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Talanta 187 (2018) 165–171

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

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,
Departamento de Patologia Básica, UFPR, Curitiba, PR, Brazil
Pós-graduação em Engenharia de Bioprocessos e Biotecnologia, UFPR, Curitiba, PR, Brazil
BioPol - Departamento de Química, UFPR, Curitiba, PR, Brazil
Pós graduação em Microbiologia, Parasitologia e Patologia, UFPR, Curitiba PR, Brazil
Pós graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil
Departamento de Bioquímica e Imunologia, UFMG, Belo Horizonte, MG, Brazil


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
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].

Although the infection is highly responsive to multidrug therapy,
Leprosy is a chronic granulomatous infection that affects the skin, physical disabilities in feet, hands, and eyes due to neural impairment
nasal mucosa, and peripheral nerves caused by the intracellular bac- are currently irreparable [4]. This fact highlights the importance of and
terial species Mycobacterium leprae also known as Hansen's bacillus [1]. need for early detection of leprosy.
In 2016, 214,783 new cases of leprosy were reported around the Diagnosis of leprosy is based on symptoms and clinical manifesta-
world, and approximately 74.8% of these patients were identified in tions. However, current bacilloscopic and pathological techniques are
India and Brazil (135,485 and 25,218 respectively) [2]. A large number unable to detect the infection in asymptomatic patients [5]. Although
of these cases involved children less than 15 years of age. Such a high nothing replaces an assessment by an experienced physician, the com-
incidence of leprosy indicates the persistent transmission from 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: (J. de Moura).
These authors have equally contributed to this work.
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. Talanta 187 (2018) 165–171

of new tools based on antigen-specific response to confirm the diag- 2.2. Coupling of peptide to carrier proteins and mouse immunization
nosis, suggest treatment, classify the clinical forms of leprosy, identify
reactional states [6], and detect the disease before the onset of symp- The peptide APDDPAWQNIFNLRRC coupled to a carrier protein
toms [7] resulting in positive clinical outcomes and control of disease (patent BR1020150047800) was obtained as previously described [23].
transmission. After approval by the Institutional Animal Care Committee (No.
Intensive efforts have been made to find specific antigens able to 93–2017 CEUA/BIO – UFPR), 6–8 week-old female BALB/c mice were
detect M. leprae infection and confirm the diagnosis such as phenolic immunized with 5 µg of keyhole limpet hemocyanin (KLH)–peptide
glycolipid I (PGL-I) [8], and recombinant proteins or semisynthetic conjugate diluted in 50 mmol L−1 phosphate buffered saline, pH 7.4
glycoproteins such as LID-1 [9], LID-ND-O [10], and antigen 85B (PBS), and emulsified with incomplete Freund's adjuvant. After the first
(Ag85B; ML2028) [11]. subcutaneous immunization, the subsequent injections were adminis-
In particular, Ag85B is recognized for its high immunogenicity and tered intraperitoneally after 3-, 2-, and 1-week intervals. The sera were
antigenicity. This antigen is part of a group of proteins which are re- collected one week after the third immunization and analyzed by ELISA
sponsible for inducing cell-mediated responses [12] and is considered a against non-coupled peptide. Control groups included preimmune mice
potential candidate in leprosy diagnosis [11]. Its immunodominant and mice injected as described above with the carrier KLH alone in the
character allowed us to select a peptide (APDDPAWQNIFNLRR) with a adjuvant. Complete Freund's adjuvant was not used because it is com-
sequence similar to a part of the amino acid sequence of Ag85B, pos- posed of inactivated mycobacteria.
sessing high serological reactivity [13], using a phage-display library. To reduce nonspecific interactions of the anticarrier antibodies
However, it was necessary to test whether the peptide had the potential without impairing antibody-epitope binding, the antiKLH-peptide
to mimic Ag85B [14], because to be considered a mimotope, the pep- mouse sera were preincubated on dots of 30 μg KLH immobilized on a
tide must not only bind to the antibody, but also induce the production PVDF membrane previously blocked with 5% (w/v) fat-free milk di-
of antibodies against the mimicked sequence [15]. luted in 0.3% (v/v) Tween 20 and PBS. Two hours post incubation of
Advances have been made in the identification and development of mouse serum (1:500), the precleaned supernatant was recovered to be
novel antigens to detect leprosy. However, although the reactivity of probed on the Ag85B spot membrane.
these antigens is high in multibacillary patients, they had limited po-
tential in identifying paucibacillary patients [16]. 2.3. Ag85B spot-synthesized peptides and anti peptide assay
This scenario would change if strategies such as biotin–streptavidin
signal amplification [17], or techniques to expose the antigen more Overlapping pentadecapeptides offset of three amino-acids scanning
efficiently to the patient's antibodies [18] are developed to enhance the the Ag85B sequence (ML2028) from M. leprae (GenBank accession
assay sensitivity. Moreover, the inclusion of flexible spacers may in- number: CAA43269.1), including the signal peptide and a random
crease the solubility as well as the flexibility of antigenic peptide targets peptide as control, were synthesized with fluorenylmethyloxycarbonyl
facing the antibody [19]. Innovative devices, such as biosensors, could (Fmoc)-protection group on cellulose membranes [24] using an auto-
also be used in leprosy serology for detection of low-titer antibodies. mated spot peptide synthesizer (Intavis Bioanalytical Instruments, Köln,
Based on pioneer studies in which synthetic peptides were coated on Germany). Nonspecific binding to the membranes was blocked by
a quartz crystal microbalance (QCM) sensor [20], the peptide APDDP- overnight agitation at 4 °C with 3% (w/v) bovine serum albumin (BSA)
AWQNIFNLRR [13] was modified by the addition of a C-terminal dissolved in PBS. Subsequently, the membranes were washed with PBS-
flexible spacer containing a sequence of serine and glycine amino-acids T (0.1% Tween 20 and PBS) and probed with preimmune, antiKLH,
(SGSG), and a C-terminal cysteine to facilitate its coating on a glass antiKLH-peptide sera, or KLH dot supernatant for 90 min at 37 °C under
slide (chip) covered with a thin layer of inert metal such as gold [20]. agitation followed by peroxidase-conjugated secondary antibodies
The QCM is a label-free device with capable of detecting alterations (1:50,000) diluted in 0.1% (w/v) BSA and PBS-T. After extensive wa-
in the resonant frequency shift (Δf) of a quartz crystal, which is pro- shes, positive spots were visualized using the ECL™ system.
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 2.4. Three-dimensional modeling of antigen 85B from M. leprae
on gold surface [20,21]. One advantage of label-free biosensors is the
requirement of a single recognition element; this feature tends to sim- The 3D structural models of the M. leprae Ag85B (GenBank acces-
plify and optimize the analysis in terms of lesser execution time and sion number: CAA43269.1) and M. bovis Ag85B (BCG strain Moreau,
reduction in reagent costs. Therefore, the QCM sensor has great po- GenBank, accession number: CCC64491.1) [25] were obtained by
tential for biomedical applications [22]. homology modeling [26] using the X-ray diffraction structure of Ag85B
In this work, in the phage-displayed peptide APDDPAWQNIFNLRR, from M. tuberculosis as template (GenBank, accession number:
characterized as an Ag85B mimotope, a spacer sequence was included CAA44269.1; PDB accession code: 1F0N) [27]. The predicted epitopes
that allowed its coating on a chip and the label-free detection of the were analyzed using Swiss-PdbViewer package [28] and PyMol soft-
antibodies of the paucibacillary leprosy patients. Taken together, the ware (PyMOL Molecular Graphics System, Version 2.0 Schrödinger,
data indicate, for the first time, that a synthetic mimotope may be LLC).
employed for the development of an innovative, inexpensive, specific,
and sensitive QCM-based immunosensor for leprosy diagnosis. 2.5. Patients

2. Materials and methods After signing an informed consent form, 132 patients with leprosy
(72 men and 60 women, mean ± SD age: 40.2 ± 20.3 years), classi-
2.1. Mycobacterium leprae mimotope-based peptide fied according to WHO, from seven municipalities in the state of Mato
Grosso (MT), Brazil, namely, Terra Nova do Norte (n = 3), Matupá
Based on the mimotope sequence of M. leprae antigens and its re- (n = 14), Novo Mundo (n = 14), Marcelândia (n = 14), Guarantã do
activity against leprosy patients’ sera by alanine scanning as previously Norte (n = 23), Peixoto Azevedo (n = 30), and Colider (n = 34) were
described [13], a linear peptide with the sequence APDDPAWQNIFN- recruited for this study. In addition, 10 patients with tuberculosis (6
LRR (patent BR00221103624550) and its homolog APDDPAWQNIFN- men and 4 women, mean ± SD age: 47.2 ± 14.2 years old) from
LRRSGSG containing a glycine/serine spacer (GSGS) were used for this Hospital Regional da Lapa São Sebastião, Lapa – Paraná (PR), Brazil,
study. Both peptides were tailored with a C-terminal cysteine and and 44 blood donors (21 men and 13 women, mean ± SD age:
synthesized by Peptide 2.0 Inc. (Chantilly, VA, USA). 33.7 ± 10.3 years old) from Hemepar, Curitiba - PR, Brazil were

J.F. de Santana et al. Talanta 187 (2018) 165–171

enrolled in this study. Peripheral blood samples were collected from the peptide dissolved in ultrapure water (500 µg mL−1) was deposited over
patients, centrifuged for 10 min at 1000 ×g and room temperature, and each crystal for 16 h at 4 °C in a humid chamber. The crystals were
stored at − 20 °C. All the experiments were conducted in accordance to rinsed in three sequential ultrapure water baths, dried at 22 °C, and
the Declaration of Helsinki Principles and approved by ethical com- blocked with 1% BSA solution for 1 h at 37 °C. After the incubation, all
mittee (No. 1080.005.11.02, CEP/SD). The diagnosis of leprosy was sample chips were washed and dried as described above. The crystals
made based on clinical symptoms and skin smear results according to were placed in a QCM flow chamber apparatus connected to a syringe
the review of each patient's medical chart. 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-
2.6. Carried peptide-based ELISA
dividual experiments. Finally, 500 µL of ultrapure water was pumped
into the system on 340 s. Each experiment was done in triplicates and
To verify the antigenicity of the APDDPAWQNIFNLRRSGSG-KLH,
the results were expressed as the average value.
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 2.8. Experimental data analysis
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% The analysis of the peptide-based ELISA was performed with the
Tween 20, the plates were blocked with Protein-Free Blocking Buffer Kruskal–Wallis test and the Dunn's multiple comparison test. The cutoff
(Thermo Fisher Scientific) for 1 h at 37 °C. After being washed again, value was determined by receiver operating characteristic (ROC) ana-
the plate was incubated with sera of leprosy, tuberculosis (TB), or ne- lysis. To maximize both, sensitivity and specificity, the Youden's index
gative control (NC) patients diluted to 1:50 in incubation buffer (0.25% (J=Sn + Sp − 1) and Euclidean distance (D = √(1-Sensitivity)2 + (1 -
casein, 0.05% Tween 20 in PBS, pH 7.4) for 1 h at 37 °C and washed. Specificity)2) parameters were applied [29]. All statistical analyses
Antibody binding was determined by antihuman IgG (Fc-specific)- were performed with GraphPad Prism version 6.0 or Microsoft Office
biotin antibody (Sigma-Aldrich) (diluted to 1:2500) in incubation 365 when necessary. P value of < 0.05 was considered statistically
buffer for 1 h at 37 °C. After washing, the plate was incubated with significant.
streptavidin-peroxidase (diluted to 1:10,000) in incubation buffer for
1 h at 37 °C. The reaction was detected by adding o-phenylenediamine
3. Results and discussion
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.
3.1. Mimotope-specific antibodies recognize an Ag85B linear peptide
Based on the absorbance values, the patients’ sera were classified as
high titer (HT), medium titer (MT), and low titer (LT) if the absorbance
Taking into consideration the importance of early leprosy detection
was two-, three-, or four-fold the cutoff, respectively. Samples with
[30]and the few advances made to improve its serological diagnosis, in
result below the cutoff were considered non-reactive.
terms of new approaches applying immunodominant peptides, a novel
phage-displayed peptide (APDDPAWQNIFNLRR) [13,31] was char-
2.7. Quartz Crystal Microbalance analysis acterized for its immunogenic and antigenic potential.
Polyclonal antibodies against KLH-conjugated peptide were pro-
The interaction of the non-coupled synthetic peptide (APDDPAW- duced in mice and assayed on an overlapping pentadecapeptide mem-
QNIFNLRRSGSGC) with the antibodies from leprosy patients, tubercu- brane of M. leprae Ag85B (Fig. 1A). The preimmune sera presented no
losis patients, or control group was analyzed in a QCM device (SRS, detectable specific antibodies (Fig. 1B) while antiKLH sera, due to high
Stanford Research Systems). Prior to the peptide immobilization, gold immunogenicity of this metalloprotein, cross-reacted with several
quartz crystals (SRS, 5 MHz) were immersed in piranha solution Ag85B spots (Fig. 1C). In both the serum samples, before (Fig. 1D) and
1:3H2O2/H2SO4 for 15 min, washed twice with absolute ethanol for after the preclearing treatment (Fig. 1E), antiKLH-peptide polyclonal
5 min, followed by three times washing with ultrapure water for 5 min, antibodies showed reactivity against the spot corresponding to the
and dried with a gentle flow of nitrogen. Afterwards, 200 µL of 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. Talanta 187 (2018) 165–171

sequence is mimicked by the phage-displayed peptide APDDPAWQNI- of M. tuberculosis and M. bovis (PSSDPAWERNDPTQQ) there are two
FNLRR. serine (S) residues at these positions (Fig. 2A).
Taking into account the fact that a mimotope is an amino-acid se- The presence of aspartic acid or asparagine residues (Fig. 2B) may
quence with physical and chemical properties similar to the original not be directly responsible for the specificity, but may influence the cis
epitope [14,15], the use of a mimotope-derived synthetic peptide as an or trans position adopted by the proline [35] and, as a consequence,
antigen may favor specific antibody binding and prevent possible cross- facilitate recognition by leprosy antibodies from patients, thus avoiding
reactions, because diagnostically irrelevant epitopes present in the nonspecific reactions with antibodies in tuberculosis patients or healthy
whole antigen are excluded [18]. These molecules also present stability volunteers who are vaccinated with BCG. Using nuclear magnetic re-
and other technical properties that can be efficiently monitored. Besides sonance (NMR), Dyson and colleagues [36] have found that when a
that, synthetic peptides can be easily engineered and produced at a low proline is followed by an aspartic acid or an asparagine in a sequence
cost [32]. YPXDV, the cis isomer is predominant in aqueous solutions and is sta-
bilized by the negatively charged side-chains of these residues.
3.2. In silico analysis of M. leprae Ag85B epitopic region and homologous
proteins 3.3. Carried peptide-based ELISA discriminates leprosy from tuberculosis
patients and healthy volunteers
Considering that Ag85B is highly conserved in mycobacterial spe-
cies, including M. tuberculosis [33], that tuberculosis is an endemic Sera from 132 patients from Mato Grosso with different leprosy
disease in developing countries [34], and M. bovis Bacillus Calmette- classifications showed reactivity to the carried peptide APDDPAWQN-
Guérin (BCG) vaccination is mandatory at birth in countries like Brazil, IFNLRRSGSGC-KLH. Besides leaving the reactive N-terminal region
the mimicked sequence of M. leprae Ag85B was compared to two other more exposed to the antibodies [13], the strategy of addition of a C-
homologous regions from M. bovis and M. tuberculosis Ag85B to ascer- terminal GSGS sequence and coupling with a carrier molecule may have
tain which features would make this region a specific antigen suitable provided higher flexibility and conformational freedom to the molecule
for leprosy diagnosis, since the identity percentages between the former [18] resulting in reduction of possible nonspecific interactions, and
two proteins and M. tuberculosis Ag85B are 85.21% and 99.65%, re- consequently, maintaining the specificity of the analysis.
spectively. According to the results obtained by the indirect ELISA (Fig. 3A)
Indeed, in silico analysis indicated that the peptide region is readily and the cutoff established by ROC curve analysis (Fig. 3B), sera of le-
accessible and structurally similar between the proteins, even though prosy patients could be classified irrespective of them being pauci- or
the mimicked sequence of M. leprae Ag85B (PPNDPAWQRNDPILQ) and multi-bacillary (Fig. 3C).
the mimotope (APDDPAWQNIFNLRR) have proline (P) as the second The area under the ROC curve (AUC) for detection of antibodies was
residue followed by asparagine (N) and aspartic acid (D), or two as- 0.95 with an optimal cutoff of 0.204, confirmed by the Youden's index
partic acid residues, respectively, while in the corresponding sequence 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

J.F. de Santana et al. Talanta 187 (2018) 165–171



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

J.F. de Santana et al. 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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