Colloids and Surfaces B: Biointerfaces 108 (2013) 72–79

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Colloids and Surfaces B: Biointerfaces

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Purification antibody by thiophilic magnetic sorbent modified with

2-mercapto-1-methylimidazol
ZhiYong Lin a , YingXue Zhang b , ChunJiao Li a , Hao Qian a,∗
a
College of Material Science and Engineering, Huaqiao University, XiaMen 362021, Fujian, China
b
College of Information Science and Engineering, Huaqiao University, XiaMen 362021, Fujian, China

a r t i c l e i n f o a b s t r a c t

Article history: An efficient strategy based on the thiophilic magnetic beads was developed to isolate immunoglobu-
Received 3 October 2012 lin G (IgG) from human plasma with high efficiency. Depending on the improved surface modification,
Received in revised form 21 February 2013 the surface density of thiophilic ligand (2-mercapto-1-methylimidazol) reached 1.67 mmol g−1 resulting
Accepted 22 February 2013
in a high adsorption capacity (qmax = 20.7 mg g−1 ) toward IgG. This purification of IgG could be carried
Available online 14 March 2013
out in some physiological conditions due to the heterocyclic structure of 2-mercapto-1-methylimidazol.
It is found that the recognition region between the thiophilic ligand and IgG was mainly focused on
Keywords:
the Fab fragments of IgG using the technology of enzyme digestion. Furthermore, the bound antibodies
Magnetic polymer beads
Immunoglobulin G
could be easily recovered without loss of their bioactivity at low salt concentration. These magnetic
Purification carries brought great potentials in large-scale antibody purification from human plasma attributing
Thiophilic to their high specificity and efficiency, stable structure, sensitive magnetic response and excellent
2-mercapto-1-methylimidazole reusability.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction hydrophobic groups, thiophilic groups and metal chelates [9–12].

The immunoglobulin G has been attracting great attentions for
its large applications in therapy and diagnosis due to its diverse
physiological functions [1,2]. The rapid demands for the highly
pure and bioactive antibodies are greatly encouraging the fur-
ther researches for the more effective and efficient purification
strategies although many methods, such as centrifugal precipi-
tation, chromatographic technologies and recombinant methods,
have been applied to purify antibodies from diversified natural
sources [3,4].
The specificity of the sorbent toward IgG plays a decisive role
in achieving highly pure antibodies. Thus, the bio-ligands are usu-
ally employed for their highly specific interaction with IgG, for
example, Protein A, Protein G, and some antigens [5–7]. However,
the disadvantages of these bio-ligands, such as high cost, diffi-
cult immobilization, easy leakage, difficulties for the sanitation
treatments and stringent storage conditions, have greatly limited
their applications [8]. Therefore, many pseudobiospecific ligands
with stable chemical structure have been employed as the alterna-
tives, such as single amino acids (e.g. histidine, tryptophan), dyes,
∗ Corresponding author. Tel.: +86 592 6166393; fax: +86 592 61662225.
E-mail address: hquqh@yahoo.com.cn (H. Qian).
On the other hand, the bound IgG should be easily released in
some mild conditions when considering the preservation of anti-
body bioactivity. Therefore, thiophilic ligands emerged for its high
specificity and capacity, excellent regeneration and low toxicity
[13].
Immunomagnetic separation based on paramagnetic beads
has been extensively applied in immunology, biotechnology and
biomedicine [14–17]. It becomes much selective and efficient in
proteins isolation because it combines the advantages of the micro-
spheres technology and magnetic separation technology [18–20].
But, the preparation of the efficient magnetic absorbent with more
thiophilic ligand becomes difficult using the conventional methods.
Therefore, the adsorption capacity and isolation efficiency were not
satisfied. In addition, the related mechanisms of selective adsorp-
tion are not clear.
In this study, the ligand of 2-mercapto-1-methylimidazole was
selected to modify the magnetic polymer beads for its ability to
capture IgG in an independent salt manner. More ligand could be
grafted on the magnetic beads depending on the improved sur-
face modification resulting in a higher adsorption capacity of IgG.
Furthermore, the recognition region of this specific interaction
between IgG and these thiophilic magnetic beads was analyzed.
The essential adsorption manner was analyzed by adsorption
isotherms. Then, the potentials of this technology in the large-scale
antibody purification were also investigated.

0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2013.02.037
 Z. Lin et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 72–79
2. Experimental
2.1. Materials
Divinylsulfone (DVS), 2-mercapto-1-methylimidazole and hex-
adecane (HD, 99%) were purchased from Alfa. Vinyl acetate (VAc)
and divinylbenzene (DVB) were distilled under reduced pressure
to remove the inhibitor and stored at 4 ◦ C prior to use. 2,2-Azobis-
(isobutyronitrile) (AIBN) was recrystallized and used as an initiator.
Poly(vinylalcohol), ferric chloride hexahydrate, ferrous chloride
tetrahydrate, ammonium hydroxide (NH3 ·H2 O, 25%, w/v), Sodium
Hydride (NaH), tetrahydrofuran (THF), citric acid and oleic acid (OA)
were all used as received.
2.2. Preparation of thiophilic magnetic adsorbents
The magnetic beads were synthesized by microsuspension poly-
merization [21]. The brief improved surface modification was as
below: The resulted magnetic beads were carefully dispersed into
10 mL THF. Then, NaH and DVS were added with continuous
stirring. This process completed after 2 h at room temperature
and the magnetic beads were thoroughly washed with DI water
to remove unreacted DVS. Then, these magnetic beads and 2-
mercapto-1-methylimidazole were coupled together by Michael
addition reaction in NaOH aqueous solution (pH 13). The grafting
reaction proceeded for 12 h at 80 ◦ C.
2.3. Isolation of IgG from human plasma
The adsorption studies were carried out in batch-wise mode
[22,23]. The experimental steps were as follows: the thiophilic
magnetic adsorbents (0.2 g) were dispersed in 10 mL phosphate
buffered saline at room temperature and incubated for 10 min.
Then, some fresh human plasma (0.2 mL) was added and mixed
on a shaking table at 25 ◦ C. But, in the measurement of adsorption
isotherm, a series of plasma concentration were employed. After
the adsorption, the magnetic beads were collected by a handhold
NdFeB magnet. Then, the magnetic beads were carefully washed
to remove the physically adsorbed proteins. Finally, the bound
IgG was released by aqueous solution of salt. Five kinds of salt
were employed to recover the bound antibody, including K2 SO4 ,
(NH4 )2 SO4 , Na2 SO4 , NaCl and KCl.
The human plasma was obtained from the local blood center
of Quanzhou city (Fujian, China). The amounts of albumin and
IgG in the plasma were 41.4 mg/ml and 15.97 mg/ml. It should be
noted that large amounts of protective agent and anticoagulant
were added into the plasma during its treatment process in blood
bank.
2.4. Enzyme digestion of IgG by papain
Preparation of F(ab’)2 and Fc’ fragments: when IgG was digested
in acidic conditions, the pH was adjusted to 4 with HCl solution.
Then activated papain were added with the ratio (w/w) of 1: 50.
After 12 h, the digestion was halted by raising the pH value to 7.0
[24].
Preparation of Fab and Fc fragments: the antibodies and papain
were firstly equilibrated with the buffer containing 20 mM sodium
phosphate, 10 mM EDTA and 20 mM Lcysteine (pH 7.0). Then, the
IgG solution and the activated papain were mixed together with the
ratio (w/w) of 1: 100 [25]. After 2.5 h, the reaction was terminated
by iodoacetamide solution (2 mmol L−1 ).
The responding experiments were all carried out at 37 ◦ C and
the products were fully dialyzed in the buffer with 25 mM Tris and
50 mM sodium chloride (pH 7.5).
73
2.5. Characterization
The composition of each sample was calculated according to
the peak area of its chromatogram depending upon the calibration
curves of IgG and albumin. The purity of desorbed proteins was
assayed by SDS-PAGE under reducing conditions using 12% sepa-
rating gel and 5% stacking gel. Then, electrophoresis was run for
4 hours at 100 V. The gel was stained for 30 min with Coomassie
Brilliant Blue G-250 (0.25% in acetic acid–methanol–water
(10:5:85, v/v/v)) with gentle shaking. Destaining was performed
in ethanol–acetic acid–water (25:10:65, v/v/v).
The amount of antibody was determined by solid phase
enzyme-linked immunosorbent kits (ELISA), which was
purchased from Bethyl Laboratories, Inc. The experimen-
tal steps were all followed with its operating instruction
(http://www.bethyl.com/product/pdf/E80-104.pdf). The amount
of total protein was determined by enhanced BCA protein assay kit,
which was purchased from Beyotime Institute of Biotechnology.
The responding experiments were performed according its manual
(http://www.beyotime.com/p0010s.htm).
The infrared spectrometry (FTIR) measurement was performed
with a Nicolet NexusTM FTIR spectrometer at a resolution of
2 cm−1 . The surface morphology of the magnetic microspheres
was observed by scanning electron microscopy (SEM, JSM-6700F,
Japan). Ligand concentration of the thiophilic magnetic adsorbents
was tested by an elemental analyser (Vario ELIII Elementar). The
content of Fe3+ was detected on inductively coupled plasma OES
spectrometer (Ultima2, Jobin Yvon). Magnetite content of the dried
samples was measured by thermogravimetric analysis (TGA, Model
TGA2050, TA Instruments). Zeta potential measurements were
carried out on the Zetasizer Nano ZS system from Malvern (Worces-
tershire, UK). Magnetization measurements were performed at
room temperature in magnetic fields up to 100 kOe using a vibrat-
ing sample magnetometer (VSM, Mpms XL-7, Quantum Design).
The specific surface area of these magnetic beads was also charac-
terized by using specific surface area analyzer (AutoChem II 2920).
All samples of corresponding supernatants were analyzed by a
HPLC analyzer (Agilent 1100) with a gel column (protein KW-803,
Shodex). An aqueous solution (pH 7.0) containing 50 mM sodium
phosphate was selected as the mobile phase. Chromatographic sep-
arations were monitored at 280 nm. The flow rate was 0.7 ml/min.
Components in human plasma were assigned according to standard
chromatograms provided by Shodex.
3. Results and discussion
3.1. Improved surface modification
A brief comparison between the thiophilic ligands and other
pesudobiospecific ligands was listed in Table 1 [26–32]. The advan-
tages of thiophilic ligands over other ligands mainly focused on
its good specificity, chemical stability and low cost. Especially, the
recovery conditions of bound protein were much mild when some
heterocyclic thiophilic ligands were employed. It meant that the
bioactivity of IgG could be preserved as much as possible.
In the Table 1, it was found that the adsorption capacity of
some ligands was much high, for example, Protein A, l-histidine,
IDA/Cu2+ , N-methacryloly-l-histidine methyl ester and Ligand
22/8(artificial SpA). But, the synthesis of these ligands was rather
complex resulting in their high cost. Although, some thiophilic lig-
ands have been employed in our previous work, the adsorption
capacity of the responding magnetic adsorbents was not much high.
Thus, some improvements were performed in this study to enhance
the adsorption capacity of these magnetic beads.
The reasons for choosing 2-mercapto-1-methylimidazole as the
functional groups are mainly based upon two aspects. The first
74 Z. Lin et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 72–79

Table 1
Brief comparison of adsorption capacities, purity and isolation conditions of different ligands.

Pseudobiospecific ligand Target anitobody Capacity mg/g Purity (%) Isolation conditions reference

Protein A Mab (mouse IgG2b) 92 98 0.1 M citric acid, pH 3.0 26

Histidine Human IgG 11.3 0.2 mol/L glycine, pH 2.3 27
l-Histidine Human IgG 44.8 2.0 M NaCl 28
IDA/Cu2+ Human IgG 171 2.0 M NaCl 8
N-methacryloly-L-histidine methyl ester Human IgG 262 1 M NaCl in 25 mM acetate buffer, pH 4.0 29
Ligand 22/8(artificial SpA) IgG 52 97–99 0.1 M glycine–HCl buffer, pH2.3–2.9 30
Thiophilic (MECH-gel) Human IgG 5.7 10 mM NaOH 31
2-Mercapto-4-mythyl-pyrimidine Human IgG 4.75 92.7 1 M NaCl 22
2-Mercapto-benzothiazole Human IgG 7.62 98.4 0.8 M KCl 32
2-Mercaptonicotinic acid Human IgG 7.3 94 0.8 M NaCl 21
2-Mercapto-1-methylimidazol Human IgG 20.7 98 0.2 M KCl This sutdy

is its heterocyclic structure of imidazole ring, which was much
important for the ligand to selectively capture antibodies (IgG) in
a salt-independent manner. The other feature is its good solubility
in the responding solvents.
The whole processes of this surface modification were demon-
strated in Scheme 1. By the conventional methods, the thiophilic
ligands were usually coupled with hydroxyl groups through DVS
in alkaline aqueous solution. But, the hydrolysis of DVS could not
be avoided resulting in a low grafting amount of thiophilic lig-
ands on the magnetic beads. In this work, the activation process
of these magnetic beads by DVS occurred in tetrahydrofuran. NaH
was used to adjust the alkalinity of tetrahydrofuran solution. Thus,
the hydrolysis of DVS could be effectively avoided.
The processes of this surface modification were recorded by
FTIR in Fig. 1. It was found that a strong adsorption peak appeared
at 585 cm−1 , which corresponded to the characteristic stretch-
ing vibration band of the magnetite (Fe3 O4 ). The sharp decreases
of peak strength were observed in the spectrum Fig. 1A (b) at
1740 cm−1 (C O) and 1250 cm−1 (C O C) in comparison with the
spectrum in Fig. 1A (a). It indicated that plenty of hydroxyl groups
were generated on the surface of these beads after the alcoholy-
sis of ester groups. Afterwards, the resulting magnetic beads were
activated by DVS in THF. The strong peaks at 1127 cm−1 , 1295 cm−1
and 1319 cm−1 appeared in Fig. 1A (c), which were assigned to the
symmetric stretching vibration and asymmetric vibration of sul-
phone groups. Finally, these activated beads were modified with
the thiophilic ligands (2-mercapto-1-methylimidazole) by Michael
addition reaction. The characteristics peak of C N on imidazole
heterocyclic ring presented at 1572 cm−1 in Fig. 1A (d).
The amount of grafted thiophilic ligand was more than ten
times than that using the conventional methods due to this opti-
mization of surface modification [23,33,34]. From the nitrogen
content (4.68%) of these thiophilic magnetic beads, the amount
of thiophilic ligand was calculated to be 1.67 mmol/g, which is
much higher than that (0.110 mmol/g) of the beads modified with
2-mercapto-4-mythyl-pyrimidine [22]. Therefore, the saturated
adsorption capacity of these magnetic beads reached 20.7 mg/g,
which is four times more than that of the previous magnetic thio-
philic beads.
The morphology was observed in Fig. 1B that the average par-
ticle size was about 1.49 ␮m. But, its size distribution is relatively
broad ranging from 0.37 ␮m to 3.94 ␮m, which is determined by
the nature of the suspension polymerization. The saturation mag-
netization of these magnetic beads reached 12.6 emu•g−1 . The
specific surface area of these magnetic beads was also charac-
terized by using specific surface area analyzer. The BET surface
area and Langmuir surface area were 16.52 and 27.32 m2 g−1 ,
respectively.

Scheme 1. Process of the surfcace modification of these magnetic beads.

 Z. Lin et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 72–79 75

Fig. 1. FTIR spectra and SEM photo of magnetic beads. A: FTIR spectra of the preparation process. a: PVAc-DVB magnetic beads; b: PVA-DVB magnetic beads after alcoholysis;
c: activated magnetic beads by DVS; d: thiophilic magnetic beads modified with 2-mercapto-1-methylimidazole. B: SEM photo of the magnetic beads.

3.2. Specific isolation of antibody by these thiophilic magnetic SDS-PAGE. However, the purity of the same sample reached 98%
beads determined by ELISA kits, which meant the isolated IgG was rather
bioactive.
The specificity of these thiophilic beads toward IgG was ana- Two adsorption isotherm models (Langmuir and Freundlich
lyzed with the size-exclusion chromatography. In Fig. 2A, two isotherms) were employed to describe the adsorption behavior in
noticeable peaks were labeled as IgG (12.3 min) and albumin Fig. 3. The Langmuir and Freundlich isotherms were expressed as
(13.1 min) according to the standard chromatogram, which was the following equations.
provided by Shodex Co. It was clear that the IgG content decreased
ce ce 1 1
greatly after the human plasma was absorbed by these thiophilic = + KE = × cL (1)
qe qmax bqmax b
magnetic beads. After the recovery, only one peak at 12.3 min
appeared, which meant that only IgG was extracted by these mag- 1
log qe = log KF + log ce (2)
netic beads and the product was free of other proteins. Meanwhile, n
the results were also confirmed by SDS-PAGE in Fig. 2B. Only ce is the equilibrium concentration of IgG in solution; cL is the con-
two major bands presented in electrophoretogram, which were centration of thiophilic ligand on the magnetic surface; qe is the
attributed to the heavy chain and light chain of IgG. The purity amount of IgG adsorbed on the beads at the equilibrium; qmax is the
of isolated IgG reached 95.8% determined by the gray intensity of maximum adsorption capacity of the thiophilic magnetic beads;

Fig. 2. Process of isolation of IgG from human serum with thiophilic magnetic beads. A: Recorded by SEC. a:dulated human plasma (×35), b: adsorption supernatant; c:
the first washing supernatant; d: the second washing supernatant; d: the third washing supernatant; e: the forth washing supernatant; f: the released IgG. B: Recorded by
SDS-PAGE. Lane 1: marker; Lane 2: human serum sample; Lane 3: adsorption supernatant; Lane 4: the sample before adsorption; Lane 5: the first washing supernatant; Lane
6: the third washing supernatant; Lane 7: extracted proteins by Na2 SO4 aqueous solution).
76 Z. Lin et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 72–79
Fig. 3. Fitting curves of IgG on the thiophilic magnetic beads by using Langmuir equation and Freundlich equation. The standard deviation of these data (SD) is below 4%.
b is the Langmuir isotherm constant and KD is the dissociation
constant; KF and n are the Freundlich constants characteristic of
the system, representing the adsorption capacity and intensity of
uptake, respectively.
It was found that the Langmuir model was better to describe
the adsorption behavior because the correlation coefficient (R2 )
of Langmuir regression equation was higher than that of Fre-
undlich model. It indicated only a monolayer of antibody was
adsorbed on these thiophilic magnetic beads. Certainly, the mono-
layer chemical adsorption could effectively guarantee the high
specificity of thiophilic ligands toward antibodies. The apparent
dissociation constant KD is a dimension of the affinity adsorption
between proteins and adsorbent. In our experiment, the KD reached
2.95 × 10−7 mol L−1 , which suggested a moderate affinity between
the adsorbent and the adsorbate.
3.3. Mechanism of the selective adsorption toward IgG
The essential reason for the high specificity of these thiophilic
magnetic beads at low salt concentration is mainly attributed to
the heterocyclic ring structure of 2-mercapto-1-methylimidazole
because the ability of electron donation of sulfur atom on
thioether was greatly strengthened by the conjugation effect
between the sulfur atom and imidazole ring [21,33]. Although
a series of thiophilic ligands, such as 2-mercaptonicotinic acid,
2-mercapto-benzothiazole, 2-mercapto-ethanol and 2-mercapto-
4-mythyl-pyrimidine have been studied in our group, the exact
region for this selective recognition between the thiophilic lig-
ands and IgG was not revealed [21,22,32]. Thus, the technology of
enzyme digestion was employed to analyze this essential mecha-
nism.
We employed the classic experiments to cut IgG into F(ab )2 or
Fab fragments by papain enzymolysis in different pH conditions
[24,25,34]. In acidic condition (pH 4.0), IgG was hydrolyzed into
F(ab )2 and Fc fragments. While in weak alkaline condition (pH
7.0), it was cut into Fab and Fc fragments. Then, these fragments
were absorbed by the thiophilic magnetic beads and the adsorp-
tion supernatant was detect be SEC. It was found in Fig. 4 the peak
at 15.46 min decreased greatly, which indicated these thiophilic
magnetic beads could specifically capture F(ab )2 fragments. When
the Fab and Fc fragments were adsorbed, these thiophilic mag-
netic beads could selectively absorbed Fab fragments. It suggested
that the recognition region on IgG for this thiophilic ligand mainly
focused on the F(ab )2 or Fab fragments, which is accord with the
results of Kumpalume’s thiophilic sorbents [24].
It is well known that ELISA kit could effectively capture anti-
body based on the specific interaction between antibody and
antigen. Thus, ELISA kits were also used to detect these fragments.
After the adsorption by these magnetic beads, no antibody was
found in the residual supernatant. But, large amount of antibody
could be detected by ELISA after the recovery of the bound pro-
teins. It indicated that F(ab )2 or Fab fragments were captured by
these magnetic beads because the recognized regions between the
antibody and antigen are mainly focused on the F(ab )2 or Fab frag-
ments.
3.4. Effects of pH value on the adsorption
Proteins adsorption is usually influenced by the environmental
pH values. In order to increase the specificity toward IgG, a series
of adsorption solution with different pH values were managed to
optimize the adsorption conditions. The results are shown in Fig. 5.
The adsorption capacity for albumin decreased down to zero as the
pH value was increased from 3 to 4. Meanwhile, the adsorption
capacity for IgG was unchanged. Because isoelectric point of albu-
min is close to 4.6, the albumin always keeps the electric neutrality
near this pH value. Therefore, the electrostatic interaction between
albumin and these thiophilic beads decreased greatly. It is reason-
able that no albumin was detected after the bound protein was
recovered when the responding adsorption was carried out at pH
4. Therefore, the strong specificity of these magnetic beads toward
IgG was expressed in some weak acidic conditions.
 Z. Lin et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 72–79 77

Fig. 4. Specific recognition between IgG and the thiophilic magnetic beads.

The thiophilic ligands are known to have mixed interaction
(hydrophobic interaction and electron donor-acceptor (EDA) inter-
action) with antibody. For the hydropholic interaction, the enthalpy
change is always small and the entropy change is the main driv-
ing force to determine the Gibbs free energy. The temperature
should have a great effect on G [35,36]. In order to distinguish
EDA interaction from hydrophobic interaction, the adsorption was
performed at 0 ◦ C and 37 ◦ C, respectively. The thermodynamic
parameters were listed in supplementary file. The negative val-
ues of G◦ for each temperature indicated that the adsorption was
a favorable process. There was no significant change of the Gibbs
free energy and the adsorption capacity for each temperature. Thus,
the driving force of the thiophilic ligands between IgG was mainly
driven by enthalpy change, which meant the adsorption was mainly
controlled by EDA effect.
3.5. Mild conditions for the recovery
A good isolation process must be a perfect combination of the
adsorption and the recovery. But, the specific adsorption and easy
desorption are conflicting in some extent. In order to achieve a
high specificity toward antibody, a highly selective interaction
between IgG and sorbent is required. However, in view of the
preservation of antibody bioactivity, it is better to release the bound
proteins in some mild conditions. It was found in the Table 1
that many conditions in some conventional methods were rather
harsh, which were not favorable to preserve the bioactivity of anti-
body.
A series of salts (NaCl, KCl, Na2 SO4 , (NH4 )2 SO4 , K2 SO4 and
Na2 CO3 ) were employed to release the bound antibodies. In Fig. 6,
it is clear that the bound proteins could be smoothly recovered

Fig. 5. Dependence of adsorption with the pH value of adsorption buffer. (A) The adsorption behavior was effected by the pH value of adsorption buffer. The standard
deviation of these data (SD) is below 7%. (B) The corresponding isolated IgG was changed with the pH value of adsorption buffer. SD < 5%) (error bars: ±SD).
78 Z. Lin et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 72–79
Fig. 6. Effect of types and concentration of different salts in elution on the recovery
of bound IgG from magnetic beads (error bars: ±SD) The standard deviation of these
data is as follows: SD(K2 SO4 ) < 5%, SD(KCl) < 4%, SD((NH4 )2 SO4 ) < 7%, SD(NaCl) < 5%,
SD(Na2 SO4 ) < 5%).
even in a low concentration (0.1 mol L−1 ). It meant this specific
interaction between IgG and thiophilic magnetic beads could be
easily broken by the disrupting effects of salt ions. The (NH4 )2 SO4
aqueous solution became more effective to recover absorbed IgG
at a low concentration (0.1 mol L−1 ). But, the amine groups are
not much friendly to antibody bioactivity. Thus, the KCl solution
(0.2 mol L−1 ) was selected as the elution buffer to release the bound
antibodies.
Thus, both the adsorption and desorption could be performed
in such mild conditions that the bioactivity of IgG was preserved
as much as possible. The ELISA assays was used to measure the
bioactivity due to the specific affinity between the antibody and
antigen. The bioactivity of isolated IgG reached 98%.
Fig. 7. Successive isolation of IgG by the thiophilic magnetic beads and its repeated use. (4 mL human plasma was isolated by 1.7 g magnetic beads. Adsorption buffer: 25 mM
PBS pH 4.5; elution buffer: 0.2 M KCl aqueous solution; a: dulated human plasma (×35); b: 1st adsorption supernatant; c: 2nd adsorption supernatant; d: 3rd adsorption
supernatant; e: 4th adsorption supernatant; f: 1st isolated IgG solution; g: 2nd isolated IgG solution; h: 3rd isolated IgG solution; i: 4th isolated IgG solution. The standard
deviation of these date is below 6%. (error bars: ±SD).
3.6. Repeated use of these magnetic absorbent
A batch separation was carried out to examine the potential of
this technology in large-scale purification and the processes were
shown in Fig. 7. The successive isolation was employed to ade-
quately isolate IgG from human plasma to decrease the residual
concentration of IgG. It meant that the plasma was repeatedly iso-
lated by the magnetic beads until IgG was completely extracted. It
was found in Fig. 7 that the peak of IgG disappeared after four runs
of successive isolation.
It took 1.5 h in our laboratory to extract 36.4 mg IgG from 4 mL
human plasma. The isolation efficiency reached 76.4%, which is
higher than that by using the customary thiophilic T-gel [15].
Another important advantage of these magnetic beads was worth
noting that these magnetic beads could be directly reused after
the bound proteins were recovered in salt aqueous solution. It
means that these magnetic beads could be repeatedly used without
any regeneration treatment. It was found in Fig. 7 that the isola-
tion efficiency decreased little after the 16 adsorption-desorption
cycles. The related errors of adsorption capacity were smaller than
1%.
The evident advantages of this technology, such as high purity
and bioactivity, easy operation, mild conditions and excellent recy-
cling utilization, were fully exhibited. Compare with the Protein A
adsorbent, these thiophilic magnetic beads become more conve-
nient and cleaner. It becomes so stable in the alkaline or the acidic
conditions that the leakage of thiophilic ligand was effectively
avoided because of the covalent linkage between the thiophilic lig-
and and magnetic beads. Thus, the drastic sanitation treatments
could be directly employed to sterilize and disinfect for these mag-
netic beads. However, this treatment could not be applied in the
Protein A adsorbent due to its protein nature. Moreover, the leakage
of magnetic beads was also investigated. No signal of Fe3+ was found
in the isolated antibody when it was measured with the inductively
coupled plasma spectrometer.
These magnetic beads are being applied on the magnetically
stabilized bed. Depending on this technology, protein purification
 Z. Lin et al. / Colloids and Surfaces B: Biointerfaces 108 (2013) 72–79
could be carried out with the continuous process, which becomes
more efficient and stabilized than the batch process [29,37]. There-
fore, this magnetic absorbent just provides a promising method to
purify antibody on large scale.
4. Conclusions
The rapidly increasing applications and requirements for
antibodies have inspired many researches for more efficient purifi-
cation technologies at lower cost. In this research, the improved
surface modification was carried out to prepare some mag-
netic beads modified with more thiophilic ligands. Then, its
adsorption capacity was more than 4 times compared with the
magnetic beads prepared with the conventional methods. The
recovery conditions were so mild that the bioactivity of iso-
lated antibody exceeded 98%. Moreover, the recognition region
of this specific interaction between thiophilic beads and IgG
was revealed that it mainly focused on the F(ab’)2 or Fab frag-
ments of IgG. So, this technology could be further employed to
isolate the F(ab’)2 or Fab fragments of IgG, which were much
useful in the fields of biotechnology, biomedicine and diagnos-
tic reagents. The excellent repeated use of these magnetic beads
was also evaluated by some batch experiments. Thanks to the
broad adaptability of thiophilic ligands, many starting materials,
including whole blood, animal serum, tissue culture medium and
ascites fluid are being fractionated by these thiophilic magnetic
beads.
Acknowledgements
The authors gratefully acknowledge financing from the Program
for New Century Excellent Talents in University of Fujian Province
(07FJRC02) and the Fundamental Research Funds for the Central
Universities (JB-GJ1007, JB-JC1002).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.colsurfb.2013.02.037.
79
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