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ISSN 00036838, Applied Biochemistry and Microbiology, 2014, Vol. 50, No. 6, pp. 683–688. © Pleiades Publishing, Inc., 2014.
Original Russian Text © P.V. Khramtsov, M.S. Bochkova, M.B. Rayev, 2014, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2014, Vol. 50, No. 6, pp. 612–618.
683
INTRODUCTION
At present, the need is growing in laboratory, clini
cal, and biotechnological practices for simplified test
ing methods that make it possible to gain accurate
results of the analysis in a very short time. The princi
ples and methods used in these tests are different:
agglutination of colored particles, immunochro
matography, immunofiltration, and dotblot. In most
cases, these tests are noninstrumental and can be used
even when there is no registration equipment or highly
qualified specialists.
The Laboratory of Ecological Immunology,
UB RAS, developed a number of such test systems. The
diagnostic reagent is based on carbon nanoparticles
covalently conjugated with affine compounds. Carbon
particles perform the function of a colored label, and
affine compounds provide specificity of conjugate
interaction with the analyte. Carbon diagnosticums
were used to create qualitative and semiquantitative
rapid analysis systems of antibodies to the heatstable
toxin of
Yersinia pseudotuberculosis
, tetanus toxoid,
α
fetoprotein (AFP) and human chorionic gonadot
ropin (HCG) [1–3]. The Gprotein of Streptococcus,
streptavidin, and monoclonal antibodies to gestation
hormones were used as the affinity component of the
diagnosticums. The sensitivity of these systems lies in
a range from 1.5 to 160 ng/mL [2, 4]. The detection
sensitivity of IgG with carbonGprotein diagnosti
cum was 80 ng/mL [5].
The potential applications of carbon conjugates are
not limited by clinical diagnosis.
Affinity chromatography is an effective, widely
used tool for obtaining immunoglobulin preparations
with a high purification degree, which are used for var
ious purposes: therapeutic, diagnostic, and research.
For affinity chromatography, sorbents and protocols,
both commercial and those developed independently
with account of special requirements and features, are
used.
The sources of antibodies are also diverse—animal
hyperimmune serum, ascite fluid, and culture
medium. The individual features of organisms—pro
ducers of antibodies—define the need for adjusting
the parameters of the chromatographic system: the
saturation rate of the sorbent with antiligands, the
sample volume applied, the volumes of elution buffers,
etc. When using commercial kits and standard tech
niques, it is possible to calculate mathematically
parameters based on known data (capacity of the sor
bent, examples of elution profile) from suppliers and
protocols with sufficient accuracy.
When using commercial sorbents for solutions of
nonstandard tasks or during independent design of a
chromatographic system (for example, for the isola
tion of antibodies to rare or synthetic antigens), it is
difficult to accurately predict the properties of the
affinity sorbent and its behavior in the experiment. A
more attractive solution is selfassessment of the
dynamics of a chromatographic process using detec
tion systems of immunoglobulins in the eluate.
Application of Diagnosticum Based on Functionalized
Carbon Nanoparticles for Monitoring
of Immunoglobulins Affinity Purification
P. V. Khramtsov, M. S. Bochkova, and M. B. Rayev
Institute of Ecology and Genetics of Microorganisms, Russian Academy of Sciences, Perm, 614081 Russia
email: khramtsovpavel@yandex.ru
Received November 16, 2013
Abstract
—
A diagnostic reagent based on carbon nanoparticles covalently functionalized with streptococcus
G protein was applied to construct a system for monitoring and optimization of the technology of affinity
purification of rabbit polyclonal antibodies to alphafetoprotein. The developed system allows a shorttime
(45 min) semiquantitative assessment of the immunoglobulins (IgG) of most higher animals and human
beings in blood serum samples and eluate. IgG detection sensitivity using the carbonGprotein diagnosti
cum was 80 ng/mL. Approaches to stabilizing the components of the analysis system, which ensures the pres
ervation of their functional properties during long storage, were developed. The storage life of the diagnosti
cum was more than 20 years, and that of immunosorbents was more than year and a half. A technique of long
term immunosorbent storage was developed. Application of the developed testsystem do not require regis
tration equipment.
DOI:
10.1134/S0003683814060064
684
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 50 No. 6 2014
KHRAMTSOV et al.
These systems include analytical methods to quan
titatively or semiquantitatively evaluate the content of
immunoglobulins of the required specificity in the ini
tial material and in eluate samples. This information
makes it possible to adjust the chromatography proto
col (change of the elution rate, the original sample vol
ume, the volume of the sorbent, etc.) and facilitates
planning subsequent experiments.
Methods for the detection of antibodies in chro
matographic samples are numerous, and instrumental
approaches based on the use of sensing devices are
widespread. These include immunoenzyme assay
(IEA) [6–9], nephelometry [10], and biosensors [9].
In most cases, IEA is used because it is a routine labo
ratory analysis method, and equipment for the method
is the most available. A noninstrumental method
based on the immunodiffusion reaction in agar gel is a
traditional method [6, 11–14].
The designed system was tested during the isolation
of rabbit polyclonal antibodies to human AFP.
The aim of this study was to develop noninstru
mental solidphase dotimmunoassay system for the
control of chromatographic immunoglobulin separa
tion using carbon nanoparticles functionalized with
streptococcus Gprotein.
MATERIALS AND METHODS
Materials and Equipment.
We used a 1.5
×
12 cm
chromatographic column (BioRad, United States), a
peristaltic pump P1, flow UV spectrometer 2138 Uvi
cord S (Pharmacia LKB, Sweden), a Soniprep 150
Plus ultrasonic disintegrator (MSE, Great Britain), a
Zetasizer NanoZS analyzer for measuring particle size
(Malvern, England), a NOVAsem 600 scanning elec
tron microscope (FEI, United States), an ultrafiltra
tion cell Amicon Stirred Cell 8050 (Millipore, Ger
many), and a 5804 R centrifuge (Eppendorf, Ger
many).
Reagents and Buffers.
The reagents were cyanogen
bromideactivated Sepharose 4B, Sepharose CL6B
(Pharmacia Fine Chemicals, Sweden), AFP (Bialexa,
Russia), white polystyrene plates for serial dilutions
Linbro (United States), agar Difco (United States),
nitrocellulose membrane with a pore size of 0.45
μ
m
(BioRad, United States), bovine serum albumin
(BSA), human IgG, streptococcus Gprotein (Sigma,
United States), glutaraldehyde (AppliChem, Ger
many), toluene (Ekros, Russia), glycerol and tween20
(Panreac, Spain).
The solutions for immunoassay and synthesis of the
diagnosticum were 0.02 M carbonatebicarbonate
buffer, pH 9.6 (CBB); 0.15 M NaCl buffered with
0.015 M Naphosphates, pH 7.25 (NaClBP); and
NaClBP containing 0.05% tween20 (NaClBPT).
The solutions for chromatography were 0.15 M
NaCl; 0.1 M glycineHCl pH 2.6; 0.5 and 0.15 M NaCl
buffered with 0.03 M Naphosphates, pH 7.25 (0.15 M
and 0.5 M PBS). All solutions were prepared using
deionized water.
Synthesis of Diagnosticum.
Carbonblack was used
as a carbon source, which was obtained through con
densation of a burning flame of toluene. It was sub
jected to multistage washing in organic solvents and
filtration to remove the underoxidized products. The
obtained amorphous carbon was a mat black powder,
insoluble in available solvents. Scanning electron
microscopy established that carbon particles were
spherical or close to spherical in shape, and the linear
dimensions were in the range of 40–70 nm.
At the next step of synthesis, 1.0 g of amorphous
ca rbo n was s usp end ed in 19 mL o f 2% BSA in N aClBP
for 1 day under vigorous stirring on a magnetic stirrer.
The carbon concentration as calculated to dry sub
stance was 5%. The resulting suspension was subjected
to an ultrasonic disintegration at 22 kHz. Total sonica
tion time was 1 h. In order to remove any remaining
large particles the sonicated suspension was centri
fuged for 5 min at 1620 g [15].
The supernatant was incubated for 40 min at room
temperature with an equal volume of 25% glutaralde
hyde solution. The mixture was then centrifuged for
5 min at 1620 g and chromatography was carried out
using a Sepharose CL6B containing column to
remove unbound BSA and glutaraldehyde. The frac
tions from the void volume fraction were combined
and concentrated to the initial volume of the carbon
suspension in an ultrafiltration cell [15].
The glutaraldehydeactivated suspension, with a
volume of 4.5 mL, was incubated with 0.5 mL of
Gprotein with a concentration of 10 mg/mL for
100 min with gentle stirring. The resulting suspension
was centrifuged for 5 min at 1620 g. The unbound
Gprotein was removed by gelchromatography on a
Sepharose CL6B column. The void volume fractions
containing the conjugate were combined, and BSA
and glycerol were added to a final concentration of
1 and 20%, respectively [15].
The particle size of the conjugate was determined
by measuring the reverse dynamic light scattering at an
angle of 173°C with the Malvern Zetasizer NanoZS
analyzer (Great Britain). For measurement the conju
gate was diluted in water to a concentration of carbon
particles of 0.01%. The diameter of 90% of the parti
cles was in the range of 70–200 nm, and particles with
a diameter of 90–100 nm dominated.
The synthesized conjugates were stored at 2–8°C.
This temperature regime ensured the stability of the
functional properties of the diagnosticum for more
than 20 years [16].
Preparation of Hyperimmune Rabbit Serum and
Determination of Titers of AFP Antibodies.
The immu
nization of rabbits was carried out according to a stan
dard method [17] using the Freund’s Complete Adju
vant (CFA) and aluminum hydroxide.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 50 No. 6 2014
APPLICATION OF DIAGNOSTICUM BASED ON FUNCTIONALIZED CARBON 685
The process was carried out according to the fol
lowing scheme:
1st day—first immunization intramuscularly with
200
μ
g of AFP with three injections of CFA;
29th day—repeated immunization intramuscu
larly with 200
μ
g of AFP with three injections with alu
minum hydroxide;
53rd day—the last blood sampling.
Before application to the column, all hyperim
mune rabbit blood sera were combined. The titer of
antibodies in the combined sera was determined by
immunodiffusion in agar gel according to the
Ouchterlony method [18]. The results were evaluated
by the presence of precipitation lines 72 h after initia
tion of the analysis.
Chromatographic Separation of Polyclonal Antibod
ies to AFP.
To isolate rabbit polyclonal antibodies to
AFP, an affinity sorbent based on Sepharose 4B was
synthesized by the method of the manufacturer using a
commercial preparation of AFP. The protein in the elu
ate was recorded using a flow UV spectrophotometer.
The sorbent in the column, having a volume of
11 mL, was washed with 100 mL of 0.5 M PBS.
Twenty mL of combined whole rabbit hyperimmune
serum was applied at a rate of 1 mL/min with three
fold recirculation. Upon completion of each of the
3 cycles of application, a sample with a volume of
300
μ
L was collected from the eluate for the analysis of
the target antibody content. The sorbent was washed
consistently with 0.15 M PBS and 0.15 M NaCl, and
the antibodies were eluted in 0.1 M glycineHCl buffer
with a pH of 2.6.
Semiquantitative Determination of Antibodies to
AFP in Eluate by SolidPhase DotImmunoassay.
Five
μ
L dots
of AFP at a concentration of 0.1 mg/mL
in CBB was placed in the bottom of polystyrene plate
wells. Human IgG and BSA were adsorbed as a posi
tive and negative control at concentrations of 0.05 and
0.1 mg/mL, respectively. After 30 min of incubation in
a wet chamber at 37°C, the plates were washed by fill
ing the wells three times with NaClBPT and immedi
ately removing it. Sensitized polystyrene plates are an
immunosorbent suitable for further detection of anti
bodies in the eluate. Sensitized plates can be stored for
long time at room temperature pretreated with 30%
aqueous sucrose solution. Under these conditions they
retain their propertis for over one and a half years [19].
Three hundred
μ
L of twofold serial dilutions of
eluate samples in NaClBPT, ranging from 1 : 2 dilution
and ending with 1 : 524 000 dilution, were added in the
sensitized plate wells. After incubation for 30 min, the
wells were washed three times with NaClBPT, and
150
μ
L of carbonGprotein conjugate diluted to
working titer was added. After 15 min of incubation
with gentle stirring, the plates were washed and dried.
The optimum period of incubation of the sample and
conjugate was selected beforehand. The analysis
results were visually evaluated according to the last vis
ible dot in serial dilutions.
Combined hyperimmune rabbit serum was simi
larly analyzed prior to its application to the immun
osorbent.
RESULTS AND DISCUSSION
Analysis of Antibodies to AFP in Combined Hyper
immune Rabbit Blood Serum.
Two methods were used
to determine the titer of polyclonal AFP antibodies in
combined rabbit hyperimmune sera. The first method
was the dotimmunoassay developed for testing serum
on AFP preparationsensitized polystyrene plate using
carbon diagnosticum. The second method was agar gel
immunodiffusion by Ouchterlony, a traditional nonin
strumental method of semiquantitative serological
analysis. In the experiment, the sensitivity and speci
ficity of the methods were compared.
In the Ouchterlony immunodiffusion reaction, the
titer of antibodies to AFP in the studied serum was 1/8.
The absence of precipitation lines opposite the well
with whole serum of intact rabbit was indicative of the
absence of a nonspecific interaction of its components
with AFP (Fig. 1).
In the dotimmunoassay testing of blood serum
using carbon diagnosticum on a polystyrene support,
the titer of antibodies to AFP in the sample was 1/8000.
The correctness of the system and its specificity were
confirmed by analysis of control samples (Fig. 2).
Comparing the obtained results, it can be con
cluded that both systems demonstrated equally high
specificity, but the sensitivity of the analysis system
constructed on the basis of functionalized carbon par
ticles was 3 orders of magnitude higher as compared to
the immunodiffusion reaction.
Control of Chromatographic Purification of Poly
clonal Rabbit Antibodies to AFP.
Polyclonal antibodies
1 : 1 1 : 2
K
1 : 8
1 : 16
1 : 4
Fig. 1
Semiquantitative determination of antibody titer to
AFP in hyperimmune rabbit serum by Ouchterlony immu
nodiffusion method. AFP at a concentration of 125
µ
g/mL
was applied into the central well. The total rabbit hyperim
mune serum in a specified dilution was introduced in wells
at edges; K is the well with the serum of an intact rabbit (neg
ative control).
686
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 50 No. 6 2014
KHRAMTSOV et al.
to AFP were isolated from rabbit hyperimmune serum
using affinity chromatography on Sepharose 4B con
jugated with AFP. Control samples of the eluate, col
lected after each application cycle, were subjected to
testing in the constructed dotimmunoassay system
for determination of the dynamics of the target anti
body titer changes.
The work was broken down into a series of experi
ments on the chromatographic separation of AFP
antibodies from a certain fraction of combined sera.
In the first series of separation, a serum sample with
a volume of 10 mL was applied to the column. The
analysis of control samples of the eluate after three
recyclings detected no antibodies of the required spec
ificity in any of them. Thus, as early as during the first
cycle of applying the preparation onto the column, all
of the containing target antibodies bound with the sor
bent. On this basis it was concluded that, with an ini
tial sample volume of hyperimmune rabbit serum
equal to 10 mL, there is no need for it to be reapplied
to the column.
Based on these results, the volume of the prepara
tion applied onto the column was increased twice, i.e.,
to 20 mL. The analysis of control samples demon
strated a steady decline in antibody titer in the eluate
with each subsequent cycle of applying the sample
onto the column (1/16 after first cycle, 1/8 after the
second, and 1/2 after the third). However, after the
first cycle, the titer of antibodies in the eluate was still
two orders lower as compared to the initial sample
(titer 1/8000) (Fig. 3). That is, even at a twofold
increase in the volume of the applied preparation, the
(а)
1:2 1:4 1:8
1:16 1:32 1:64 1:128
1:20001:10001:512
1:256
1:4000 1:8000 1:16000 1:32000
K
AFP
BSA
lgG
(b)
Fig. 2.
Semiquantitative determination of antibody titer to AFP in hyperimmune rabbit serum by solidphase dotimmunoassay
using (a) carbon diagnosticum with Gprotein and (b) scheme of applying antiligands to plate wells. (a) dilutions of the sample
are indicated near the wells; K is the well in which the whole serum of an intact rabbit (negative external control) was applied, and
the arrow marks the last visible dot in a series of dilutions. (b) IgG is the zone of the well in which human IgG adsorbed at a con
centration of 0.05 mg/mL (internal positive control); AFP is the zone in the well in which AFP adsorbed at a concentration of
0.1 mg/mL; BSA is the zone in the well in which BSA adsorbed at a concentration of 0.1 mg/mL.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 50 No. 6 2014
APPLICATION OF DIAGNOSTICUM BASED ON FUNCTIONALIZED CARBON 687
capacity of the affinity sorbent (upon maintaining
other parameters of application unchanged) was found
to be sufficient for adsorption of the vast majority of
target antibodies contained in the initial sample. Fur
ther adsorption of the sample during subsequent cycles
proceeded much slower.
Hence, the constructed system is a convenient tool
for the optimization and monitoring of the chromato
graphic purification of antibodies. In this case, its use
could make it possible to dispense with additional
cycles of application of the preparation as inessential
to obtaining the desired product and to draw conclu
sions about the possibility of increasing the initial sam
ple volume.
Carbon diagnosticum based on Gprotein can sim
ilarly be used successfully to detect antibodies of any
specificity isolated from the majority of higher animals
used in technological processes (goats, rabbits, mice,
guinea pigs, etc.) and humans. Another positive fea
ture of the developed control system is the ability of
the carbon conjugate to retain functional properties
when stored for over 20 years.
The constructed control system based on carbon
diagnosticums is enough efficient and can be used
under certain conditions to monitor chromatographic
process in real time (for example, at large volumes of
sorbent, low elution rates, etc.).The short analysis
time is an additional advantage in retrospective analy
sis, especially when a large number of series of chro
matographic purification of immunoglobulins are per
formed in a short time.
1:2 1:4 1:8
1:16 1:32 1:64 1:128
K
(a)
1:2 1:4 1:8
1:16 1:32 1:64 1:128
K
1:2 1:4 1:8
1:16 1:32 1:64 1:128
K
(b)
(c)
Fig. 3.
Semiquantitative determination of antibody titer to AFP in eluate samples using the constructed solidphase dotimmu
noassay system based on carbon diagnosticum after (a) first, (b) second, and (c) third cycles of applying the preparation onto the
column. Arrows indicate the last visible dot in serial dilutions of the eluate sample; dilutions of the sample are indicated close to
the wells. K is the well wherein the whole serum of an intact rabbit was applied (external negative control).
Scheme
of antiligand application onto the wells is as in Fig. 2b.
688
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 50 No. 6 2014
KHRAMTSOV et al.
ACKNOWLEDGMENTS
This study was supported by a grant for young sci
entists and graduate students of the Ural Branch, Rus
sian Academy of Sciences (No.134NP571).
REFERENCES
1. Rayev, M., Ambrosov, I., and Briko, N., in
Streptococci
and the Host. Advances in Experimental Medicine and
Biology
, Thea Horaud at al., Eds., New York: Plenum
Press, 1997, vol. 418, pp. 327–329.
2. Rayev, M.B.,
Nanobiotekhnologii v neinstrumental’noi
immunoanalitike
(Nanobiotechnology in NonInstru
mental Immunoanalytics), Demakov, V.A., Ed., Yekat
erinburg: UrO RAN, 2012.
3. Rayev, M.B., Timchenko, N.F., Bochkova, M.S.,
Nedashkovskaya, E.P., and Andryukov, B.G.,
Dokl.
Biochem. Biophys.
, 2013, vol. 451, pp. 221–224.
4. Rayev, M.B., Bochkova, M.S., Timganova, V.P.,
Khramtsov, P.V., and Tyulenev, A.V.,
Vestn. Ural. Med.
Akad. Nauki
, 2011, vol. 38, no. 4/1, pp. 146–147.
5. Bochkova, M.S., Timganova, V.P., and Rayev, M.B.,
Dokl. Biochem. Biophys.
, 2013, vol. 449, pp. 63–65.
6. Staak, C., Salchow, F., Clausen, P.H., and Luge, E.,
J. Immunol. Meth.
, 1996, vol. 194, no. 2, pp. 141–146.
7. Proll, G., Kumpf, M., Mehlmann, M., Tschmelak, J.,
Griffith, H., Abuknesha, R., and Gauglitz, G.,
J. Immunol. Meth.
, 2004, vol. 292, nos. 1–2, pp. 35–42.
8. Griffith, H.M.T. and Abuknesha, R.A.,
J. Chromatogr.
B
, 2005, vol. 827, no. 2, pp. 182–192.
9. Yang, H., Gurgel, P.V., and Carbonell, R.G.,
J. Chro
matogr. A
, 2009, vol. 1216, no. 6, pp. 910–918.
10. de Souza, M.C.M., Bresolin, I.T.L., and Bueno, S.M.A.,
J. Chromatogr. B
, 2010, vol. 878, nos. 5–6, pp. 557–566.
11. Miller, T.J. and Stone, H.O.,
J. Immunol. Methods
,
1978, vol. 24, nos. 1–2, pp. 111–125.
12. Shadduck, R.K., Waheed, A., Pigoli, G., Boegel, F.,
and Higgins, L.,
Blood
, 1979, vol. 53, no. 6, pp. 1182–
1190.
13. Ansari, A.A. and Chang, T.S.,
American J. Veterinary
Res.
, 1983, vol. 44, no. 5, pp. 901–906.
14. Etienne, J., Noe, L., Millot, F., Laruelle, P., and
Debray, J.,
Atherosclerosis
, 1987, vol. 64, nos. 2–3,
pp. 201–209.
15. RF Patent No. 2089912, 1993.
16. Timganova, V.P., Bochkova, M.S., Rayev, M.B.,
Khramtsov, P.V., and Tyulenev, A.V.,
Vestn. Ural. Med.
Akad. Nauk
, 2012, vol. 41, no. 4, p. 167.
17.
Immunologicheskie metody
(Immunological Methods),
Frimelya, G., Ed., Moscow: Meditsina, 1987.
18.
Antibodies: A Practical Approach
, Catty, D., Ed.,
Oxford, Washington, DC: IRL Press, 1989.
19. RF Patent No. 2327992, 2007.
Translated by M. Novikova
