Tetanus toxoid paper

Journal of Biotechnology 238 (2016) 15–21
Contents lists available at ScienceDirect
Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec
Specific detection of tetanus toxoid using an aptamer-based matrix
Harshvardhan B. Modh1
, Ankan K. Bhadra2
, Kinjal A. Patel, Rajeev K. Chaudhary3
,
Nishant K. Jain4
, Ipsita Roy∗
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India
a r t i c l e i n f o
Article history:
Received 13 July 2016
Received in revised form 30 August 2016
Accepted 12 September 2016
Available online 13 September 2016
Keywords:
ALISA (aptamer-linked immobilized
sorbent assay)
Aptamers
ELISA (enzyme-linked immunosorbent
assay)
Tetanus toxoid
a b s t r a c t
Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous moni-
toring at all stages of the production process. A large number of animals are employed for risk assessment
of biologicals, which has low ethical and economic acceptability. Research is now focussed on the val-
idation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked
immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus
toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modi-
fiability, may have low stability and result in irreproducibility. We have developed a method using highly
specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first
identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers
were employed as capture-detection ligands in a sandwich-ALISA (aptamer-linked immobilized sorbent
assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate
well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity
as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was sub-
jected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions.
The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar
to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to
develop a more robust detection tool for tetanus toxoid.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
Vaccines have played an unambiguous role in the success of
various global immunization programmes. Sporadic failures may
result from the denaturation of the vaccine when exposed to
stress conditions. Thus, stability of the vaccine during production,
transport and storage is a major concern. Lower potency of the vac-
cine may lead to inadequate protection and require re-vaccination
(PATH, 2010; Centers for Disease Control and Prevention, 2012).
Downstream processing of biologicals leads to a lot of variation in
Abbreviations: ALISA, (aptamer-linked immobilized sorbent assay); ELISA,
(enzyme linked immunosorbent assay).
∗ Corresponding author.
E-mail address: ipsita@niper.ac.in (I. Roy).
1
Present address: Institut für Technische Chemie, Callinstrasse 5, 30167, Han-
nover, Germany.
2
Present address: Department of Cell Biology & Physiology, Washington Univer-
sity, School of Medicine, St. Louis, MO, 63110, USA.
3
Present address: Sun Pharma Advanced Research Company (SPARC Ltd.), Baroda,
390020, India.
4
Present address: Pfizer, Inc, San Diego, CA, 92121, USA.
product characteristics among different batches (Alahmad et al.,
2010; Rustichelli et al., 2013; Oliveira et al., 2014). Quality con-
trol of various batches is thus an important issue. The testing and
retesting of these batches at the level of the manufacturer and
the regulatory authorities require a large number of laboratory
animals. Although no precise figures are available, it is generally
assumed that numbers exceed 15% of total animals used in biomed-
ical research (Hendriksen, 2006, 2007; Schiffelers et al., 2007). In
vaccine research and development, laboratory animals are used for
a wide range of purposes, such as adjuvant selection, for testing
immunogenicity, immunokinetics and safety of the selected anti-
gen components (Stickings et al., 2011). Animals are also used for
batch release testing so there is no variation remaining in the final
product. Apart from ethical issues, use of animals for such studies is
also expensive and time consuming. Potency testing of vaccines has
been a highlight of the so-called 3Rs research (reduction, refine-
ment and replacement of animals used in research and testing).
According to this, the protective antibody response is estimated
by an in vitro method. In case of tetanus toxoid, for example, the
method of choice is ELISA (enzyme-linked immunosorbent assay)
or the toxin binding inhibition assay. Testing the antigenic purity of
http://dx.doi.org/10.1016/j.jbiotec.2016.09.004
0168-1656/© 2016 Elsevier B.V. All rights reserved.

16 H.B. Modh et al. / Journal of Biotechnology 238 (2016) 15–21
tetanus toxoid by ELISA has now become a standard method of anal-
ysis (Determan et al., 2006; Matejtschuk et al., 2009; Stickings et al.,
2011; Tierney et al., 2011; Jain et al., 2013; Jetani et al., 2014; Juan-
Giner et al., 2014; Lockyer et al., 2015). Its application in quality
testing in the manufacturing process of tetanus toxoid is limited by
the sensitivity of the assay. Since minor differences among batches
can result in a vaccine losing its potency, the sensitivity of the assay
needs to be high enough to detect minor changes in the antigenicity
of the toxoid. The major problem with any antibody-based diagnos-
tic technique is the availability and stability of the antibody itself,
especially in tropical countries where variation in temperature and
moisture may result in denaturation of the antibody ligand.
Aptamers are short, single-stranded DNA or RNA sequences
which recognize their targets on the basis of shape complemen-
tarity with high degree of specificity and affinity. Aptamers have
been selected against a wide variety of targets such as proteins like
growth and clotting factors, cell-surface proteins, cancer cells, small
molecules such as nucleotides, antibiotics, organic dyes, cofactors,
sugars, amino acids, etc. They have been used in fields as var-
ied as drug discovery and development, target validation, analysis,
diagnostics, etc. (Kaur and Roy, 2008; Cerchia and de Franciscis,
2010; Keefe et al., 2010; Kim et al., 2010; Hu et al., 2014; Ma et al.,
2015). Aptamers have often been referred to as ‘chemical antibod-
ies’ because of the high affinity that they exhibit for their targets.
In contrast to antibodies, however, they are easier to handle and
manipulate since they are synthesized in vitro and can be selected
against self or toxic targets. They have been able to substitute anti-
bodies in almost all the areas where the latter are used and in some
cases, have also overtaken the application of antibodies (Keefe et al.,
2010; Lollo et al., 2014; McConnell et al., 2014). One such applica-
tion is that of ELISA.
We have earlier selected specific RNA aptamers against tetanus
toxoid, which bind to the protein with high affinity and stabilize the
toxoid against aggregation when exposed to different stress con-
ditions (Jain et al., 2013; Jetani et al., 2014). Thus, they have been
referred to as ‘universal stabilizers’ of proteins. We report here that
some of these nucleic acid sequences bind to different ‘aptatopes’
on the toxoid and hence can be employed as capture-detection pair
in a diagnostic assay by replacing antibodies in the traditional for-
mat of the in vitro measurement. This approach will minimize the
use of biological reagents and improve the robustness of the assay.
2. Materials and methods
Deoxyribonucleotides (dNTPs), ribonucleotides (rNTPs), ribonu-
clease A, Corning® 96-well plates (catalogue no. 3590, 3912 and
3925) and aluminium hydroxide gel (13 mg/ml, AlhydrogelTM,
Cat. No. A8222) were purchased from Sigma-Aldrich, Bangalore,
India. RNase free DNase I, T7 RNA polymerase and yeast inorganic
pyrophosphatase were purchased from Fermentas Inc., Maryland,
USA. RNaseOUT was obtained from Invitrogen Corporation, Califor-
nia, USA. GoTaq® Flexi DNA polymerase and PCR Clean-Up System
were obtained from Promega Corporation, Madison, USA. Fluores-
cein RNA labeling mix was obtained from Roche Applied Science,
Mumbai, India. Mouse anti-tetanus toxoid monoclonal antibody
(HYB 278-01, raised against full length formaldehyde inactivated
tetanus toxoid) was obtained from Santa Cruz Biotechnology, Inc.,
California, USA. All other reagents and chemicals used were of ana-
lytical grade or higher.
2.1. Synthesis and purification of RNA aptamers
RNA sequences showing high affinity for tetanus toxoid (Table 1)
were selected earlier by SELEX (sequential evolution of ligands
by exponential enrichment), an iterative selection process (Jain
Table 1
RNA aptamers selected against tetanus-toxoid (Jain et al., 2013) and used in this
work. Values represent mean ± s.e.m. of three independent experiments.
Designation Length of sequence Kd, nM
TT-13 97 bp 142 ± 22
TT-17 97 bp 271 ± 11
TT-20 97 bp 224 ± 25
TT-23 97 bp 223 ± 22
TT-24 97 bp 181 ± 40
et al., 2013). Glycerol stocks of five different clones (named as TT-
13, TT-17, TT-20, TT-23 and TT-24) were inoculated in 10 ml of
Luria bertani (LB) media containing ampicillin (100 ␮g/ml), incu-
bated at 37 ◦C with shaking at 200 rpm overnight. Grown cells
were harvested by centrifugation. Plasmid DNA was extracted by
alkaline lysis method (Sambrook and Russell, 2001). The isolated
plasmids were subjected to polymerase chain reaction (PCR) using
the primers and conditions described earlier (Jain et al., 2013).
In vitro transcription was carried out with the amplified product
and the transcribed product was purified by 8% urea denaturing
polyacrylamide gel electrophoresis.
2.2. Displacement assay
Dot blot assay was carried out to check RNA-protein inter-
action. Tetanus toxoid (100 nM) was added in the SELEX buffer
(50 mM phosphate buffer, pH 7.4 containing 150 mM NaCl and 3
or 4 mM MgCl2). A constant amount (70 ng) of fluorescein labelled
and different amounts of unlabelled RNA (in increasing order) were
incubated in a reaction volume of 50 ␮l for 2 h at 25 ◦C. Sam-
ples were filtered through an activated PVDF membrane (0.45 ␮m)
using a 96-well vacuum filtration manifold (Whatman-Biometra,
Goettingen, Germany). The membrane was washed with SELEX
buffer and dried between folds of filter papers. Fluorescence inten-
sity of the retained RNA-protein complex was measured using an
image scanner (Typhoon Trio, GE Healthcare) in the fluorescence
mode.
2.3. Aptamer-linked immobilized sorbent assay (ALISA)
Unlabelled aptamer (0.5–2 ng/␮l, in SELEX buffer) or mouse
anti-tetanus toxoid monoclonal antibody (2–4 ng/␮l, in 50 mM car-
bonate buffer, pH 9.6) was coated on a 96-well microtitre plate for
14 h at 24 ◦C with shaking at 300 rpm. Unbound aptamer/antibody
was removed by washing with SELEX buffer. Unbound sites in wells
were blocked with 0.2 M glycine for 30 min at 24 ◦C, followed by
washing with SELEX buffer. Non-specific binding was eliminated
by blocking with 2% BSA for 6 h, followed by washing with SELEX
buffer. Tetanus toxoid prepared in SELEX buffer was added to the
wells and incubated for 2 h at 24 ◦C, 300 rpm. Unbound protein
was removed by washing with SELEX buffer. Fluorescein-labelled
aptamer (0.7 ng/␮l) was added to each well and incubated for 2 h
at 24 ◦C. Wells were washed with SELEX buffer and the fluores-
cence intensity in the wells was read at 526 nm, using an excitation
wavelength of 488 nm.
For direct ALISA, increasing amounts of tetanus toxoid were
coated on a 96-well microtitre plate for 18 h at 24 ◦C. Unbound tox-
oid was removed by washing with SELEX buffer. Unreacted sites
were blocked with glycine and BSA, as before. Labelled aptamer
(0.7 ng/␮l) was added to each well and incubated for 2 h at 24 ◦C.
Wells were washed with SELEX buffer and the fluorescence inten-
sity in the wells was read at 526 nm, using an excitation wavelength
of 488 nm.

H.B. Modh et al. / Journal of Biotechnology 238 (2016) 15–21 17
Fig. 1. Displacement assay was carried out to determine the overlap of binding site of the aptamers on the surface of the toxoid. Densitometric analysis of the retained
fluorescence intensity of the RNA-protein complex was carried out using ImageQuantTL software (GE Healthcare). Results are shown for increasing amounts of unlabelled
(A) TT-24, (B) TT-23, (C) TT-13 and (D) TT-17 aptamers, in the presence of constant amount (0.7 ng/␮l) of fluorescein labelled TT-13 (cross), TT-17 (triangle), TT-20 (circle),
TT-23 (square) and TT-24 (star). In each case, duplicate pairs are avoided. The fluorescence intensity of the RNA-protein complex in the absence of any unlabelled aptamer
has been arbitrarily assigned a value of 100% in each case. Values shown are mean ± s.e.m. of three independent experiments.
2.4. Exposure of adjuvanted tetanus toxoid to stress conditions
Tetanus toxoid was adsorbed on alumina as described earlier
(Solanki et al., 2011, 2012; Jetani et al., 2014). Adsorbed toxoid
was subjected to thermal stress by incubating the preparation at
50 ◦C with mild shaking for 150 min. Mechanical stress was admin-
istered by agitating the samples at 300 rpm for 2 h at 37 ◦C. The
adjuvanted preparation was also subjected to freeze-thawing stress
by five cycles of incubation at −20 ◦C for 12 h, followed by 2 h in a
water bath at 37 ◦C. Details of stress conditions have been described
earlier (Jetani et al., 2014). In each case, after completion of stress
exposure, the suspensions were centrifuged at 500g for 3 min, the
pellet was resuspended in 10 mM sodium phosphate buffer, pH 7.4
and capture ALISA was performed to determine residual antigenic-
ity of the adsorbed toxoid, as described above.
2.5. Enzyme-linked immunosorbent assay (ELISA)
After immobilizing tetanus toxoid on a microtitre plate (cata-
logue no. CLS3590) as described above, mouse anti-tetanus toxoid
monoclonal antibody (1:5000) was used as the primary anti-
body and horseradish peroxidase (HRP)-conjugated anti-mouse
antibody (1:3000) was used as the secondary antibody. Tetanus
toxoid was detected after addition of tetramethyl benzidine/H2O2
(TMB/H2O2) as the substrate for HRP. The absorbance of solution in
the wells was measured at 450 nm after terminating the reaction
with 0.02 N H2SO4 (Determan et al., 2006; Jain et al., 2013; Jetani
et al., 2014).
3. Results
3.1. Displacement assay
In order to determine the binding specificities of the aptamers,
i.e. whether they bind at the same or different sites on tetanus
toxoid, displacement studies were carried out by dot blot assay.
For this, labelled and unlabelled aptamers were added to tetanus
toxoid in a reaction mixture and incubated for 2 h at 25 ◦C. The
amount of labelled aptamer was constant and the amount of unla-
belled aptamer was varied. If both aptamers bind at the same site on
tetanus toxoid, as the amount of unlabelled aptamer is increased,
it will displace the labelled aptamer and the fluorescence intensity
of the complex retained on the membrane will be reduced. If the
aptamers bind to different sites, no significant change in fluores-
cence intensity would be observed upon increase in concentration
of the unlabelled aptamer.
In order to validate the displacement assay, labelled TT-24 was
allowed to bind to tetanus toxoid and challenged with unlabelled
TT-24. As equilibrium is set up between the labelled aptamer
and tetanus toxoid leading to the formation of protein-RNA com-
plex, increasing the amount of the unlabelled aptamer will lead to
the displacement of the labelled aptamer from the surface of the
protein surface. This will result in reduction in the fluorescence
intensity of the protein-RNA complex retained on the membrane.
As the amount of unlabelled RNA (TT-24) was increased, the inten-
sity of the retained complex decreased on the membrane in a
concentration-dependent manner (Fig. 1A). Thus, this positive con-
trol validates the use of displacement assay in this work.
Next, constant amounts of different labelled aptamers (TT-13,
TT-14, TT-20 and TT-23) were mixed with increasing amounts
of unlabelled TT-24 and incubated with tetanus toxoid. The flu-
orescence intensity of the protein-RNA complex retained on the
membrane was determined by densitometry. The fluorescence
intensity of the protein-RNA complex with labelled TT-17 was
found to decrease with increasing amounts of unlabelled TT-24
(Fig. 1A). Thus, TT-24 was able to displace TT-17 from the protein
surface, indicating that these two aptamers probably bind to the
same site on the toxoid. Similarly, the fluorescence intensity of the
protein-RNA complex with labelled TT-20 was found to decrease
with increasing amounts of unlabelled TT-24 (Fig. 1A), although not
to the same extent as with labelled TT-17, showing that TT-20 and
TT-24 recognize overlapping regions on the surface of the toxoid. A
similar result was seen with labelled TT-13-protein complex. With
increasing amount of unlabelled TT-24, the fluorescence inten-
sity of the protein-RNA complex was found to decrease (Fig. 1A),
although the reduction was much less than with TT-17. Thus, TT-
13 and TT-24 may partially share binding site on the surface of
the protein. No significant difference in fluorescence intensity of
the protein-RNA complex was observed with labelled TT-23 when
challenged with increasing amounts of unlabelled TT-24 (Fig. 1A).
Thus, TT-24 was not able to displace TT-23 from the protein surface,

Fig. 2. Standardization of conditions for carrying out ALISA. (A) Summary of bind-
ing results obtained in Fig. 1. The table shows the retention of fluorescence intensity
(in%) when increasing amounts of unlabelled (UL) aptamer is added to a constant
amount of labelled (L) aptamer complexed with tetanus toxoid. Pairs which showed
≥80% retention of fluorescence intensity on the PDVF membrane were assumed
to bind to distinct sites on the surface of the protein and were considered to be
suitable for sandwich ALISA. N.D. not determined; Duplicate implies the reverse
pair (labelled/unlabelled) was analyzed. (B) ALISA was carried out with three dif-
ferent Corning®
96-well plates. Features of the plates are described in Table 2.
Values shown are mean ± s.e.m. of three independent experiments. (C) Specificity
of the assay was confirmed by incubating tetanus toxoid on the blocked plate in
the presence (sample) or absence (control) of labelled capture aptamer (TT-23). The
fluorescence intensity of the well in the presence of the capture aptamer has been
arbitrarily assigned a value of 100% in each case. Values shown are mean ± s.e.m. of
three independent experiments.
indicating that these two aptamers bind to different regions of the
toxoid.
Similar comparative measurements were carried out with other
aptamer pairs. With increasing amount of unlabelled TT-23, signif-
icant reduction in fluorescence intensity of protein-RNA complex
in case of labelled TT-13 and TT-17 was observed (Fig. 1B), demon-
strating the partial displacement of TT-13 and TT-17 by TT-23 from
the surface of the toxoid. On the other hand, unlabelled TT-23 was
not able to displace labelled TT-20 (Fig. 1B). Thus, it is likely that
TT-13, TT-17 and TT-23 may share binding sites on tetanus toxoid.
It is probable that TT-20 binds to a different site on the protein. On
being tested with increasing amounts of unlabelled TT-13, only par-
tial reduction in fluorescence intensity of the protein-RNA complex
was seen with labelled TT-17 and TT-20 (Fig. 1C). This confirms that
TT-13, TT-17 and TT-20 partially share the ‘aptatope’ on the tox-
oid. Similar overlapping recognition of the toxoid surface was seen
Table 2
Properties of Corning®
96-well plates used in this work.
Catalog No.
(Sigma)
Propertiesa
CLS3590 High binding surface binds medium (>10 kD) and large
biomolecules that possess ionic groups and/or
hydrophobic regions
CLS3912 Not treated (or medium binding) polystyrene surface is
hydrophobic in nature and binds biomolecules through
passive interactions. White microplates enhance
luminescent signals and have low background
luminescence and fluorescence.
CLS3925 High binding surface is capable of binding medium
(>10 kD) and large biomolecules that possess ionic groups
and/or hydrophobic regions. Black microplates have low
background fluorescence and minimize light scattering.
a
As per information available at the company website.
with partial displacement of labelled TT-20 by unlabelled TT-17 too
(Fig. 1D).
3.2. Aptamer-linked immobilized sorbent assay (ALISA)
Five monoclonal aptamer sequences were selected which have
high affinity and selectivity towards tetanus toxoid (Jain et al.,
2013). In displacement studies, it was found that two pairs of
aptamers were not able to displace each other while the remain-
ing pairs could displace each other from the surface of the protein
(tetanus toxoid) (Fig. 2A). Thus, the aptamer pairs belonging to the
first category (with ≥80% retention of fluorescence intensity) were
not able to displace each other and presumably bound to different
regions on the protein. These could be probable candidates to carry
out sandwich ALISA. The other aptamers would be suitable candi-
dates for detection of tetanus toxoid, either alone (direct ALISA), or
when paired with tetanus toxoid-specific monoclonal antibody, if
the latter does not bind to the same epitope as the aptamer.
Aptamers TT-20 and TT-23 were shown to bind to different
regions on tetanus toxoid (Fig. 1B). Hence these were chosen as
capture and detection pair for sandwich ALISA. Unlabelled aptamer
TT-23 was used as the capture aptamer and labelled aptamer TT-
20 was used as the detection aptamer. The capture aptamer was
immobilized on three different plates (Table 2), followed by addi-
tion of different amounts of the analyte (tetanus toxoid) and a
constant amount of the labelled detection aptamer. Detectable and
reproducible results could be obtained only with Corning® plate
(catalogue no. CLS3912) (Fig. 2B) and this plate was used for fur-
ther studies. The specificity of the test was confirmed by running
a control. In this case, the unlabelled (capture) aptamer was not
coated on the wells. Tetanus toxoid (5 ng) was added to the well
after blocking the unreacted sites in the well with glycine followed
by BSA, as described in the Methods section, and was quantified
using labelled (detection) aptamer. Negligible fluorescence inten-
Fig. 3. Standardization of ALISA conditions was carried out at (A) higher and (B) lower amounts of tetanus toxoid as the analyte. TT-23 was used as the capture aptamer and
labelled TT-20 was used as the detection aptamer. Values shown are mean ± s.e.m. of three independent experiments.

Fig. 4. Validation of sandwich ALISA. (A) The experiments were repeated using mouse anti-tetanus toxoid monoclonal antibody (solid lines) as the capture agent. Results
with TT-23 as the capture agent (dashed lines) are shown for comparison. In both cases, labelled TT-20 was used as the detection agent. (B) The experiment was also repeated
using unlabelled TT-24 (2 ng/␮l) as the capture agent and labelled TT-23 (0.7 ng/␮l) as the detection agent. Values shown are mean ± s.e.m. of three independent experiments.
Fig. 5. Comparison of aptamers with antibody for detection of tetanus toxoid by direct measurement. (A) Lower and (B) higher amounts of tetanus toxoid were immobilized
on microtitre plate (Sigma catalogue no. CLS3590 for measurement of absorbance using antibody and CLS3912 for measurement of fluorescence using aptamers, as described
in sections 2.3 and 2.5)) wells. Capture ELISA was carried out using mouse monoclonal antibody (diamond, dashed line) and detection of absorbance was carried out at
450 nm (secondary axis). Capture ALISA was carried out using 1 ng/␮l of labelled TT-23 (triangle, solid line) or TT-24 (circle, solid line). Values shown are mean ± s.e.m. of
three independent experiments.
sity was observed in this case as compared to coating the well with
the capture aptamer (Fig. 2C). No toxoid was detected in the wash-
ings in the latter case showing that all the protein added could be
‘captured’ by the aptamer. Thus, the increase in fluorescence inten-
sity observed with increasing amounts of tetanus toxoid (Fig. 2B)
was due to the detection of the toxoid by the sandwich pair and not
due to non-specific adsorption of the protein on the well.
In order to optimize the amount of capture aptamer, different
amounts of the unlabelled aptamer TT-23 were used to coat the
wells. With 0.25 ng/␮l of the aptamer, the resultant fluorescence
intensity was found to be quite low (Fig. 3A). With 0.5 or 1 ng/␮l of
the capture aptamer, the fluorescence intensity was found to be in
the linear range. No significant difference in fluorescence intensity
could be observed between the two cases, indicating that 0.5 ng/␮l
of the capture aptamer is sufficient to bind to the highest amount
of the toxoid (100 ng). Surprisingly, with 2 ng/␮l of the capture
aptamer, the fluorescence intensity was found to be saturated at
higher amounts of the toxoid (Fig. 3A). In order to determine the
sensitivity of the assay, the experiment was repeated with lower
amounts of tetanus toxoid. At lower amounts, both 1 ng/␮l and
2 ng/␮l of the capture aptamer showed linear curves over the whole
range, i.e. till 5 ng toxoid (Fig. 3B). Thus, the signal intensity is lin-
ear at lower amounts of the analyte but reaches saturation at higher
amounts with higher amounts of the capture aptamer.
Next, we compared the use of mouse anti-tetanus toxoid mon-
oclonal antibody with unlabelled TT-23 as the capture reagent.
Labelled TT-20 was used as the detection aptamer in both cases.
At a lower concentration of the antibody (2 ng/␮l), the fluorescence
intensity of detection was lower while the signal output was higher
with a higher concentration of the antibody (4 ng/␮l) (Fig. 4A). Thus,
the signal intensity of response did not show any difference irre-
spective of whether the monoclonal antibody or the aptamer was
used as the capture reagent.
As the aptamer pair TT-23 and TT-24 was shown to bind to
different regions on the toxoid (Figs. 1A and 2A), the above experi-
ment was repeated with unlabelled TT-24 (2 ng/␮l) as the capture
aptamer and labelled TT-23 as the detection aptamer. This pair was
also shown to exhibit reproducible and linear curve with increasing
amounts of the analyte (1–5 ng) (Fig. 4B). At higher amounts of the
toxoid (5–20 ng), the curve for fluorescence intensity was found to
reach saturation (Fig. 4B), similar to the other pair (TT-20 and TT-
23) (Fig. 3A). Thus, the method described here is reproducible and
not limited to a single pair of aptamers being used for detection of
the analyte.
3.3. Direct ALISA
We repeated the conventional protocol of capture ELISA
(Determan et al., 2006; Jain et al., 2013; Jetani et al., 2014) by
replacing the antibody with the aptamer for detection. Increasing
amounts of tetanus toxoid (1–5 ng) were coated on the walls of
the wells. Detection was carried out with labelled TT-23 and TT-
24 (0.7 ng/␮l). Curves obtained with both of the aptamers were
found to be linear (Fig. 5A) at lower amounts of the analyte and
were found to saturate at higher amounts (Fig. 5B). Using mouse
anti-tetanus toxoid monoclonal antibody as the detection agent
(Determan et al., 2006; Jain et al., 2013; Jetani et al., 2014) con-
firmed that there was no reduction in sensitivity of detection upon
using aptamers (Fig. 5A).
3.4. Exposure of adjuvanted tetanus toxoid to stress conditions
We have shown earlier that when exposed to stress condi-
tions, adjuvanted tetanus toxoid is partially desorbed from the
matrix (Jetani et al., 2014). The residual adsorbed toxoid is par-
tially inactive, as determined by measurement of antigenicity by

Table 3
Comparison of ALISA in detecting changes in antigenicity of tetanus toxoid. Adjuvanted tetanus toxoid was subjected to thermal, mechanical and freeze-thawing stress as
already described. Antigenicity of the toxoid retained on the matrix was determined using ALISA. In both cases, 100% antigenicity refers to the antigenicity of the adsorbed
tetanus toxoid which was incubated at 4 ◦
C for the same time period as the stress condition. Values shown are mean ± s.e.m. of three independent experiments.
Stress Residual antigenicity, % p-value
Antibody (Jetani et al., 2014) Aptamer
Thermal 63.40 ± 1.30 71.18 ± 4.16 0.148
Mechanical 50.73 ± 2.09 46.69 ± 3.30 0.294
Freeze-thawing 24.54 ± 3.07 31.46 ± 6.10 0.368
ELISA. In order to determine whether the method reported here
has broad applicability, we subjected adjuvanted tetanus toxoid
to three stress conditions, viz. thermal, mechanical and freeze-
thawing, and determined the residual antigenicity of the adsorbed
toxoid by ALISA using unlabelled TT-23 as the capture aptamer and
labelled TT-20 as the detection aptamer. In all cases, loss of anti-
genicity of the adsorbed toxoid was observed (Table 3). Comparison
with antigenicity values reported by ELISA [13] showed that there
was no significant difference between the measurements using
antibodies or aptamers as ligands (Table 3). As exposure to acceler-
ated stress conditions leads to changes in the native conformation
of tetanus toxoid (Solanki et al., 2011, 2012), our results show that
the selected aptamers are capable of discriminating between anti-
genically active and inactive forms of tetanus toxoid. Thus, the
method developed here can measure changes in antigenicity of
tetanus toxoid occurring during the production, storage and trans-
port steps and has comparable sensitivity to conventional ELISA.
Thus, it may replace the antibody in the analytical measurement.
4. Discussion
Potency of vaccines is confirmed by in vivo assays. These assays
are approved by the European Pharmacopoeia and World Health
Organization (WHO). In these assays, multiple dilutions of a refer-
ence and test preparations are required which leads to the need
of a large number of animals (Hendriksen et al., 1987; Hendriksen,
2006, 2007; Schiffelers et al., 2007; Council of Europe, 2013, 2014).
The collaborative studies done by National Institute for
Biological Standards and Control (NIBSC) and the European Phar-
macopoeia have led to the validation of serological methods used
for testing the potency of tetanus vaccine (Stickings et al., 2011).
These studies validated a correlation between the results obtained
by animal challenge and in vitro analysis methods including ELISA
and the toxin neutralization test (TNT) assays for checking the
potency of testing of vaccines.
In order to decrease the use of animals, the Interagency Coor-
dinating Committee on the Validation of Alternative Methods
(ICCVAM) and the National Toxicology Program Interagency Center
for the Evaluation of Alternative Toxicological Methods (NICEATM)
are promoting alternative methods that will help in reduce, refine
(less pain and distress) and replace animal use in testing the vac-
cines. The primary objective of this work was to develop a method
which will replace animal testing, minimize the use of antibodies
from biological sources and thus reduce batch-to-batch variation
in the toxoid production process.
For detection of hVEGF (human vascular endothelial growth fac-
tor) ELISA method has LOD (limit of detection) of 10 pg but in case
of aptamer, the LOD is 100 fg (Yoshida et al., 2009). The aptamer-
based assay was able to detect 1 ng of tetanus toxoid, which was
the same as with ELISA. Lower concentrations of capture aptamers
(0.25 ng/␮l) were able to detect the maximum amount of analyte
although the fluorescence readout was low. This problem could
be solved with using marginally higher concentrations (0.5 ng/␮l)
of the aptamers. A diagnostic tool with a pair of aptamers (and no
antibody) was recently reported for the detection of Escherichia coli
O157:H7 (Wu et al., 2015). Detection was however based on a cou-
pled amplification step. Some of the aptamer pairs, which were
found to bind to different sites on the surface of the toxoid, were
used for sandwich ALISA. Some of the aptamers were used for direct
ALISA for detection of tetanus toxoid. Two pairs of aptamers [(TT-
20 and TT-23) and (TT-23 and TT-24)] were confirmed to bind at
different positions on the surface of tetanus toxoid. They were fur-
ther used for development of sandwich ALISA. They were able to
detect 1 ng tetanus toxoid. Sensitivity of ELISA for tetanus toxoid
was also confirmed. 1 ng of tetanus toxoid could be determined by
ELISA. A major drawback of the use of aptamers is their nuclease-
instability during in vivo administration (Cerchia and de Franciscis,
2010; Keefe et al., 2010). In the present case, this is not a limiting
factor as the application envisaged is in vitro analysis of batch-to-
batch variation of the toxoid during production.
5. Conclusion
Aptamers are selected by an in vitro evolution process such
that they exhibit very high specificity and selectivity for their
target molecule. They possess several advantages over antibod-
ies which make them suitable for use in diagnostic and clinical
applications. Their in vitro synthesis and amenability to chemical
modification for improved stability make them attractive ligands
for any binding/inhibitions studies where antibodies could poten-
tially be employed (Jayasena, 1999; Keefe et al., 2010; Lollo et al.,
2014; McConnell et al., 2014). Reproducibility of antibody genera-
tion protocols in cell cultures or polysera, especially for large-scale
production, introduces additional variables. The main purpose of
this study was to establish a wholly antibody-less aptamer-linked
immobilized sorbent assay (ALISA) for detection of tetanus toxoid.
We have shown earlier that the selected aptamers bind specifi-
cally to tetanus toxoid and do not show affinity for other proteins
with similar properties (Jain et al., 2013). Our developed method
matches the sensitivity of the present gold standard method for
detection of tetanus toxoid. Complete replacement of antibod-
ies with aptamers makes this protocol less susceptible to adverse
environmental conditions and reduces the loss of reproducibility
commonly associated with the use of antibodies.
Acknowledgements
Tetanus toxoid was obtained as a gift from Shantha Biotech-
nics Ltd., Hyderabad, India. This work was partially supported by
Indian Council of Medical Research (ICMR). AKB, KAP, RKC and
NKJ acknowledge the award of senior research fellowships from
DST-INSPIRE programme, Department of Biotechnology, ICMR and
Council for Scientific and Industrial Research, respectively. The
funding agency had no role in study design; in the collection, anal-
ysis and interpretation of data; in the writing of the report; and in
the decision to submit the article for publication.

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