Talanta 187 (2018) 165–171

Contents lists available at ScienceDirect

Talanta
journal homepage: www.elsevier.com/locate/talanta

Engineered biomarkers for leprosy diagnosis using labeled and label-free T

analysis
Juliana F. de Santanaa,b,1, Mariângela R.B. da Silvaa,b,1, Guilherme F. Pichethc,
Isabel B. Yamanakaa,d, Rafaela L. Fogaçaa,b, Vanete Thomaz-Soccolb,d,
Ricardo A. Machado-de-Avilae, Carlos Chávez-Olórteguif, Maria Rita Sierakowskic,
⁎
Rilton Alves de Freitasc, Larissa M. Alvarengaa,b,d, Juliana de Mouraa,b,d,
a
Departamento de Patologia Básica, UFPR, Curitiba, PR, Brazil
b
Pós-graduação em Engenharia de Bioprocessos e Biotecnologia, UFPR, Curitiba, PR, Brazil
c
BioPol - Departamento de Química, UFPR, Curitiba, PR, Brazil
d
Pós graduação em Microbiologia, Parasitologia e Patologia, UFPR, Curitiba PR, Brazil
e
Pós graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil
f
Departamento de Bioquímica e Imunologia, UFMG, Belo Horizonte, MG, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: The biotechnological evolution towards the development of antigens to detect leprosy has been progressing.
Leprosy diagnosis However, the identification of leprosy in paucibacillary patients, based solely on the antigen-antibody interac-
Ag85B tion still remains a challenge. The complexity of clinical manifestations requires innovative approaches to im-
Mimotope prove the sensitivity of assays to detect leprosy before the onset of symptoms, thus avoiding disabilities and
Synthetic peptide
contributing, indirectly, to reduce transmission. In this study, the strategies employed for early leprosy diagnosis
Immunosensor
QCM
were: i. using a phage-displayed mimotope (APDDPAWQNIFNLRR) which mimics an immunodominant se-
quence (PPNDPAWQRNDPILQ) of an antigen of Mycobacterium leprae known as Ag85B; ii. engineering the
mimotope by adding a C-terminal flexible spacer (SGSG-C); iii. conjugating the mimotope to a carrier protein to
provide better exposure to antibodies; iv. amplifying the signal using biotin–streptavidin detection system in an
ELISA; and v. coating the optimized mimotope on a quartz crystal microbalance (QCM) sensor for label-free
biosensing. The ELISA sensitivity increased up to 91.7% irrespective of the immunological profile of the 132
patients assayed. By using comparative modeling, the M. tuberculosis Ag85B was employed as a template to
ascertain which features make the mimotope a good antigen in terms of its specificity. For the first time, a
sensitive QCM-based immunosensor to detect anti M. leprae antibodies in human serum was used. M. leprae
antibodies could also be detected in the sera of paucibacillary patients; thus, the use of a mimotope-derived
synthetic peptide as bait for antibodies in a novel analytical label-free immunoassay for leprosy diagnosis ex-
hibits great potential.

1. Introduction untreated, undiagnosed, or subclinical patients [3].

Leprosy is a chronic granulomatous infection that affects the skin,
nasal mucosa, and peripheral nerves caused by the intracellular bac-
terial species Mycobacterium leprae also known as Hansen's bacillus [1].
In 2016, 214,783 new cases of leprosy were reported around the
world, and approximately 74.8% of these patients were identified in
India and Brazil (135,485 and 25,218 respectively) [2]. A large number
of these cases involved children less than 15 years of age. Such a high
incidence of leprosy indicates the persistent transmission from
Although the infection is highly responsive to multidrug therapy,
physical disabilities in feet, hands, and eyes due to neural impairment
are currently irreparable [4]. This fact highlights the importance of and
need for early detection of leprosy.
Diagnosis of leprosy is based on symptoms and clinical manifesta-
tions. However, current bacilloscopic and pathological techniques are
unable to detect the infection in asymptomatic patients [5]. Although
nothing replaces an assessment by an experienced physician, the com-
plexity of clinical manifestations of leprosy demands the development

⁎
Corresponding author at: UFPR – Setor de Ciências Biológicas, Departamento de Patologia Básica, Jardim das Américas, CEP 81531-990, Curitiba, Paraná, Brazil.
E-mail address: julianademoura@ufpr.br (J. de Moura).
1
These authors have equally contributed to this work.

https://doi.org/10.1016/j.talanta.2018.05.027
Received 25 March 2018; Received in revised form 7 May 2018; Accepted 7 May 2018
Available online 09 May 2018
0039-9140/ © 2018 Elsevier B.V. All rights reserved.
J.F. de Santana et al.
of new tools based on antigen-specific response to confirm the diag-
nosis, suggest treatment, classify the clinical forms of leprosy, identify
reactional states [6], and detect the disease before the onset of symp-
toms [7] resulting in positive clinical outcomes and control of disease
transmission.
Intensive efforts have been made to find specific antigens able to
detect M. leprae infection and confirm the diagnosis such as phenolic
glycolipid I (PGL-I) [8], and recombinant proteins or semisynthetic
glycoproteins such as LID-1 [9], LID-ND-O [10], and antigen 85B
(Ag85B; ML2028) [11].
In particular, Ag85B is recognized for its high immunogenicity and
antigenicity. This antigen is part of a group of proteins which are re-
sponsible for inducing cell-mediated responses [12] and is considered a
potential candidate in leprosy diagnosis [11]. Its immunodominant
character allowed us to select a peptide (APDDPAWQNIFNLRR) with a
sequence similar to a part of the amino acid sequence of Ag85B, pos-
sessing high serological reactivity [13], using a phage-display library.
However, it was necessary to test whether the peptide had the potential
to mimic Ag85B [14], because to be considered a mimotope, the pep-
tide must not only bind to the antibody, but also induce the production
of antibodies against the mimicked sequence [15].
Advances have been made in the identification and development of
novel antigens to detect leprosy. However, although the reactivity of
these antigens is high in multibacillary patients, they had limited po-
tential in identifying paucibacillary patients [16].
This scenario would change if strategies such as biotin–streptavidin
signal amplification [17], or techniques to expose the antigen more
efficiently to the patient's antibodies [18] are developed to enhance the
assay sensitivity. Moreover, the inclusion of flexible spacers may in-
crease the solubility as well as the flexibility of antigenic peptide targets
facing the antibody [19]. Innovative devices, such as biosensors, could
also be used in leprosy serology for detection of low-titer antibodies.
Based on pioneer studies in which synthetic peptides were coated on
a quartz crystal microbalance (QCM) sensor [20], the peptide APDDP-
AWQNIFNLRR [13] was modified by the addition of a C-terminal
flexible spacer containing a sequence of serine and glycine amino-acids
(SGSG), and a C-terminal cysteine to facilitate its coating on a glass
slide (chip) covered with a thin layer of inert metal such as gold [20].
The QCM is a label-free device with capable of detecting alterations
in the resonant frequency shift (Δf) of a quartz crystal, which is pro-
portional to the changes in mass or thickness of the chip that occurs due
to a molecular interaction between an analyte and its target adsorbed
on gold surface [20,21]. One advantage of label-free biosensors is the
requirement of a single recognition element; this feature tends to sim-
plify and optimize the analysis in terms of lesser execution time and
reduction in reagent costs. Therefore, the QCM sensor has great po-
tential for biomedical applications [22].
In this work, in the phage-displayed peptide APDDPAWQNIFNLRR,
characterized as an Ag85B mimotope, a spacer sequence was included
that allowed its coating on a chip and the label-free detection of the
antibodies of the paucibacillary leprosy patients. Taken together, the
data indicate, for the first time, that a synthetic mimotope may be
employed for the development of an innovative, inexpensive, specific,
and sensitive QCM-based immunosensor for leprosy diagnosis.
2. Materials and methods
2.1. Mycobacterium leprae mimotope-based peptide
Based on the mimotope sequence of M. leprae antigens and its re-
activity against leprosy patients’ sera by alanine scanning as previously
described [13], a linear peptide with the sequence APDDPAWQNIFN-
LRR (patent BR00221103624550) and its homolog APDDPAWQNIFN-
LRRSGSG containing a glycine/serine spacer (GSGS) were used for this
study. Both peptides were tailored with a C-terminal cysteine and
synthesized by Peptide 2.0 Inc. (Chantilly, VA, USA).
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Talanta 187 (2018) 165–171
2.2. Coupling of peptide to carrier proteins and mouse immunization
The peptide APDDPAWQNIFNLRRC coupled to a carrier protein
(patent BR1020150047800) was obtained as previously described [23].
After approval by the Institutional Animal Care Committee (No.
93–2017 CEUA/BIO – UFPR), 6–8 week-old female BALB/c mice were
immunized with 5 µg of keyhole limpet hemocyanin (KLH)–peptide
conjugate diluted in 50 mmol L−1 phosphate buffered saline, pH 7.4
(PBS), and emulsified with incomplete Freund's adjuvant. After the first
subcutaneous immunization, the subsequent injections were adminis-
tered intraperitoneally after 3-, 2-, and 1-week intervals. The sera were
collected one week after the third immunization and analyzed by ELISA
against non-coupled peptide. Control groups included preimmune mice
and mice injected as described above with the carrier KLH alone in the
adjuvant. Complete Freund's adjuvant was not used because it is com-
posed of inactivated mycobacteria.
To reduce nonspecific interactions of the anticarrier antibodies
without impairing antibody-epitope binding, the antiKLH-peptide
mouse sera were preincubated on dots of 30 μg KLH immobilized on a
PVDF membrane previously blocked with 5% (w/v) fat-free milk di-
luted in 0.3% (v/v) Tween 20 and PBS. Two hours post incubation of
mouse serum (1:500), the precleaned supernatant was recovered to be
probed on the Ag85B spot membrane.
2.3. Ag85B spot-synthesized peptides and anti peptide assay
Overlapping pentadecapeptides offset of three amino-acids scanning
the Ag85B sequence (ML2028) from M. leprae (GenBank accession
number: CAA43269.1), including the signal peptide and a random
peptide as control, were synthesized with fluorenylmethyloxycarbonyl
(Fmoc)-protection group on cellulose membranes [24] using an auto-
mated spot peptide synthesizer (Intavis Bioanalytical Instruments, Köln,
Germany). Nonspecific binding to the membranes was blocked by
overnight agitation at 4 °C with 3% (w/v) bovine serum albumin (BSA)
dissolved in PBS. Subsequently, the membranes were washed with PBS-
T (0.1% Tween 20 and PBS) and probed with preimmune, antiKLH,
antiKLH-peptide sera, or KLH dot supernatant for 90 min at 37 °C under
agitation followed by peroxidase-conjugated secondary antibodies
(1:50,000) diluted in 0.1% (w/v) BSA and PBS-T. After extensive wa-
shes, positive spots were visualized using the ECL™ system.
2.4. Three-dimensional modeling of antigen 85B from M. leprae
The 3D structural models of the M. leprae Ag85B (GenBank acces-
sion number: CAA43269.1) and M. bovis Ag85B (BCG strain Moreau,
GenBank, accession number: CCC64491.1) [25] were obtained by
homology modeling [26] using the X-ray diffraction structure of Ag85B
from M. tuberculosis as template (GenBank, accession number:
CAA44269.1; PDB accession code: 1F0N) [27]. The predicted epitopes
were analyzed using Swiss-PdbViewer package [28] and PyMol soft-
ware (PyMOL Molecular Graphics System, Version 2.0 Schrödinger,
LLC).
2.5. Patients
After signing an informed consent form, 132 patients with leprosy
(72 men and 60 women, mean ± SD age: 40.2 ± 20.3 years), classi-
fied according to WHO, from seven municipalities in the state of Mato
Grosso (MT), Brazil, namely, Terra Nova do Norte (n = 3), Matupá
(n = 14), Novo Mundo (n = 14), Marcelândia (n = 14), Guarantã do
Norte (n = 23), Peixoto Azevedo (n = 30), and Colider (n = 34) were
recruited for this study. In addition, 10 patients with tuberculosis (6
men and 4 women, mean ± SD age: 47.2 ± 14.2 years old) from
Hospital Regional da Lapa São Sebastião, Lapa – Paraná (PR), Brazil,
and 44 blood donors (21 men and 13 women, mean ± SD age:
33.7 ± 10.3 years old) from Hemepar, Curitiba - PR, Brazil were
J.F. de Santana et al.
enrolled in this study. Peripheral blood samples were collected from the
patients, centrifuged for 10 min at 1000 ×g and room temperature, and
stored at − 20 °C. All the experiments were conducted in accordance to
the Declaration of Helsinki Principles and approved by ethical com-
mittee (No. 1080.005.11.02, CEP/SD). The diagnosis of leprosy was
made based on clinical symptoms and skin smear results according to
the review of each patient's medical chart.
2.6. Carried peptide-based ELISA
To verify the antigenicity of the APDDPAWQNIFNLRRSGSG-KLH,
an indirect ELISA was conducted using human sera according to the
procedure described below. Each microtitration plate (Nunc, Roskilde,
Denmark) was coated with 10 μg mL−1 of carried peptide or KLH in
50 mmol L−1 sodium carbonate/bicarbonate buffer, pH 9.6, and kept
overnight at 4 °C. After washing with saline solution containing 0.05%
Tween 20, the plates were blocked with Protein-Free Blocking Buffer
(Thermo Fisher Scientific) for 1 h at 37 °C. After being washed again,
the plate was incubated with sera of leprosy, tuberculosis (TB), or ne-
gative control (NC) patients diluted to 1:50 in incubation buffer (0.25%
casein, 0.05% Tween 20 in PBS, pH 7.4) for 1 h at 37 °C and washed.
Antibody binding was determined by antihuman IgG (Fc-specific)-
biotin antibody (Sigma-Aldrich) (diluted to 1:2500) in incubation
buffer for 1 h at 37 °C. After washing, the plate was incubated with
streptavidin-peroxidase (diluted to 1:10,000) in incubation buffer for
1 h at 37 °C. The reaction was detected by adding o-phenylenediamine
dihydrochloride. After 15 min, the reaction was interrupted with 20 µL
of 2 mol L−1 H2SO4, and the absorbance was measured at 492 nm.
Based on the absorbance values, the patients’ sera were classified as
high titer (HT), medium titer (MT), and low titer (LT) if the absorbance
was two-, three-, or four-fold the cutoff, respectively. Samples with
result below the cutoff were considered non-reactive.
2.7. Quartz Crystal Microbalance analysis
The interaction of the non-coupled synthetic peptide (APDDPAW-
QNIFNLRRSGSGC) with the antibodies from leprosy patients, tubercu-
losis patients, or control group was analyzed in a QCM device (SRS,
Stanford Research Systems). Prior to the peptide immobilization, gold
quartz crystals (SRS, 5 MHz) were immersed in piranha solution
1:3H2O2/H2SO4 for 15 min, washed twice with absolute ethanol for
5 min, followed by three times washing with ultrapure water for 5 min,
and dried with a gentle flow of nitrogen. Afterwards, 200 µL of the
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Talanta 187 (2018) 165–171
peptide dissolved in ultrapure water (500 µg mL−1) was deposited over
each crystal for 16 h at 4 °C in a humid chamber. The crystals were
rinsed in three sequential ultrapure water baths, dried at 22 °C, and
blocked with 1% BSA solution for 1 h at 37 °C. After the incubation, all
sample chips were washed and dried as described above. The crystals
were placed in a QCM flow chamber apparatus connected to a syringe
pump with a 100 µL min−1 flow rate (KD Scientific). An initial hydra-
tion step with 500 µL of ultrapure water was done, and 250 µL of each
sample (HT, MT, LT, TB, and NC) (diluted to 1:50) was injected in in-
dividual experiments. Finally, 500 µL of ultrapure water was pumped
into the system on 340 s. Each experiment was done in triplicates and
the results were expressed as the average value.
2.8. Experimental data analysis
The analysis of the peptide-based ELISA was performed with the
Kruskal–Wallis test and the Dunn's multiple comparison test. The cutoff
value was determined by receiver operating characteristic (ROC) ana-
lysis. To maximize both, sensitivity and specificity, the Youden's index
(J=Sn + Sp − 1) and Euclidean distance (D = √(1-Sensitivity)2 + (1 -
Specificity)2) parameters were applied [29]. All statistical analyses
were performed with GraphPad Prism version 6.0 or Microsoft Office
365 when necessary. P value of < 0.05 was considered statistically
significant.
3. Results and discussion
3.1. Mimotope-specific antibodies recognize an Ag85B linear peptide
Taking into consideration the importance of early leprosy detection
[30]and the few advances made to improve its serological diagnosis, in
terms of new approaches applying immunodominant peptides, a novel
phage-displayed peptide (APDDPAWQNIFNLRR) [13,31] was char-
acterized for its immunogenic and antigenic potential.
Polyclonal antibodies against KLH-conjugated peptide were pro-
duced in mice and assayed on an overlapping pentadecapeptide mem-
brane of M. leprae Ag85B (Fig. 1A). The preimmune sera presented no
detectable specific antibodies (Fig. 1B) while antiKLH sera, due to high
immunogenicity of this metalloprotein, cross-reacted with several
Ag85B spots (Fig. 1C). In both the serum samples, before (Fig. 1D) and
after the preclearing treatment (Fig. 1E), antiKLH-peptide polyclonal
antibodies showed reactivity against the spot corresponding to the
amino-acid sequence PPNDPAWQRNDPILQ, indicating that the Ag85B
Fig. 1. Mycobacterium leprae Ag85B peptide is recognized
by anti mimotope antibodies. A) Overlapping pentadeca-
peptides covering the M. leprae Ag85B sequence were
synthesized on a cellulose membrane which was incubated
with B) preimmune, C) antiKLH, D) antiKLH-peptide
(APDDPAWQNIFNLRRC coupled to KLH), and E) antiKLH-
peptide mouse sera after preclearing with immobilized
KLH. The spot corresponding to mimicked sequence
(PPNDPAWQRNDPILQ) found in D) and E) is indicated by
an arrow.
J.F. de Santana et al.
sequence is mimicked by the phage-displayed peptide APDDPAWQNI-
FNLRR.
Taking into account the fact that a mimotope is an amino-acid se-
quence with physical and chemical properties similar to the original
epitope [14,15], the use of a mimotope-derived synthetic peptide as an
antigen may favor specific antibody binding and prevent possible cross-
reactions, because diagnostically irrelevant epitopes present in the
whole antigen are excluded [18]. These molecules also present stability
and other technical properties that can be efficiently monitored. Besides
that, synthetic peptides can be easily engineered and produced at a low
cost [32].
3.2. In silico analysis of M. leprae Ag85B epitopic region and homologous
proteins
Considering that Ag85B is highly conserved in mycobacterial spe-
cies, including M. tuberculosis [33], that tuberculosis is an endemic
disease in developing countries [34], and M. bovis Bacillus Calmette-
Guérin (BCG) vaccination is mandatory at birth in countries like Brazil,
the mimicked sequence of M. leprae Ag85B was compared to two other
homologous regions from M. bovis and M. tuberculosis Ag85B to ascer-
tain which features would make this region a specific antigen suitable
for leprosy diagnosis, since the identity percentages between the former
two proteins and M. tuberculosis Ag85B are 85.21% and 99.65%, re-
spectively.
Indeed, in silico analysis indicated that the peptide region is readily
accessible and structurally similar between the proteins, even though
the mimicked sequence of M. leprae Ag85B (PPNDPAWQRNDPILQ) and
the mimotope (APDDPAWQNIFNLRR) have proline (P) as the second
residue followed by asparagine (N) and aspartic acid (D), or two as-
partic acid residues, respectively, while in the corresponding sequence
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Talanta 187 (2018) 165–171
of M. tuberculosis and M. bovis (PSSDPAWERNDPTQQ) there are two
serine (S) residues at these positions (Fig. 2A).
The presence of aspartic acid or asparagine residues (Fig. 2B) may
not be directly responsible for the specificity, but may influence the cis
or trans position adopted by the proline [35] and, as a consequence,
facilitate recognition by leprosy antibodies from patients, thus avoiding
nonspecific reactions with antibodies in tuberculosis patients or healthy
volunteers who are vaccinated with BCG. Using nuclear magnetic re-
sonance (NMR), Dyson and colleagues [36] have found that when a
proline is followed by an aspartic acid or an asparagine in a sequence
YPXDV, the cis isomer is predominant in aqueous solutions and is sta-
bilized by the negatively charged side-chains of these residues.
3.3. Carried peptide-based ELISA discriminates leprosy from tuberculosis
patients and healthy volunteers
Sera from 132 patients from Mato Grosso with different leprosy
classifications showed reactivity to the carried peptide APDDPAWQN-
IFNLRRSGSGC-KLH. Besides leaving the reactive N-terminal region
more exposed to the antibodies [13], the strategy of addition of a C-
terminal GSGS sequence and coupling with a carrier molecule may have
provided higher flexibility and conformational freedom to the molecule
[18] resulting in reduction of possible nonspecific interactions, and
consequently, maintaining the specificity of the analysis.
According to the results obtained by the indirect ELISA (Fig. 3A)
and the cutoff established by ROC curve analysis (Fig. 3B), sera of le-
prosy patients could be classified irrespective of them being pauci- or
multi-bacillary (Fig. 3C).
The area under the ROC curve (AUC) for detection of antibodies was
0.95 with an optimal cutoff of 0.204, confirmed by the Youden's index
and Euclidean distance, with a sensitivity of 91.7% and specificity of
Fig. 2. Ag85B epitope sequence analysis. A)
Mimicked M. leprae Ag85B peptide aligned to
the corresponding sequences of M. tuberculosis
and M. bovis Ag85B. Bold letters show identical
residues and the dash indicates a gap to allow a
better alignment. B) The Ag85B epitope re-
cognized by anti-mimotope antibodies is the
sequence in red and the homologous sequence
in M. tuberculosis Ag85B is highlighted in
green.
J.F. de Santana et al. Talanta 187 (2018) 165–171

A B

C D

Fig. 3. KLH-conjugated peptide-based ELISA can identify leprosy patients. A) Carried peptide-based ELISA illustration using antihuman IgG-biotin (b) as antibody
and streptavidin-HRP (S) as signal amplifier. B) ROC curve indicating the cutoff value (A492 = 0.204). C) Patients grouped according to the reactivity against the
carried peptide, and D) immunological profile. NC: negative control; NR: non-reactive serum; LT, IT, and HT: low, intermediate, and high titer sera, respectively. PB,
MB, and IND: paucibacillary, multibacillary, and indeterminate leprosy, respectively. **** P < 0.0001.

100%.
The magnitude of antibody reactivity against the synthetic peptide
was significantly higher in sera of patients with leprosy, even in those
with low titer (LT), in comparison to 44 samples from healthy volun-
teers (negative control, NC) (x̅ = 0.149 ± 0.029) (P < 0.0001)
(Fig. 3C). As expected, almost half of the patients (48%) had their sera
grouped as low titer (0.204 ≤ A492 < 0.408), while 26% and 17% were
classified as intermediate (IT) (0.408 ≤ A492 < 0.612) and high titer
(HT) (0.612 ≤ A492), respectively. Eleven samples of sera (8.3%) did
not reach the cutoff absorbance values (x̅ = 0.143 ± 0.030) (Fig. 3C
and D).
Our data indicate that this biomarker presents an analytical ad-
vantage, i.e., it has the capacity of detecting leprosy in patients from
different regions [9]; the mimotope APDDPAWQNIFNLRR was obtained
by bioselection from a phage display library using antibodies from le-
prosy patients form Curitiba, Paraná state, in Southern Brazil [13]
which is located about 2500 km far from north of Mato Grosso state,
where the samples were collected for analysis.
3.4. Ag85B mimotope-derived peptide sensor detects leprosy patients’
antibodies
Although the ELISA results were promising, the open question was
whether it was possible to detect low-titer antibodies, i.e., antibodies in
paucibacillary patients, using a synthetic peptide-based sensor [20].
Despite the fact that the QCM has been extensively exploited in the
field of public health because of its sensitivity, specificity, low cost, and
portability [22,37], most of the QCM devices allow searching for the
etiological agent by coating specific antibodies on the quartz crystal
transducer [38], while very few studies are designed to detect anti-
bodies in patient samples [39].
According to QCM frequency monitoring, antibodies from leprosy-
positive serum revealed an irreversible attachment towards the peptide-
coated surface and no desorption was observed after the washing steps.
In contrast, the frequency signal returned to the original value in case of
negative sera due to desorption of nonspecific antibodies during
washing (Fig. 4A and B).
When leprosy patients’ sera with different titers (assessed by ELISA)
were assayed against the mimotope on the electrode, the frequency
shift was proportional to the titer of each serum, i. e., Δf
= 277 ± 70 Hz, 196 ± 45 Hz, and 123 ± 59 Hz for high, inter-
mediate, and low titer sera (Table 1), respectively, confirming that
QCM reveals mass variation per unit area [21]. Therefore, our results
indicated that the antibodies from positive sera interacted strongly with
the mimotope, based on the irreversible adsorption on the gold sensor;
whereas in the negative sera, the nonspecific antibodies desorbed after
washing (Fig. 4C).
Our data indicate that the strategy of implementing a label-free
device bound to a highly antigenic molecule is likely to be an effective
way to detect positive patients for leprosy even when they exhibit un-
detectable antibodies under ELISA.

169
J.F. de Santana et al.
Fig. 4. Mimotope coated QCM biosensor assays. Schematic illustration of fre-
quency shifts (Δf) after mimotope immobilization on coated sensor surface,
washing, and exposure to positive A) and negative control B) samples. The QCM
results C) indicate the mimotope's ability to detect leprosy in patients with
different antibody titers and its specificity compared to tuberculosis patients’
and healthy volunteers’ samples. NC: negative control; LT, IT, and HT: low,
intermediate, and high antibody titer sera, respectively, classified according to
KLH-conjugated peptide-based ELISA.
Table 1
Measured resonant frequency shifts (Δf) of the QCM-based Ag85B mimotope
sensor for different samples after rinsing step.
Samples HT IT LT TB NC
Δf (Hz) − 277 ± 70 − 196 ± 45 − 123 ± 59 50 ± 28 17 ± 13
Note: HT = high titer, IT = intermediate titer, and LT = low titer sera; TB
= tuberculosis patients’ sera; NC = negative control sera of healthy volunteers.
4. Conclusion
An optimized peptide sequence representing a mimotope of M. le-
prae Ag85B was efficient in distinguishing antibodies of leprosy patients
from tuberculosis and BCG vaccinated volunteers using ELISA. ELISA's
failure in detecting low titer antibodies in paucibacillary patients was
suppressed when the synthetic peptide was assayed on a QCM sensor.
Although further studies are required, the results obtained with this
label-free mass-sensitive technique demonstrate, for the first time, the
sensitivity of this promising biotechnological platform for the early
detection of leprosy.
Acknowledgments
This study was supported by CNPq (445136/2014-6). We would like
to thank Alessandra Becker-Finco for her skillful technical
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Talanta 187 (2018) 165–171
contributions, and Marilde Lernen and Veroni Pansera for collecting
patients’ samples. We are also grateful to the following institutions for
their support to the respective authors: CAPES (JFS, GFP, IBY, RLF, and
MRS) and CNPq (VTS, CCO, MRS, RAF, LMA, and JM).
Conflicts of interest
The authors declare that there is no conflict of interest.
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