognize epitopes which are
otherwise incompatible with classical antibodies [9]. In
addition, amino acid substitutions in VHHs can explain
the high stability of their corresponding nanobodies in
expressing Escherichia coli, where they can be produced
economically as soluble and non-aggregating proteins
[10,11]. Nanobodies, because of their single domain
nature, offer several advantages for biotechnological and
medical applications.
Nanobodies are procured by cloning their genetic
repertoire from B cells circulating in the blood of an
immunized camel, constructing a cDNA library and
panning by phage display [10,12]. Several published
reports described the involvement of HCAbs in camelid
immunity, especially in the response to pathogenic
antigens [4,13,14]. Hence, after camel immunization with
the antigen of interest, and during antibody purification,
three fractions containing IgGs of distinct molecular weight
can be isolated from the dromedary serum by differential
adsorption on protein-A and protein-G columns [15]. The
so-called IgG-1 subclass contains the conventional
antibodies comprising two heavy and two light chains. The
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
than the heavy chain of conventional antibodies [4]. The
percentage of HCAbs and conventional IgG in the sera of
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
the antigen-inducing immune response, as manifested in
antigen recognition in ELISA, is an important indication
of a good camel immunizing process, since nanobodies
are derived from the variable domain of these subclasses.
Such information is indispensable before constructing an
expensive and laborious nanobody immune library.
A limiting factor for investigations into camel immunity
has been a shortage of well-characterized, isotype-
specific reagents. One of the key components in ELISA and
in various detection systems during this procedure is the
specific antibody used against camel IgGs to evaluate the
raised immune response and the participation of HCAbs.
The production of several monoclonal antibodies specific
to different subclasses of llama IgGs have been described,
but failed in detecting camel IgGs [17]. Furthermore,
testing retrieved nanobodies from the library against
their antigens requires specific antibodies. Generally,
camel IgGs are detected by anti-camel anti-serum
produced mainly from rabbit (Bethyl Laboratories, Inc.
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and antibodies-online). Moreover, nanobodies are usually
detected by mean of recombinant tags, added at one of
their ends like 6His, c-Myc, glutathione S-transferase
(GST) or hemagglutinin (HA) [18]. Most of these tags have
commercialized antibodies which differ certainly in their
optimal working conditions and concentrations. Here,
we have characterized a purified polyclonal rabbit anti-
camel antibody which represents an interesting tool for
detecting all camel IgG subclasses as well as their derived
nanobodies.
2 Materials and Methods
2.1 Purification of Rabbit polyclonal
antibodies
Polyclonal rabbit antibodies (rIgGs) were purified
from 2 ml of commercial rabbit serum by affinity
chromatography on a 5 ml HiTrap protein A column (GE
Life Science) according to the manufacturers instructions.
Binding was performed in 0.02 M sodium phosphate (pH
7), and rabbit IgG was eluted with 0.1 M citric acid (pH
3). Eluted IgG was collected and immediately neutralized
to physiological (pH 9), with 1 M Tris-base buffer. After
on a Vivaspin concentrator with a molecular mass cutoff
of 50 kDa (Vivascience) to 1 mg/ml and stored at -20C in
phosphate buffer containing 50% glycerol.
2.2 ELISA
An indirect ELISA format was employed for the analysis
of commercial immunized rabbit sera and the detection
of camel antibody subclasses. Maxisorp 96-well plates
(Nunc) were coated overnight at 4C with conventional
IgG-1, heavy chain camel antibodies IgG-2 and IgG-3
(0.2-1g/well), and nanobody mix (0.25 g/well) diluted in
carbonate buffer. After coating, ELISA plates were washed
3 times with washing buffer TBST (20 mM Tris-base,
150 mM NaCl, 0.05% Tween-20, pH 7.5). Residual protein
binding sites in the wells were blocked for 1 h at 37C with
5 blocking buffer (3% skimmed milk and 1% BSA) in
TBS-T. After the removal of blocking buffer, the indicated
dilutions of purified rabbit anti-camel rIgG or anti-llama
(kindly provided by Prof. Serge Muyldermans, Vrije
Universiteit Brussel), were prepared in 1 blocking buffer
and added in the wells for 1 hour at RT. After 3 washes,
detection of rabbit polyclonals was performed by 1 h
incubation at RT with goat anti-rabbit conjugated to HRP
  Purification of polyclonal IgG specific for Camelids antibodies and their recombinant nanobodies
(Bethyl Laboratories Inc.) at 1:3000 in 1 blocking buffer.
After 5 additional washes, bound conjugate was detected
with 3,3,5,5tetramethylbenzidine (TMB) substrate
(Sigma), the reaction was stopped after 10 minutes with
the addition 1 M H2SO4. The spectroscopic absorbance of
the enzymatic reaction was measured in an automated
plate reader at a wavelength of 450 nm.
2.3 Fractionation of IgG subclasses
An automated protocol to separate different IgG
subclasses from the 5 ml camel plasma was setup using
the AKTAprime (GE Healthcare) fast protein liquid
chromatography system (FPLC). This was performed
by differential adsorption on Hitrap protein-A and
Hitrap protein-G columns (GE Healthcare) as described
previously [4,19]. Briefly, 5 ml camel serum was diluted
with an equal volume of PBS and loaded at a flow rate
of 5 ml/min on the protein-G column (5 ml) equilibrated
with PBS. The flow-through was collected as this part
contained the IgG-2 subclass of antibodies that do not
adsorb on protein-G. After a column-volume washing with
PBS, IgG-3 was eluted with buffer A (150 mM NaCl, 0.58%
acetic acid, pH 3.5), IgG-1 was then eluted with buffer B
(100 mM glycineHCl, pH 2.7). The column was washed
intensively with PBS and the flow through was reloaded
on the column to remove the residual IgG-1 and IgG-3 of
the flow through (as all IgGs from camel bind to protein
A). The flow through was captured and loaded on PBS
equilibrated protein A column, IgG-2 was eluted from the
column using buffer A. Monitoring the UV absorption at
280 nm facilitated fractionation and sample collection.
Once eluted, all IgG fractions were neutralized with
1.0 M Tris pH 9.0, dialyzed with PBS, quantified, diluted
to 1 mg/ml, aliquoted and stored at 20C. The integrity
and the purity of IgG fractions were verified by loading
a sample of 2 g protein (denatured and reduced) onto a
15% polyacrylamide SDS gel, followed by coomassie blue
staining.
2.4 Immunoblotting and coomassie blue
staining of SDS-PAGE
SDS-PAGE was performed using Bio-Rad mini-Protean
II system following the manufacturers instructions.
Gels were prepared using stacking gel 5% and running
gel 15%. After electrophoresis, the gel was stained with
coomassie blue for 2 h followed by destaining in 5% acetic
acid and 10% methanol. For immuno-blotting, separation
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3
was carried out to 0.25 g of different camel subclasses
antibodies (IgG1, IgG2, IgG3) and pure nanobodies mixture
was then blotted onto 0.45 m nitrocellulose membranes
(BioRad) using 1 blotting buffer (25 mM Tris-base, 200 mM
glycine, 0.1% SDS and 20% methanol). After incubation
in blocking buffer, membranes were incubated with the
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
(Bethyl Laboratories Inc.) at dilution 1:2000 for 1 h at RT.
Bands revelation was achieved by adding chromogen
substrate (0.05% NBT and 0.025% BCIP; Sigma) in AP
buffer (100 mM Tris-base, 100 mM NaCl, 5 mM MgCl2, pH
9.5).
2.5 Nanobody detection by competitive
ELISA
Apparent detection affinity was determined by competitive
indirect ELISA testing. In brief, nanobody mixture (0.2g/
well) was prepared in carbonate buffer and coated in
96-well ELISA plate at 4C overnight. After blocking and
washing, nanobody titrations (1 to 100 ng/ml), previously
incubated with rabbit anti-camel rIgG (1:1000) for 1 h at
RT, were added (100 l) to the wells and incubated for an
additional 1 h at RT. Plates were washed and bound rabbit
anti-camel antibodies were detected with a goat anti-
rabbit-HRP conjugate antibody (1:3000). The absorption
at 450 nm was measured 15 min after adding the enzyme
substrate TMB for peroxidase conjugates.
2.6 Dot blotting
Different antigens of recombinant human growth
hormone (rhGH) from previous work [12] were
immobilized on nitrocellulose membrane by spotting
2 l (1 g) of the samples at the center of the grid, and
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
different antibodies: R-anti-camel rIgG (1:1000), R-anti-
6His (Bethyl Laboratories Inc., 1:5000), R-anti-GFP
(homemade, 1:3000) [20], and R-anti-GH (homemade,
1:3000) [21]. After three washes with TBS-T, primary
antibodies were detected with secondary conjugated AP
goat anti-rabbit (1:3000). Spots revelation was achieved
by adding chromogen substrate NBT/BCIP in AP buffer as
described previously.
4 M. Haddad, et al.

3 Results prepared as 1 mg/ml stock (data not shown). Furthermore,

indirect ELISA showed that purified rIgG was reactive
toward immobilized cIgGt (Fig. 2A). Interestingly, rIgG
3.1 Purification of rabbit anticamel IgG
was able to detect similarly, but to a lesser extent, an
immobilized mixture of pure nanobodies, since they were
One interesting and commercial product for the detection
originally derived from the variable region of camel HCAb
of camel IgG (cIgG) that are bound to their inducing
(Fig. 2A). Detection sensitivity of rIgG was tested toward
antigen during immune response evaluation, is a rabbit
anti-camel serum from a commercial source (Bethyl
Laboratories Inc.). It represents an efficient tool for the
detection of cIgG with a titer exceeding 1/100000 (data not
shown). Rabbit IgG (rIgG) from this serum was purified
by affinity chromatography using protein-A column. An
automated purification procedure was established using
AKTA prime fast liquid protein chromatography (FPLC)
system, allowing a direct and confirmed purification
of total they from 2 ml rabbit serum (Fig. 1A). A second
purification step on protein-A column is necessary to
assure the total purification of rIgGs from the serum flow-
through (Fig. 1B).

Fig. 1. Purification diagram of rabbit anti-camel IgG (A) Rabbit

3.2 Reactivity of purified rabbit anti-camel
IgG
In order to test the reactivity of purified rIgG, total IgG
was purified from 5 ml camel serum (cIgGt) using the
same previous affinity purification method and was also
serum was injected onto a protein A column and washed away with
phosphate buffer (void) to remove the unbound proteins from the
column (flowthrough), before eluting pure IgGs (eluate). (B) Flow
through from the previous purification was reinjected onto the
same column to confirm the total purification of serum IgGs. Conti-
nuous line represents the absorbance of the eluate and dashed line
represents its conductivity.

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
decreasing concentrations (ng/ml) of immobilized cIgGt
or nanobodies by indirect ELISA (Fig. 2B). Using this
method, rIgG was able to detect low concentrations of
cIgGt (linear range from 3 to 100 ng/ml) and nanobodies
at a concentration ten times greater.
3.3 Purification of camel IgG subclasses for
testing against rIgG
In order to evaluate the reactivity of rIgG for the different
subclasses of camel cIgG, rIgG were prepared from 5 ml
blood serum of an immunized camel with rhGH. The cIgG
subclasses (IgG-1, 2 and 3) were fractionated from the
serum sample by differential adsorption on protein-G and
protein-A columns (Fig. 3A). IgG-1 and IgG-3 can be purified
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
Fig. 3. Fractionation of camel immunoglobulin subclasses by
affinity chromatography (A) Purification diagram of camel IgG-1,
IgG-2 and IgG-3 using protein-G and protein-A columns is shown.
Pure camel immunoglobulins and the mixture of purified nano-
bodies were separated by SDS-PAGE and then revealed by either
blue coomassie staining (B) or with immunoblotting (C) using rIgG
(1:1000 v:v). The location of camel heavy (HC) and light (LC) chains
was indicated. The protein molecular weight ladder was in the first
lane (M).
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column is necessary to assure the total purification of these
two cIgGs from the serum flow-through (Fig. 3A, middle
panel), before further purification of cIgG-2 on protein-A
column from this flow-through (Fig. 3A, right panel). The
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
showed two distinct bands: one of the heavy chains
(~55kDa) and a smaller one of the light chains (~25kDa),
whereas, HCAbs (IgG-2 and 3) showed smaller single
bands related to their heavy chains which lack the CH1
domain. The band of cIgG-2 (~50 kDa) was notably bigger
than that of cIgG-3 (~45kDa) because of the long hinge
region characterizing this subclass of antibodies (Fig 3B).
SDS-PAGE-loaded nanobody mixture appeared as a single
small band of ~15 kDa (Fig. 3B).
3.4 Reactivity of rIgG against camel subclas-
ses and nanobodies
As a polyclonal antibody, rIgG was able to detect all
different chains from the three subclasses when incubated
with the nitrocellulose blot of the transferred samples
from SDS-PAGE in the same order as shown in the first
blue-stained gel (Fig. 3C). In confirmation of our previous
result in ELISA, rIgG detected nanobodies, despite being
denatured during sample preparation before loading
on the gel. Furthermore, using the same amount for
immobilization, rIgG was able to detect similarly all
three subclasses of cIgG in indirect ELISA and, to a lesser
extent, the nanobody mixture (Fig. 4A). The reactivity of
our anti-camel rIgG was higher than anti-llama rIgG which
recognized equally all three different cIgG subclasses but
not the nanobodies (Fig. 4A).
3.5 Quantitation of nanobodies using rIgG in
a competitive ELISA
To test whether anti-camel rIgG can be used in nanobody
quantification, a competitive indirect ELISA was
conducted by its incubation with serial logarithmic
dilutions of free nanobodies (fNb). Bound rIgG/fNb
complexes were then added to a 96-well microplate,
pre-coated with immobilized nanobodies. The more free
nanobodies in the reaction, the less rIgG will be available
to bind to immobilized nanobodies in the wells, hence a
weaker signal in ELISA, and vice versa (Fig. 4B).
6 M. Haddad, et al.
3.6 Detection of antigen-bound cIgG and
nanobodies using rabbit anti-camel rIgG
Finally, indirect ELISA was performed to test the capacity
of anti-camel rIgG to efficiently detect different subclasses
of cIgG and the nanobodies when they are bound to
their specific antigen. For this aim, the microplate was
coated with rhGH (0.25 g/ml) before adding diluted
(1/10000) crude serum samples from before (S0) and after
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)-
-a GH specific (Nb+) and negative control nanobody
(Nb-)--were all added to the precoated wells and then
Fig. 4. Immunodetection of the different immunoglobulin sub-
classes using pure rabbit anti-camel IgG (A) Indirect ELISA for the
detection of immobilized (0.25 g/well) IgG1, IgG2, IgG3, a mixture
of nanobodies (Nb) or empty wells (No Ag) using rIgG (1:1000 v:v),
rabbit anti-llama IgG (1:250) or left in the absence of primary anti-
bodies (No Ab). (B) Indirect competitive ELISA using rIgG (1:1000)
incubated with serial concentrations of nanobody mixture (ng/ml).
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incubated in the presence of rIgG (Fig. 5A). Interestingly,
rabbit anti-camel rIgG succeeded in similarly recognizing
the antigen-bound cIgG in the positive crude serum (after
immunization) and in all pure subclasses, recognizing at
the same time the bound specific nanobody and not the
control. Impressively, the background signals in control
or empty wells were very weak using pure rIgG for the
detection of cIgG as compared with rabbit anti-camel
crude serum (data not shown).
3.7 Testing the background of nanobody
detection using dot blotting
To confirm the utility of the anti-camel rIgG in the
detection of nanobody bound to its specific recombinant
Fig. 5. Detection of antigen-bound antibodies and nanobodies
using rabbit anti-camel IgG (A) Indirect ELISA for the detection of
immobilized rhGH (0.25 g/well) using camel serum (1/10000) from
before (S0) and eight weeks (S8) after being immunized with rhGH,
dilutions (1/500) of IgG1, IgG2, IgG3, NbGH01 (Nb+), NbGFP01 (Nb-)
or left without antibodies (No Ab). Revelation was performed using
rIgG (1/500). (B) Dot-blotting membranes using different antigens
(below). Detection was performed in the presence or the absence
of NbGH01. Several detecting polyclonal antibodies were used (to
the right).
  Purification of polyclonal IgG specific for Camelids antibodies and their recombinant nanobodies
antigen comparing with a commercial anti-6His
antibody, a dot blot experiment was carried out. For this
purpose, different recombinant antigens were spotted on
a nitrocellulose membrane: rhGH, His-GH, GFP-GH, GFP,
GFP-TEV, with PBS used as a negative control (Fig. 5 B).
After blocking, spots were incubated with an anti-GH
nanobody (NbGH01), and bound nanobodies were
detected either by anti-6His antibody or the purified anti-
camel rIgG. In another case, spots were directly treated
with these antibodies in the absence of nanobodies. As
expected, the two methods for nanobody detection were
able to detect bound NbGH01 to GH in its different forms.
However, one confusing spot appeared using GFP-TEV as
antigen when anti-6His antibody was used for detection.
This spot was not related to the nanobody as it was also
observed in the absence of nanobody. It is explained by the
capacity of this antibody to detect c-terminal 6His tags in
the recombinant proteins (like the nanobody itself), and
GFP-TEV is the only immobilized antigen that possess the
His tag at the c-terminal.
4 Discussion
It seems that the paratopes of HCAbs and conventional
antibodies recognize different antigenic sites on their
target. It is therefore possible that HCAbs have been
selected and maintained in the camelid species for
complementary function in their immune response [22].
Although knowledge of the exact roles and functions of
the various camelid IgG subclasses is still in its infancy, an
infected or vaccinated animal raises an immune response
in the three isotype fractions (to various extents in
different animals, depending on the actual immunogen).
In a previous study, monoclonal antibodies (MAbs),
specific for dromedary (Camelus dromedarius) IgG1 and
IgM, have been produced and characterized to monitor
the camel immune response [23]. Remarkably, the llama
IgG1 and IgG3 neutralize West Nile virus, whereas IgG2
seems less effective in this respect [24].
The antigen-binding fragments of HCAbs are
comprised in a single-domain, referred to as VHH or
Nanobody. They have a broad range of applications in
biotechnical and therapeutic uses due to their small
size, simple production and high affinity [25]. Nanobody
production technology requires several checkpoints
in order to evaluate the correct advancement of the
procedure. For example, at the end of camel immunization,
two questions are routinely asked, one regarding the rise
of specific and powerful immune response against used
antigen and the other about the participation of HCAbs
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in this immune response, since these last are the true
representatives of nanobodies in camel blood. A simple
ELISA on the immobilized antigen could provide the
answers for these questions using several dilutions of total
camel serum or its IgG subclasses purified by differential
affinity chromatography. One key element in such ELISA
is the anti-camel detecting antibody, which must be very
reactive and specific towards all camel IgG subclasses. We
showed in this work that rabbit anti-camel antibody from
Bethyl Laboratories Inc. might represent an interesting
choice. Unfortunately, this antibody is commercialized
as total rabbit serum, which imposes a first step of IgG
purification. For the purification of rabbit IgG from serum
we used protein-A sepharose affinity chromatography
since protein-A column is the best candidate to isolate
monoclonal and polyclonal IgG from ascites, serum,
tissue culture and bioreactor supernatants. Protein A
is a recombinant cell wall protein from the bacterium
Staphylococcus aureus with an important binding affinity
for the constant domains (CH) in the Fc fragment of
immunoglobulins from different species [26,27]. Also,
protein-A and protein-G affinity chromatography are the
fastest methods for purifying antibodies and protein-A
purification is recommended for rabbit antibodies [28].
Anti-camel rIgG may be considered as an alternative
tool for the detection of nanobodies since all used
secondary antibodies against nanobodies target their
recombinant tags like anti-6His tag antibody. Moreover,
certain applications of nanobodies necessitate the
elimination of these tags because of their undesirable side
effects or their immunogenicity, making the detection of
these nanobodies unachievable using regular antibodies.
In addition, replacement of rIgG with a commercial rabbit
anti-6His tag, in ELISA detection of bound nanobody,
was futile since immobilized recombinant antigen (rhGH)
possesses the same tag as well (data not shown). This
provides an interesting example for applying rIgG in
nanobody detection when regular anti-tag antibodies are
unusable.
The calculated EC50 for anti-camel rIgG against
cIgG was estimated of about 0.2 g/ml (1/5000 dilution)
and this value was ten time higher against immobilized
nanobodies. Interestingly, rIgG was able to detect very
low amounts (3 ng/well) of nanobodies by simple and
competitive indirect ELISA. The last method has great
potential in certain applications for quantitation of impure
nanobodies within protein samples like total cell extracts.
Furthermore, humanized and untagged nanobodies are
the only formulations approved for medical administration
in humans, thus anti-nanobody antibody that recognize
the main polypeptide sequence of the protein, and not
8 M. Haddad, et al.
the additive tags, is the only remaining method for the
detection and the real quantitation of these nanobodies
during manufacturing. The small portion of the anti-camel
rIgG that recognizes nanobodies could be extracted using
affinity chromatography on a special type of columns
containing covalently immobilized nanobodies as
capturing ligands. Furthermore, by a covalent conjugating
of anti-camel rIgG to HRP or AP enzymes, the final step
of immunoassays which requires conjugated secondary
antibodies can be omitted [29].
In summary, detection of camel conventional and
heavy chain IgGs, as well as their derivative nanobodies,
could be achieved using one single reagent: the anti-camel
rIgG. Such a reagent has very important applications in
nanobody technology, first in evaluating camel immune
response, then in assessing HCAb participation in this
response, and finally in testing the isolated nanobodies
against their antigens using several immunoassays.
Acknowledgements: The authors would like to thank
the Director General of the Atomic Energy Commission
of Syria and the head of the Molecular Biology and
Biotechnology department for their continuous support
throughout this work.
Conflict of interest: The authors report no potential
conflicts of interest in this work and have nothing to
disclose.
References
[1] Taussig M.J., Stoevesandt O., Borrebaeck C.A., Bradbury A.R.,
Cahill D., Cambillau C., et al., ProteomeBinders: planning
a European resource of affinity reagents for analysis of the
human proteome, Nat. Methods, 2007, 4, 13-17
[2] Padlan E.A., Anatomy of the antibody molecule, Mol. Immunol.,
1994, 31, 169-217
[3] Ward E.S., Gussow D., Griffiths A.D., Jones P.T., Winter G.,
Binding activities of a repertoire of single immunoglobulin
variable domains secreted from Escherichia coli, Nature, 1989,
341, 544-546
[4] Hamers-Casterman C., Atarhouch T., Muyldermans S., Robinson
G., Hamers C., Songa E.B., et al., Naturally occurring antibodies
devoid of light chains, Nature, 1993, 363, 446-448
[5] Nguyen V.K., Hamers R., Wyns L., Muyldermans S., Loss of
splice consensus signal is responsible for the removal of the
entire C(H)1 domain of the functional camel IGG2A heavy-chain
antibodies, Mol. Immunol., 1999, 36, 515-524
[6] Woolven B.P., Frenken L.G., van der Logt P., Nicholls P.J., The
structure of the llama heavy chain constant genes reveals a
mechanism for heavy-chain antibody formation, Immuno-
genetics, 1999, 50, 98-101
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Download Date | 5/7/16 6:51 AM
[7] Su C., Nguyen V.K., Nei M., Adaptive evolution of variable
region genes encoding an unusual type of immunoglobulin in
camelids, Mol Biol Evol, 2002, 19, 205-215
[8] Vu K.B., Ghahroudi M.A., Wyns L., Muyldermans S.,
Comparison of llama VH sequences from conventional and
heavy chain antibodies, Mol. Immunol., 1997, 34, 1121-1131
[9] Lauwereys M., Arbabi Ghahroudi M., Desmyter A., Kinne J.,
Holzer W., De Genst E., et al., Potent enzyme inhibitors derived
from dromedary heavy-chain antibodies, EMBO J., 1998, 17,
3512-3520
[10] Nguyen V.K., Desmyter A., Muyldermans S., Functional
heavy-chain antibodies in Camelidae, Adv. Immunol., 2001, 79,
261-296
[11] Arbabi-Ghahroudi M., Tanha J., MacKenzie R., Prokaryotic
expression of antibodies, Cancer Metastasis Rev., 2005, 24,
501-519
[12] Abbady A.Q., Al-Shemali R., Mir Assaad J., Murad H.,
Generation and characterization of nanobodies against rhGH
expressed as sfGFP fusion protein, Gen. Comp. Endocrinol.,
2014, 204, 33-42
[13] Stijlemans B., Conrath K., Cortez-Retamozo V., Van Xong H.,
Wyns L., Senter P., et al., Efficient targeting of conserved cryptic
epitopes of infectious agents by single domain antibodies.
African trypanosomes as paradigm, J. Biol. Chem., 2004, 279,
1256-1261
[14] van der Linden R., de Geus B., Stok W., Bos W., van
Wassenaar D., Verrips T., et al., Induction of immune
responses and molecular cloning of the heavy chain
antibody repertoire of Lama glama, J Immunol Methods,
2000, 240, 185-195
[15] Abbady A.Q., Al-Mariri A., Zarkawi M., Al-Assad A.,
Muyldermans S., Evaluation of a nanobody phage display
library constructed from a Brucella-immunised camel, Vet.
Immunol. Immunopathol., 2011, 142, 49-56
[16] Blanc M.R., Anouassi A., Ahmed Abed M., Tsikis G., Canepa
S., Labas V., et al., A one-step exclusion-binding procedure
for the purification of functional heavy-chain and mammalian-
type gamma-globulins from camelid sera, Biotechnol. Appl.
Biochem., 2009, 54, 207-212
[17] Daley L.P., Gagliardo L.F., Duffy M.S., Smith M.C., Appleton
J.A., Application of monoclonal antibodies in functional and
comparative investigations of heavy-chain immunoglobulins
in new world camelids, Clin. Diagn. Lab. Immunol., 2005, 12,
380-386
[18] Pardon E., Laeremans T., Triest S., Rasmussen S.G., Wohlkonig
A., Ruf A., et al., A general protocol for the generation of
Nanobodies for structural biology, Nat. Protoc., 2014, 9,
674-693
[19] Srdic-Rajic T., Jurisic V., Andrejevic S., Bonaci-Nikolic B.,
Bowker T., Concas D., et al., Naturally occurring V region
connected antibodies inhibit anti-dsDNA antibody reactivity
with dsDNA, Immunobiology, 2012, 217, 111-117
[20] Al-Homsi L., Al-Assad J.M., Kweider M., Al-Okla S., Abbady
A.Q., Construction of pRSET-sfGFP plasmid for fusion-protein
expression, purification and detection, Jordan J. Biol. Sci.,
2012, 5, 279-288
[21] Murad H., Ali B., Makeya R., Abbady A.Q., Prokaryotic overex-
pression of TEV-rhGH and characterization of its polyclonal
antibody, Gene, 2014, 542, 69-76
  Purification of polyclonal IgG specific for Camelids antibodies and their recombinant nanobodies 9

[22] Flajnik M.F., Deschacht N., Muyldermans S., A case of
convergence: why did a simple alternative to canonical
antibodies arise in sharks and camels?, PLoS Biol., 2011, 9,
e1001120
[23] Azwai S.M., Carter S.D., Woldehiwet Z., Monoclonal antibodies
against camel (Camelus dromedarius) IgG, IgM and light
chains, Vet. Immunol. Immunopathol., 1995, 45, 175-184
[24] Daley L.P., Kutzler M.A., Bennett B.W., Smith M.C., Glaser
A.L., Appleton J.A., Effector functions of camelid heavy-chain
antibodies in immunity to West Nile virus, Clin. Vaccine
Immunol., 2010, 17, 239-246
[25] Muyldermans S., Nanobodies: natural single-domain
antibodies, Annu. Rev. Biochem., 2013, 82, 775-797
[26] Goudswaard J., van der Donk J.A., Noordzij A., van Dam R.H.,
Vaerman J.P., Protein A reactivity of various mammalian
immunoglobulins, Scand J Immunol, 1978, 8, 21-28
[27] Forsgren A., Sjoquist J., Protein A from S. aureus. I. Pseudo-
immune reaction with human gamma-globulin, J Immunol,
1966, 97, 822-827
[28] Andrew S.M., Titus J.A., Purification of immunoglobulin G, Curr
Protoc Immunol, 2001, Chapter 2, Unit 2 7
[29] Winston S.E., Fuller S.A., Evelegh M.J., Hurrell J.G., Conjugation
of enzymes to antibodies, Curr. Protoc. Mol. Biol., 2001,
Chapter 11, Unit11 11

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