Open Journal of Applied Sciences, 2012, 2, 135-138
doi:10.4236/ojapps.2012.23019 Published Online September 2012 (http://www.SciRP.org/journal/ojapps)
Investigating Cytokine Binding Using a Previously
Reported TNF-Specific Aptamer
James D. Fisher1,2, Morgan V. DiLeo1,3, William J. Federspiel1,2,3,4*
1McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, USA
2Departments of Chemical Engineering, University of Pittsburgh, Pittsburgh, USA
3Departments of Bioengineering, University of Pittsburgh, Pittsburgh, USA
4Departments of Surgery, University of Pittsburgh, Pittsburgh, USA
Received May 27, 2012; revised June 28, 2012; accepted July 14, 2012
ABSTRACT
Cytokines are of chief importance in the pathophysiology of sepsis and other systemic inflammatory response syn-
dromes. We are designing and testing an extracorporeal cytokine adsorption device (CAD) that can remove cytokines
via adsorption on biocompatible, microporous beads. The goal of this study was to determine whether a previously re-
ported TNF binding DNA aptamer, 5’-GCGGCCGATA AGGTCTTTCC AAGCGAACGA ATTGAACCGC-3’, could
be immobilized our hemoadsorption polymer surface to increase the removal rate of TNF. A reservoir consisting of
horse serum spiked with a known concentration of TNF was perfused through our CAD packed with aptamer modified
or unmodified (control) polymer beads. The binding affinity of the TNF aptamer was characterized using an en-
zyme-linked oligonucleotide assay (ELONA). As a positive control a well-established DNA aptamer that binds PDGF
BB was also subjected to the same ELONA to validate the assay. TNF capture using the CAD showed no TNF removal
over four hours for both the aptamer modified and unmodified control beads. Additionally, the results of the ELONA
showed no binding of TNF to the reported aptamer; however the PDGF BB aptamer did bind PDGF BB. Based on these
results we are able to conclude that the reported TNF specific aptamer does not bind TNF. These results will be of im-
portance to other studies exploring aptamers for specific binding of TNF.
Keywords: Aptamers; Sepsis; Hemoadsorption; TNF
1. Introduction
Sepsis is defined as systemic inflammation in the pres-
ence of infection. The worldwide prevalence of this dis-
ease and the lack of efficient treatment options have
made sepsis one of the leading causes of death in the
world and the most common cause of death in adult, non-
coronary intensive care units [1]. Systemic inflammation
results in the excessive production of pro-inflammatory
cytokines such as tumor necrosis factor (TNF) and inter-
leukin-6 (IL-6) and anti-inflammatory cytokines such as
interleukin-10 (IL-10). Overproduction of cytokines along
with their interactions with one another contribute to the
pathological process of sepsis [2].
One emerging therapy aimed at treating sepsis is
hemofiltration. The aim of this therapy is to remove pro-
and anti-inflammatory cytokines from circulation using
convection, thus reestablishing a physiological balance.
Hemofiltration studies with sepsis have shown an initial
decrease in cytokine removal followed by a gradual de-
crease as the filter becomes saturated [3]. However, Kel-
lum and Dishart (2002) were able to show evidence that
the primary mechanism responsible for interleukin-6
(IL-6) removal during hemofiltration is most likely due
to adsorption onto the membrane rather than filtration
from plasma [4]. Thus, newer therapies now focus on
hemoadsorption in addition to hemofiltration. Our group
is developing a cytokine capture device that consists of a
column packed with microporous polymer beads through
which whole blood is perfused [5]. The polymer beads
used in the cytokine adsorption device (CAD) have a
high surface area (850 m2/g), making them ideal for ad-
sorption-based therapy.
Our device removes a variety of middle-molecular
weight proteins in the 10-30 kDa range including cyto-
kines generally considered of clinical relevance to sepsis
such as IL-6, IL-10, and TNF. While our device has been
effective at removing both IL-6 and IL-10 in both in vitro
and ex vivo animal studies, the removal rate of TNF has
been considerably slower than that of IL-6 and IL-10 [6].
To address this issue, we have begun studying specific
capture of TNF by immobilized ligands on the outer sur-
face of the polymer beads currently used in the CAD as a
*Corresponding author.

J. D. FISHER ET AL.
136
means to increase the removal rate of TNF. An ongoing
focus in our group has been immobilizing antibodies as
specific capture ligands for TNF. Antibodies have sev-
eral drawbacks, including the substantial cost associated
with coating several grams of polymer beads with TNF
antibodies, in addition to their limited shelf life. Another
concern is the potentially harmful immune response that
may occur if antibodies leach off of the beads.
A novel alternative to antibodies are aptamers, short
strands of oligonucleotides. Aptamers fold into a unique
three dimensional structure (similar to antibodies) which
allows them to specifically bind to a variety of bio-
molecules with affinity constants comparable to that of
antibodies (~10–9 M). These nucleic acid oligomers are
synthesized via an iterative in vitro selection process
called SELEX (systematic evolution of ligands by expo-
nential enrichment) [7,8]. Aptamers possess several ad-
vantages over their antibody counterparts as they are less
expensive, synthesized in vitro, have longer shelf lives
and are less likely to elicit immunogenicity than antibod-
ies [9,10]. Moreover, various chemical functionalities
can be added to the 5’ and 3’ ends of the aptamer to al-
low for easier conjugation to a surface.
In this work, our group investigated the TNF specific
aptamer sequence, 5’-GCGGCCGATA AGGTCTTTCC
AAGCGAACGA ATTGAACCGC-3’, reported in the
patent submitted by Zhang et al. [11]. We immobilized
this aptamer on the surface of porelesspolystyrene-divi-
nylbenzene (PSDVB) beads and tested its ability to bind
TNF in the CAD and subsequently deplete it from the
circulating serum solution. Poreless PSDVB beads were
used since the aptamers, unlike antibodies, are small
enough to diffuse into the porous network of our standard
porous PSDVB beads. In addition, the specificity of this
aptamer sequence to human TNF alpha was evaluated
using the enzyme-linked oligonucleotide assay (ELONA)
methodologies of Yan et al. [12].
2. Methods
Carboxyl groups were incorporated onto the surface of
the poreless PSDVB beads to provide a functionalized
surface for aptamer coupling, using a modified polysty-
rene oxidation protocol [13]. Batches of 2 grams of pore-
less PSDVB beads were incubated in manganese (VII)
oxide in H2SO4 at 65˚C for 1 hour. The surface concen-
tration of carboxyl groups was measured to be 24 nmol/g
polymer using a para-nitrophenol colorimetric assay [14].
Beads were washed with 6 N hydrochloric acid and DI
water. Functional groups were activated by incubating the
beads with a 1 mg/ml solution of 1-ethyl-3-(3-dimethy-
laminopropyl) carbodiimide (EDC) in 2-(N-morpholino)
ethanesulfonic acid (MES) buffer (pH 4.5) for 1 hour.
The beads were washed with MES buffer followed by DI
water. Aptamers were coupled to the beads by adding 11
μg/ml aptamer solution in sodium phosphate buffer (pH
7.0) and incubating at room temperature for 2 hours. Ap-
tamers used in this step were functionalized with an
amine group at the 5’ end for coupling. Beads were
washed with a .05% Tween solution followed by DI water.
For TNF capture experiments, 1.5 grams of aptamer-
immobilized beads or unmodified beads were packed in
an unused CAD and connected in series with a peristaltic
pump. Inlet and outlet tubes were connected to an 8 ml
reservoir of horse serum spiked with TNF at a concentra-
tion of ~1200 pg/ml. The reservoir was perfused through
the CAD at a flow rate of 0.8 ml/min and samples were
taken at t = 0, 15, 30, 60, 90, 120, 180, 240 min. TNF
concentrations were measured using a Biosource en-
zyme-linked immunosorbent assay (ELISA) (Invitrogen)
according to the manufacturer’s instructions.
The ELONA technique was performed as follows.
Recombinant human TNF (ThermoFisher) was diluted to
5 μg/ml using coating buffer (0.05 M Sodium Carbonate
pH 9.76), and 100 μl of this solution was incubated over-
night at 4˚C in a polystyrene microplate. As a negative
control, recombinant human IL-6 (Thermo) at the same
concentration was used. The plate was washed with a
0.15 M NaCl buffer, pH 7.4, containing 0.1% Tween 20,
and remaining adsorption sites were then blocked with
100 μl of 1% bovine serum albumin (BSA) in phosphate-
buffered saline (PBS) for 2 h at 37˚C. Wells were once
again washed followed by addition of 100 μl of TNF-
specific DNA aptamers at concentrations of 1.10 × 107,
1.10 × 105, 1104, 552, and 276 pg/ml. 100 μl of the 1104
pg/ml aptamer solution was added to the IL-6 coated well
as a negative control, and 100 μl of biotinylated TNF
antibody (Biosource) was used a positive control. The
aptamer/antibody solutions were incubated at 37˚C for 1 h
and then washed. 100 μl of streptavidin-conjugated horse-
radish peroxidase (Biosource) was added to each well and
incubated at 37˚C for 1h. The wells were washed for the
final time, after which 100 μl of tetramethylbenzidine
(TMB) substrate solution was added to each well. After
20 min, the optical density was measured at 450 nm on a
MultiSkan Plus microplate reader (ThermoFisher).
We also evaluated the affinity of a well-established
aptamer for its target ligand using ELONA to ensure that
the technique was being done correctly. Green et al. pub-
lished a DNA aptamer sequence, 5’-CAGGCTACGG-
CACGTAGAGCATCACCATGATCCTG-3’, which ex-
hibited high binding affinity towards platelet-derived
growth factor BB (PDGF-BB) [15]. The methods used in
the PDGF-BB ELONA were the same as those used in
the TNF ELONA. Wells were coated with PDGF-BB and
the PDGF-BB aptamer was the target analyte.
3. Results
Figure 1 shows the results of TNF capture for horse se-
rum perfused through the CAD packed with aptamer-

J. D. FISHER ET AL.
137
immobilized poreless PSDVB beads and unmodified
poreless PSDVB beads (control).
Neither the aptamer-immobilized nor the unmodified
beads were able to significantly decrease the circulating
concentration of TNF.
The results of the human TNF and PDGF-BB ELONAs
are shown in Figures 2 and 3, respectively. The TNF
ELONA data indicates that the TNF aptamer exhibited
no binding affinity toward recombinant TNF. The posi-
tive control, a biotinylated human TNF antibody, showed
a significant amount of binding affinity toward TNF rela-
tive to the negative control and test wells. The PDGF-BB
ELONA, however, demonstrated that the PDGF-BB ap-
tamer did have significant binding affinity to PDGF-BB
relative to the control wells. The wells of the PDGF-BB
ELONA corresponding to concentrations 4.51 × 108,
4.51 × 106, and 4.5 × 104 pg/ml did not show a decrease
in signal as these concentrations were beyond the detec-
tion limit of the plate reader.
However, the subsequent concentrations showed a de-
crease in signal with a decrease in concentration of ap-
tamer, ruling out the possibility of non-specific binding.
Figure 1. TNF capture with aptamer-immobilized and unmodified PSDVB beads.
Figure 2. TNF aptamer ELONA.
Figure 3. Platelet derived growth factor B B ELONA.

J. D. FISHER ET AL.
138
4. Discussion
The ability of CADs packed with aptamer-immobilized
or unmodified beads to capture TNF from horse serum
was tested. The results in Figure 1 show that TNF cap-
ture with unmodified and aptamer-immobilized beads
was negligible. The aptamer-immobilized on the surface
of the PSDVB beads was reported to specifically bind
TNF, therefore we expected that the aptamer-immobi-
lized beads would display a significantly higher ability to
capture TNF than the control beads. Based on the surface
density of carboxyl groups on the beads, we calculated
that if successfully coupled there would be at least a 10
molar excess of aptamer to TNF. Therefore, one possible
explanation is that the TNF aptamer was not successfully
coupled to the surface of the PSDVB beads. This is
unlikely however, as we have successfully coupled anti-
bodies to the PSDVB beads using the same chemistry.
Another possible explanation was that the reported ap-
tamer did not bind to TNF. To characterize the affinity of
the published aptamer for TNF, we utilized a previously
reported enzyme-linked oligonucleotide assay (ELONA)
[12].
From the ELONA data we are able to conclude that
the TNF aptamer sequence does not specifically bind
TNF. There are several possible explanations for this
finding. The discrepancies in data could be a result of
differences in the protein at which the aptamer was tar-
geted. Our group used commercially available recombi-
nant human TNF from ThermoFisher Scientific, but the
recombinant protein used by Zhang et al. was produced
in their laboratory. The target proteins were synthesized
in different environments, which may suggest that the
three dimensional structure of the proteins may have dif-
fered enough to impact the aptamer’s affinity toward
TNF. The TNF used in our work was in its correct three-
dimensional shape, as evidenced by our positive control,
a TNF antibody, being able to bind TNF in the ELONA.
A TNF antibody was not used as a positive control in the
group’s patent or published description of the RNA ap-
tamer [11,12].
The aptamer sequence, 5’-GCGGCCGATA AGGTC-
TTTCC AAGCGAACGA ATTGAACCGC-3’, reported
by Zhang and coworkers does not appear to bind com-
mercially available recombinant TNF. While this result is
a negative finding, we believe that this correspondence
provides important data to other investigators who may
be studying specific ligands for TNF.
REFERENCES
[1] M. Schoenberg, M. Weiss and P. Radermacher, “Out-
come of Patients with Sepsis and Septic Shock after ICU
Treatment,” Langenbeck’s Archives Surgery, Vol. 383,
No. 1, 1998, pp. 44-48. doi:10.1007/s004230050090
[2] J. A. Kellum, L. Kong, M. P. Fink, L. A. Weissfield, D.
M. Yealy, M. R. Pinsky, J. Fine, A. Krichevsky, R. L.
Delude and D. C. Angus, “Understanding the Inflamma-
tory Cytokine Response in Pneumonia and Sepsis,” Ar-
chives of International Medicine, Vol. 167, No. 15, 2007,
pp. 1655-1663. doi:10.1001/archinte.167.15.1655
[3] J. Kellum, “Immunomodulation in Sepsis: The Role of
Hemofiltration,” Minerva Anestesiologica, Vol. 65, No. 6,
1999, pp. 410-418.
[4] J. A. Kellum and M. K. Dishart, “Effect of Hemofiltration
Filter Adsorption on Circulating IL-6 Levels in Septic
Rats,” Critical Care, Vol. 6, No. 5, 2002, pp. 429-433.
doi:10.1186/cc1528
[5] M. Song, J. Winchester, R. L. Albright, V. J. Capponi, M.
D. Choquette and J. A. Kellum, “Cytokine Removal with
a Novel Adsorbent Polymer,” Blood Purification, Vol. 22,
No. 5, 2004, pp. 428-434. doi:10.1159/000080235
[6] M. V. DiLeo, J. Kellum and W. J. Federspiel, “A Simple
Mathematical Model of Cytokine Capture Using a He-
moadsorption Device,” Annals of Biomedical Engineer-
ing, Vol. 37, No. 1, 2009, pp. 222-229.
doi:10.1007/s10439-008-9587-8
[7] R. Stoltenburg, C. Reinemann and B. Strehlitz, “SELEX—
A (r)Evolutionary Method to Generate High-Affinity Nu-
cleic Acid Ligands,” Biomolecular Engineering, Vol. 24,
No. 4, 2007, pp. 381-403. doi:10.1016/j.bioeng.2007.06.001
[8] S. J. Klug and M. Famulok, “All You Wanted to Know
about SELEX,” Molecular Biology Reports, Vol. 20, No.
2, 1994, pp. 97-107. doi:10.1007/BF00996358
[9] X. Yang and D. G. Gorenstein, “Progress in Thioaptamer
Development,” Current Drug Targets, Vol. 5, No. 8, 2004,
pp. 705-715. doi:10.2174/1389450043345074
[10] J. W. Guthrie, C. L. A. Hamula, H. Zhang and X. C. Le,
“Assays for Cytokines Using Aptamers,” Methods, Vol. 38,
No. 4, 2006, pp. 324-330. doi:10.1016/j.ymeth.2006.01.001
[11] Z. Zhang, X. Yan and C. Xu, “Oligonuecleotide Antago-
nist for Human Tumor Necrosis Factor A (TNF-a),” In-
stitute for Viral Disease Control and Prevention Chinese
Center for Disease Control and Prevention, United States,
2007.
[12] X. Yan, X. Gao and Z. Zhang, “Isolation and Characteri-
zation of 2’-Amino-Modified RNA Aptamers for Human
TNFa,” Genomics Proteomics & Bioinformatics, Vol. 2,
No. 1, 2004, pp. 32-42.
[13] N. Zammatteo, C. Girardeaux, D. Delforge, J.-J. Pireaux
and J. Remacle, “Amination of Polystyrene Microwells:
Application to the Covalent Grafting of DNA Probes for
Hybridization Assays,” Analytical Biochemistry, Vol. 236,
No. 1, 1996, pp. 85-94. doi:10.1006/abio.1996.0135
[14] M. Matteucci and M. Caruthers, “Synthesis of Deoxyoli-
gonucleotides on a Polymer Support,” Journal of the Ameri-
can Chemical Society, Vol. 103, No. 11, 1981, pp. 3185-
3191. doi:10.1021/ja00401a041
[15] L. S. Green, D. Jellinek, R. Jenison, A. Ostman, C.-H.
Heldin and N. Janjic, “Inhibitory DNA Ligands to Plate-
let-Derived Growth Factor B-Chain,” Biochemistry, Vol.
35, No. 45, 1996, pp. 14413-14424.
doi:10.1021/bi961544+