Accepted Manuscript Generation and application of ssDNA aptamers against glycolipid antigen ManLAM of Mycobacterium tuberculosis for TB diagnosis Xiao-lei Tang, Shi-Min Wu, Yan Xie, Neng Song, Qing Guan, Chunhui Yuan, Xiang Zhou, Xiao-Lian Zhang PII:
S0163-4453(16)00031-1
DOI:
10.1016/j.jinf.2016.01.014
Reference:
YJINF 3669
To appear in:
Journal of Infection
Received Date: 11 August 2015 Revised Date:
20 December 2015
Accepted Date: 14 January 2016
Please cite this article as: Tang X-l, Wu S-M, Xie Y, Song N, Guan Q, Yuan C, Zhou X, Zhang X-L, Generation and application of ssDNA aptamers against glycolipid antigen ManLAM of Mycobacterium tuberculosis for TB diagnosis, Journal of Infection (2016), doi: 10.1016/j.jinf.2016.01.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Generation and application of ssDNA aptamers against glycolipid antigen
2
ManLAM of Mycobacterium tuberculosis for TB diagnosis
3
Xiao-lei Tang1,2, Shi-Min Wu1,3, Yan Xie1, Neng Song1, Qing Guan1, Chunhui
5
Yuan1, Xiang Zhou4, Xiao-Lian Zhang1 *
RI PT
4
6 7
1
8
Immunology, Medical Research Institute, and Department of Immunology Wuhan
9
University School of Medicine, Donghu Road 165#, Wuhan 430071, Hubei Province,
M AN U
SC
State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and
10
China
11
2
12
Wuhu 241000, China
13
3
14
Huazhong University of Science and Technology, China
15
4
16
Wuhan 430072, China
TE D
Wuhan Center for Clinical Laboratory, Puai Hospital, Tongji Medical College,
EP
College of Chemistry and Molecular Sciences, Wuhan University, Hubei Province,
AC C
17
Department of Clinical Laboratory, the Second Hospital of Wuhu, Anhui Province,
18
Xiao-lei Tang and Shi-Min Wu contributed equally to this paper
19
*
20
University School of Medicine, Wuhan 430071, China. E-mail addresses:
21
[email protected].
22
Running title: ManLAM aptamers for TB antigen diagnosis
Corresponding author: Xiao-Lian Zhang, Department of Immunology, Wuhan
1
ACCEPTED MANUSCRIPT ABSTRACT
2
The development of effective Mycobacterial antigen diagnostic reagents remains a
3
high priority. Mannose-capped lipoarabinomannan (ManLAM) is a lipoglycan serving
4
as a major cell wall component. ManLAM is also an early released antigen in the
5
blood circulation system during Mycobacteria tuberculosis (M.tb) infection and is a
6
perfect target antigen for TB diagnosis. In this study, ssDNA aptamers “antibodies”
7
against ManLAM of the predominant clinical epidemic M.tb Beijing genotype strains
8
were generated by the Systematic Evolution of Ligands by Exponential Enrichment
9
(SELEX) technique. The selected single aptamer T9 demonstrated the highest
10
specificity and binding affinity, with an equilibrium dissociation constant (Kd) of
11
668±159 nmol/L. We further detected ManLAM antigens in serum and sputum
12
samples from active pulmonary tuberculosis (aPTB) patients, extrapulmonary TB
13
(EPTB) patients and healthy donors by using a T9 based enzyme-linked
14
oligonucleotide assay (ELONA). The results showed that the specificity and
15
sensitivity were 95.31% and 83.00% (for 100 aPTB serum samples), 98.70% and
16
92.71% (for 96 aPTB sputum samples), and 94.44% and 88.71% (for 62 EPTB serum
17
samples), respectively. A good correlation was observed between the T9
18
aptamer-based ELONA and the clinical T-SPOT.TB. Thus, T9 based ELONA has
19
potentials for diagnosis of TB, including inactive TB, smear-negative TB, EPTB, and
20
TB with immunodeficiency, and assist the diagnosis of LTBI albeit it could not
21
distinguish LTBI and active TB.
AC C
EP
TE D
M AN U
SC
RI PT
1
22 2
ACCEPTED MANUSCRIPT 1
KEY WORDS: aptamer, diagnosis, mannosylated lipoarabinomannan (ManLAM),
2
Mycobacteria tuberculosis (M.tb), tuberculosis (TB), enzyme-linked oligonucleotide
3
assay (ELONA)
4
INTRODUCTION
6
The World Health Organization (WHO) reported that there were 9 million incident
7
cases of tuberculosis (TB) in 2013 with approximately 1.1 million TB cases being
8
co-infected with human immunodeficiency virus (HIV) and 1.5 million TB deaths. 1, 2
9
The emergence of multidrug-resistant tuberculosis (MDR-TB) makes TB control even
10
more urgent.3-5 Mycobacteria tuberculosis (M.tb) usually infects the lungs and causes
11
pulmonary TB, but it can also infect lymph nodes, bone, and other tissues causing
12
extrapulmonary TB (EPTB). M.tb can also exist in host body without causing any
13
clinical symptoms (latent TB infection, LTBI), thus could form new infection sources
14
when latent M.tb bacteria were reactivated. In recent years the Beijing genotype of
15
M.tb has attracted special attention because of its global emergence and
16
drug-resistance potential.6 The occurrence of Beijing genotype strains has been
17
documented in several parts of the world, including Asia, former Soviet Union,
18
Europe, Africa, and the United States, and significantly associated with MDR and
19
responsible for outbreaks of MDR-TB.6-8 Thus far, rapid and accurate diagnosis of TB,
20
especially caused by the predominant clinical epidemic M.tb Beijing genotype strain,
21
remains elusive.
22
AC C
EP
TE D
M AN U
SC
RI PT
5
Mannosylated lipoarabinomannan (ManLAM) is a major and structurally 3
ACCEPTED MANUSCRIPT important component of M.tb cell wall. ManLAM is constantly released from
2
metabolically active or degrading bacterial cells but not dead cells, and its presence
3
was demonstrated in human serum, urine, cerebrospinal fluid and the sputum of TB
4
infected individuals, which makes it a perfect candidate biomarker for TB diagnostic
5
tests.9
RI PT
1
Nucleic acid aptamers are short, single-stranded oligonucleotides that fold into
7
particular structures to bind to target molecules such as small chemicals, sugars, lipids,
8
proteins, or cells. High affinity aptamers for specific target molecules can be isolated
9
from a random nucleic acid library in vitro using the systematic evolution of ligands
10
by exponential enrichment (SELEX).10-12 SELEX involves sequential rounds of
11
selection and application to generate ligands (aptamers) that bind with high affinity to
12
target molecules.10 Aptamers are analogous to antibodies but possess several key
13
advantages: higher specificity and affinity than antibodies; easier chemical
14
modifications and greater stability; smaller molecular weight; and easier and more
15
economical production methods, making aptamers as ideal tools for disease
16
diagnosis13, 14
M AN U
TE D
EP
AC C
17
SC
6
The aim of this study was to screen and generate ssDNA aptamers against
18
ManLAM from the clinical widespread M.tb Beijing genotype. We further applied the
19
selected aptamer for TB antigen diagnosis with serum and sputum samples by an
20
enzyme-linked oligonucleotide assay (ELONA).
21 22
MATERIALS AND METHODS 4
ACCEPTED MANUSCRIPT Ethics Statement
2
The study was approved by the ethics committee of the Wuhan University School of
3
Medicine and Wuhan Medical Treatment Center. Written consent was obtained from
4
all of the participants including children from parents or legal guardians.
RI PT
1
5
Participant inclusion criteria
7
Culture-proven active pulmonary tuberculosis (aPTB) patients，EPTB patients，
8
inactive TB patients and non-tuberculous mycobacteria (NTM) infected patients were
9
prospectively enrolled from Wuhan Medical Treatment Center of Hubei province, the
10
First Affiliated Hospital of Wannan Medical Hospital of Anhui Province and the
11
Third People’s Hospital of Shenzhen of Guangdong Province, and HDs were recruited
12
from Wuhan University Zhongnan Hospital. The diagnosis of TB was based on the
13
following criteria: clinical symptoms of TB/abnormal chest X-ray findings, and
14
positive
15
culture-confirmed samples were included in the aTB group. The LTBI high risk
16
subjects were the front-line health care workers enrolled from Wuhan Medical
17
Treatment Center based on known contact with TB patients and the occupational
18
history of work in a high risk setting. The inactive TB patients were subjects who had
19
a history of previous tuberculous disease, with negative bacteriologic studies, and no
20
clinical evidence of current disease15. The NTM patients were subjects infected with
21
mycobacteria not causing tuberculosis and leprosy. NTM was identified by
22
para-nitrobenzoic acid (PNB) test and Mycobacteria Species Identification Genetic
TE D
M AN U
SC
6
culture
results
(current
“golden
standard”).
Only
AC C
EP
mycobacteria
5
ACCEPTED MANUSCRIPT Detection Kit (Yaneng Bio, China)16. The HDs were determined based on a lack of
2
symptoms of TB, no TB presentation on the X-ray examination, and an
3
epidemiologically low risk of TB infection (no contact with TB). HDs did not include
4
TB suspects later ruled out for TB. All of the subjects were unrelated Chinese of the
5
Han nationality.
RI PT
1
6
Bacterial strains
8
M.tb Beijing genotype strain and M. bovis were kindly provided by Professor Aizhen
9
Guo.17 All mycobacteria were maintained Middlebrook 7H9 medium supplemented
10
with Middlebrook OADC (BD Diagnostics, Sparks, MD, USA), and harvested while
11
in the exponential phase of growth. Bacteria used for aptamer specificity analysis
12
were propagated from laboratory stocks (School of Medicine, Wuhan University,
13
Wuhan, China) included M.tb H37Rv [strain ATCC 27294], BCG (Pasteur strain
14
ATCC 35734), M. smegmatis (strain ATCC 19420), M. avium (strain ATCC25291), E.
15
coli (strain ATCC 25922), S. aureus (strain ATCC25923), S. epidermidis (strain
16
14490); E. faecalis (strain ATCC 19433), P. vulgaris (strain ATCC 6380), S.
17
pneumonia (strain ATCC49619), S. pyogenes /S. hemolyticus (strain ATCC21059), K.
18
pneumonia (strain ATCC700603), P. aeruginosa (strain ATCC27853), and N.
19
gonorrhoeae (strain ATCC49981). All bacteria were heat inactivated (65°C for one
20
hour) and bacterial cultures before use and were carried out in the Animal Biosafety
21
Level 3 Laboratory (ABSL-III) of the Wuhan University School of Medicine.
AC C
EP
TE D
M AN U
SC
7
22 6
ACCEPTED MANUSCRIPT In vitro selection of aptamer against ManLAM by SELEX
2
ManLAM was extracted and purified from delipidated M.tb Beijing genotype strain
3
according to a previous report.18 The aptamers against ManLAM were screened by
4
SELEX using the method previously described.19
5
Briefly, the random ssDNA library (~1014 aptamers) contained the 68-mer
6
oligonucleotides
7
5’-GCGGAATTCAACAGTCCGAGCC-N30- GGGTCAATGCGTCATA-3’ (68 nt),
8
where the central N30 represented random oligonucleotides based on equal
9
incorporation of A, G, C, and T at each position. Primers P1, P2 and bio-P2
the
following
sequence:
P2)
M AN U
SC
with
RI PT
1
10
(biotin-labeled
11
(P1:5’-GCGGAATTCAACAGTCCGAGCC-3’,
12
recognition
13
specifies the BamHI recognition site).
14
At the beginning of in vitro selection, 10 µg of ManLAM was coated on a 96-well
15
plate and incubated at 37 °C for 2 hours. The wells were then washed five times and
16
subsequently blocked with 200 µl of salmon sperm DNA (100 µg/ml) at 37 °C for 1
17
hour. SsDNAs were mixed with yeast transfer RNA (5 µM) and incubated at 37 °C for
18
30 minutes. After washing, 50 µl of double-distilled H2O was added and heated at
19
99 °C for 10 minutes. The supernatant was used as the template for asymmetric
20
polymerase chain reaction amplification (PCR), and then the ssDNA products were
21
used in the next round of selection, with the ManLAM used decreased gradually to
22
0.5 µg to improve the selection efficacy. We applied the counter selection step to
P2:
used
underline
for specifies
PCR the
5’-GCGGGATCCTATGACGCATTGACCC-3’,
EcoRI
underline
AC C
EP
TE D
site;
were
7
ACCEPTED MANUSCRIPT improve aptamer specificity by the use of BSA and healthy donor serum from the 5th
2
to the 12th round of selection, following the positive selection by ManLAM. SsDNAs
3
from the 13th round of selection were amplified by PCR to obtain double-stranded
4
DNAs. Double-stranded DNAs were digested with BamHI and EcoRI and subcloned
5
into a pUC19 vector, which was used to transform E. coli DH5α bacteria. Selected
6
clones were sequenced. The identified single-clone aptamers were named as T1-T36.
7
The binding affinities of individual 36 aptamers were assessed as follows: 1 µg
8
ManLAM was coated into microplate. After blocking with 200 µl salmon sperm DNA
9
(100 µg/ml) at 37 °C for 1 hour, 0.3 µM biotin-labeled ssDNA aptamer was added and
10
incubated at 37 °C for 2 hours. Horseradish peroxidase (HRP)-conjugated streptavidin
11
(1:1000) was added and incubated for 30 minutes at 37 °C. After adding substrate and
12
stop buffer, absorbance was determined at 450 nm by a microplate reader. To evaluate
13
the binding specificity of aptamer, 20 g/mL of ManLAM was coated onto wells of
14
microplates, and biotin-labeled aptamer and soluble ManLAM was added to each well.
15
The absorbance at 450 nm was determined following by adding HRP-conjugated
16
streptavidin and substrate. The apparent Kd and Ki values were determined by
17
nonlinear regression for on-site binding using GraphPad Prism version 5.0 (GraphPad
18
Software, Inc.).
SC
M AN U
TE D
EP
AC C
19
RI PT
1
20
Detection of ManLAM antigen in serum or sputum samples with an
21
aptamer-based sandwich ELONA
22
The ELONA was performed according to our previous publications.19, 20 The mouse 8
ACCEPTED MANUSCRIPT anti-ManLAM pAb (1:3000 dilution) was used to coat a 96-well microplate at 4 °C
2
overnight. The human serum or sputum (sputum samples were pretreated with a half
3
volume of 3% NaOH, and then neutralized with an equal volume of 0.8 M HCl)
4
samples were added into the anti-ManLAM pAb-coated wells and then incubated at
5
37 °C for 2 hours. The biotin-labeled aptamer T9 (300 nM) was then added and
6
incubated at 37 °C for 2 hours. Horseradish Peroxidase (HRP)-conjugated streptavidin
7
(1:2000 dilution) was then added and incubated for 30 min at 37 °C. Color
8
development
9
tetramethylbenzidine (TMB) chromogen-substrate (Sigma). After adding stop buffer
10
(2 M H2SO4), the absorbance values at 450 nm (OD450) were determined using an
11
ELISA microplate reader.
achieved
adding
100
µl/well
of
ready-to-use
TE D
12
by
M AN U
was
SC
RI PT
1
Comparison between the aptamer-based ELONA and clinical T-SPOT.TB for TB
14
diagnosis
15
Fresh human blood samples from 100 TB patients collected from Wuhan Medical
16
treatment center were used in both the aptamer-based ELONA and clinical
17
T-SPOT.TB assay on the same day. The aptamer-based ELONA assay was performed
18
as mentioned above. The clinical T-SPOT.TB assay (Oxford Immunotec, Oxford,
19
United Kingdom) was performed according to the instruction manual. The
20
interpretation of positive, negative, and indeterminate outcomes followed the criteria
21
recommended by the manufacturer.
AC C
EP
13
22 9
ACCEPTED MANUSCRIPT Confocal microscopy
2
To observe the binding abilities between aptamer T9 and different M.tb strains, each
3
Rhodamine B (BD Diagnostics, Sparks, Maryland, USA)-lableled heat-inactivated
4
M.tb (at 65 °C for 30 minutes) bacterium (1 × 104 CFUs) was coated onto slides at
5
37 °C for 30 minutes. After washing with PBS, samples were incubated with
6
FAM-labeled T9 aptamer (5 µM) at 37 °C for 2 hours. After extensive washing with
7
PBS, samples were observed using confocal microscopy.
SC
Flow cytometry analysis
M AN U
8 9
RI PT
1
To assess the binding specificity of T9 aptamer, 1 × 107 CFUs of M.tb Beijing
11
genotype strain, M.tb H37Rv, BCG, and M. smegmatis (M.smeg) were fixed with 70%
12
ethanol for 10 minutes and then incubated with FAM-labeled T9 (5 µM) at 37 °C for 1
13
hour and then analyzed using a BD Accuri C6 flow cytometer (BD Biosciences).
14
TE D
10
Preparation of mouse anti-ManLAM polyclonal antibody (pAb)
16
BALB/c mice (7 week old) were obtained from the Wuhan University Center for
17
Animal Experiment/A3-Lab (Wuhan, China). Purified ManLAM (10 µg) emulsified
18
with Freund’s incomplete adjuvant was injected at five or six different sites
19
subcutaneously in the dorsal region and the footpad of the mice every week for four
20
weeks. One week after the final injection, blood was collected via retroorbital sinus
21
under anesthesia. Sera were isolated by centrifugation and stored at -80°C. The pAb
22
titer was evaluated by ELISA in ManLAM-coated microtiter plates, with serial
AC C
EP
15
10
ACCEPTED MANUSCRIPT dilutions of the mice serum as the primary antibody and HRP-conjugated goat
2
anti-mouse IgG as the secondary antibody. Serum collected before immunization was
3
used as a negative control. The maximum antibody dilution that fulfilled the criteria
4
(OD450positvie/OD450negative > 2.1) was considered as the antibody titer.
RI PT
1
5
Detection of the ManLAM antigen in serum samples with an indirect ELISA
7
using anti-ManLAM
8
The microplates were coated with ManLAM or serum samples and incubated
9
overnight at 4 °C. After blocking with 200 µl of salmon sperm DNA (100 µg/ml) at
10
37 °C for 1 hour, mouse anti-ManLAM pAb (1:3000 dilution) was added and
11
incubated at 37 °C for 2 hours. The absorbance at 450 nm was determined as
12
described above.
M AN U
TE D
13
SC
6
Detection of ManLAM antigen in serum or sputum samples with an
15
aptamer-based indirect ELONA
16
The serum samples from 100 aPTB patients and 64 HDs were coated onto microplates
17
(overnight at 4 °C), and detected with biotin-labeled T9 aptamer as a detection agent
18
(300 nM, 37 °C for 2 hours) and HRP-streptavidin conjugate (1:2000 dilution, 30 min
19
at 37 °C). The absorbance at 450 nm was determined as described above.
AC C
EP
14
20 21
Molecular typing for a panel of mycobacteria species
22
A real-time PCR-based molecular typing method adopted from a previous study21 was 11
ACCEPTED MANUSCRIPT used for the molecular typing using specific primers for Beijing genotype strain: BjF:
2
5’-CGAACTCGAGGCTGCCTACTAC- 3’, BjR: 5’-CTTGGCAGCTTCCTCGAT- 3’
3
and TaqMan probe (BjTm): 5’FAM-AACGCCAGAGACCAGCCGCCGGCT-BHQ
4
3’, under reaction conditions of 95°C for 4 min and 40 two-step cycles consisting of
5
95°C for 15s and 60°C for 35s.
RI PT
1
6
Determination of aptamer reactivity with a panel of clinical isolates
8
Sixty-five clinical isolated strains from three different geographic locations were
9
collected, and submitted to aptamer ELONA assay. Five clinically isolated Samonella
10
strains and PBS were used as negative control. Primary isolation, cultivation, and
11
species identification were performed with standard mycobacteriological methods.22
12
All bacteria were heat inactivated (65°C for one hour) before use. The microplates
13
were coated with each kinds of bacteria (1 × 105 CFUs) and incubated overnight at
14
4 °C, and detected with biotin-labeled T9 aptamer as a detection agent. The
15
absorbance at 450 nm was determined as described above.
M AN U
TE D
EP
AC C
16
SC
7
17
Statistical analysis
18
SPSS 13.0 software was used for all statistical analysis. Mann-Whitney U test were
19
used to evaluate the statistical differences in optical density (OD) values between
20
different groups. A p value of less than 0.05 was considered significant. Receiver
21
operating characteristic (ROC) curves of the OD values for ELONA assay were
22
plotted and the area under the curves (AUC) and 95% confidence intervals (95% CIs) 12
ACCEPTED MANUSCRIPT were calculated. The optimal cutoff values were chosen as when Youden’s index (YI =
2
sensitivity + specificity - 1) was maximum. The strength of the agreement between
3
ELONA and T-SPOT.TB assays was evaluated using Cohen's kappa coefficient, with
4
values of >0.75 representing excellent agreement beyond chance, 0.40-0.75
5
representing fair to good agreement beyond chance, and
S0163-4453(16)00031-1
DOI:
10.1016/j.jinf.2016.01.014
Reference:
YJINF 3669
To appear in:
Journal of Infection
Received Date: 11 August 2015 Revised Date:
20 December 2015
Accepted Date: 14 January 2016
Please cite this article as: Tang X-l, Wu S-M, Xie Y, Song N, Guan Q, Yuan C, Zhou X, Zhang X-L, Generation and application of ssDNA aptamers against glycolipid antigen ManLAM of Mycobacterium tuberculosis for TB diagnosis, Journal of Infection (2016), doi: 10.1016/j.jinf.2016.01.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Generation and application of ssDNA aptamers against glycolipid antigen
2
ManLAM of Mycobacterium tuberculosis for TB diagnosis
3
Xiao-lei Tang1,2, Shi-Min Wu1,3, Yan Xie1, Neng Song1, Qing Guan1, Chunhui
5
Yuan1, Xiang Zhou4, Xiao-Lian Zhang1 *
RI PT
4
6 7
1
8
Immunology, Medical Research Institute, and Department of Immunology Wuhan
9
University School of Medicine, Donghu Road 165#, Wuhan 430071, Hubei Province,
M AN U
SC
State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and
10
China
11
2
12
Wuhu 241000, China
13
3
14
Huazhong University of Science and Technology, China
15
4
16
Wuhan 430072, China
TE D
Wuhan Center for Clinical Laboratory, Puai Hospital, Tongji Medical College,
EP
College of Chemistry and Molecular Sciences, Wuhan University, Hubei Province,
AC C
17
Department of Clinical Laboratory, the Second Hospital of Wuhu, Anhui Province,
18
Xiao-lei Tang and Shi-Min Wu contributed equally to this paper
19
*
20
University School of Medicine, Wuhan 430071, China. E-mail addresses:
21
[email protected].
22
Running title: ManLAM aptamers for TB antigen diagnosis
Corresponding author: Xiao-Lian Zhang, Department of Immunology, Wuhan
1
ACCEPTED MANUSCRIPT ABSTRACT
2
The development of effective Mycobacterial antigen diagnostic reagents remains a
3
high priority. Mannose-capped lipoarabinomannan (ManLAM) is a lipoglycan serving
4
as a major cell wall component. ManLAM is also an early released antigen in the
5
blood circulation system during Mycobacteria tuberculosis (M.tb) infection and is a
6
perfect target antigen for TB diagnosis. In this study, ssDNA aptamers “antibodies”
7
against ManLAM of the predominant clinical epidemic M.tb Beijing genotype strains
8
were generated by the Systematic Evolution of Ligands by Exponential Enrichment
9
(SELEX) technique. The selected single aptamer T9 demonstrated the highest
10
specificity and binding affinity, with an equilibrium dissociation constant (Kd) of
11
668±159 nmol/L. We further detected ManLAM antigens in serum and sputum
12
samples from active pulmonary tuberculosis (aPTB) patients, extrapulmonary TB
13
(EPTB) patients and healthy donors by using a T9 based enzyme-linked
14
oligonucleotide assay (ELONA). The results showed that the specificity and
15
sensitivity were 95.31% and 83.00% (for 100 aPTB serum samples), 98.70% and
16
92.71% (for 96 aPTB sputum samples), and 94.44% and 88.71% (for 62 EPTB serum
17
samples), respectively. A good correlation was observed between the T9
18
aptamer-based ELONA and the clinical T-SPOT.TB. Thus, T9 based ELONA has
19
potentials for diagnosis of TB, including inactive TB, smear-negative TB, EPTB, and
20
TB with immunodeficiency, and assist the diagnosis of LTBI albeit it could not
21
distinguish LTBI and active TB.
AC C
EP
TE D
M AN U
SC
RI PT
1
22 2
ACCEPTED MANUSCRIPT 1
KEY WORDS: aptamer, diagnosis, mannosylated lipoarabinomannan (ManLAM),
2
Mycobacteria tuberculosis (M.tb), tuberculosis (TB), enzyme-linked oligonucleotide
3
assay (ELONA)
4
INTRODUCTION
6
The World Health Organization (WHO) reported that there were 9 million incident
7
cases of tuberculosis (TB) in 2013 with approximately 1.1 million TB cases being
8
co-infected with human immunodeficiency virus (HIV) and 1.5 million TB deaths. 1, 2
9
The emergence of multidrug-resistant tuberculosis (MDR-TB) makes TB control even
10
more urgent.3-5 Mycobacteria tuberculosis (M.tb) usually infects the lungs and causes
11
pulmonary TB, but it can also infect lymph nodes, bone, and other tissues causing
12
extrapulmonary TB (EPTB). M.tb can also exist in host body without causing any
13
clinical symptoms (latent TB infection, LTBI), thus could form new infection sources
14
when latent M.tb bacteria were reactivated. In recent years the Beijing genotype of
15
M.tb has attracted special attention because of its global emergence and
16
drug-resistance potential.6 The occurrence of Beijing genotype strains has been
17
documented in several parts of the world, including Asia, former Soviet Union,
18
Europe, Africa, and the United States, and significantly associated with MDR and
19
responsible for outbreaks of MDR-TB.6-8 Thus far, rapid and accurate diagnosis of TB,
20
especially caused by the predominant clinical epidemic M.tb Beijing genotype strain,
21
remains elusive.
22
AC C
EP
TE D
M AN U
SC
RI PT
5
Mannosylated lipoarabinomannan (ManLAM) is a major and structurally 3
ACCEPTED MANUSCRIPT important component of M.tb cell wall. ManLAM is constantly released from
2
metabolically active or degrading bacterial cells but not dead cells, and its presence
3
was demonstrated in human serum, urine, cerebrospinal fluid and the sputum of TB
4
infected individuals, which makes it a perfect candidate biomarker for TB diagnostic
5
tests.9
RI PT
1
Nucleic acid aptamers are short, single-stranded oligonucleotides that fold into
7
particular structures to bind to target molecules such as small chemicals, sugars, lipids,
8
proteins, or cells. High affinity aptamers for specific target molecules can be isolated
9
from a random nucleic acid library in vitro using the systematic evolution of ligands
10
by exponential enrichment (SELEX).10-12 SELEX involves sequential rounds of
11
selection and application to generate ligands (aptamers) that bind with high affinity to
12
target molecules.10 Aptamers are analogous to antibodies but possess several key
13
advantages: higher specificity and affinity than antibodies; easier chemical
14
modifications and greater stability; smaller molecular weight; and easier and more
15
economical production methods, making aptamers as ideal tools for disease
16
diagnosis13, 14
M AN U
TE D
EP
AC C
17
SC
6
The aim of this study was to screen and generate ssDNA aptamers against
18
ManLAM from the clinical widespread M.tb Beijing genotype. We further applied the
19
selected aptamer for TB antigen diagnosis with serum and sputum samples by an
20
enzyme-linked oligonucleotide assay (ELONA).
21 22
MATERIALS AND METHODS 4
ACCEPTED MANUSCRIPT Ethics Statement
2
The study was approved by the ethics committee of the Wuhan University School of
3
Medicine and Wuhan Medical Treatment Center. Written consent was obtained from
4
all of the participants including children from parents or legal guardians.
RI PT
1
5
Participant inclusion criteria
7
Culture-proven active pulmonary tuberculosis (aPTB) patients，EPTB patients，
8
inactive TB patients and non-tuberculous mycobacteria (NTM) infected patients were
9
prospectively enrolled from Wuhan Medical Treatment Center of Hubei province, the
10
First Affiliated Hospital of Wannan Medical Hospital of Anhui Province and the
11
Third People’s Hospital of Shenzhen of Guangdong Province, and HDs were recruited
12
from Wuhan University Zhongnan Hospital. The diagnosis of TB was based on the
13
following criteria: clinical symptoms of TB/abnormal chest X-ray findings, and
14
positive
15
culture-confirmed samples were included in the aTB group. The LTBI high risk
16
subjects were the front-line health care workers enrolled from Wuhan Medical
17
Treatment Center based on known contact with TB patients and the occupational
18
history of work in a high risk setting. The inactive TB patients were subjects who had
19
a history of previous tuberculous disease, with negative bacteriologic studies, and no
20
clinical evidence of current disease15. The NTM patients were subjects infected with
21
mycobacteria not causing tuberculosis and leprosy. NTM was identified by
22
para-nitrobenzoic acid (PNB) test and Mycobacteria Species Identification Genetic
TE D
M AN U
SC
6
culture
results
(current
“golden
standard”).
Only
AC C
EP
mycobacteria
5
ACCEPTED MANUSCRIPT Detection Kit (Yaneng Bio, China)16. The HDs were determined based on a lack of
2
symptoms of TB, no TB presentation on the X-ray examination, and an
3
epidemiologically low risk of TB infection (no contact with TB). HDs did not include
4
TB suspects later ruled out for TB. All of the subjects were unrelated Chinese of the
5
Han nationality.
RI PT
1
6
Bacterial strains
8
M.tb Beijing genotype strain and M. bovis were kindly provided by Professor Aizhen
9
Guo.17 All mycobacteria were maintained Middlebrook 7H9 medium supplemented
10
with Middlebrook OADC (BD Diagnostics, Sparks, MD, USA), and harvested while
11
in the exponential phase of growth. Bacteria used for aptamer specificity analysis
12
were propagated from laboratory stocks (School of Medicine, Wuhan University,
13
Wuhan, China) included M.tb H37Rv [strain ATCC 27294], BCG (Pasteur strain
14
ATCC 35734), M. smegmatis (strain ATCC 19420), M. avium (strain ATCC25291), E.
15
coli (strain ATCC 25922), S. aureus (strain ATCC25923), S. epidermidis (strain
16
14490); E. faecalis (strain ATCC 19433), P. vulgaris (strain ATCC 6380), S.
17
pneumonia (strain ATCC49619), S. pyogenes /S. hemolyticus (strain ATCC21059), K.
18
pneumonia (strain ATCC700603), P. aeruginosa (strain ATCC27853), and N.
19
gonorrhoeae (strain ATCC49981). All bacteria were heat inactivated (65°C for one
20
hour) and bacterial cultures before use and were carried out in the Animal Biosafety
21
Level 3 Laboratory (ABSL-III) of the Wuhan University School of Medicine.
AC C
EP
TE D
M AN U
SC
7
22 6
ACCEPTED MANUSCRIPT In vitro selection of aptamer against ManLAM by SELEX
2
ManLAM was extracted and purified from delipidated M.tb Beijing genotype strain
3
according to a previous report.18 The aptamers against ManLAM were screened by
4
SELEX using the method previously described.19
5
Briefly, the random ssDNA library (~1014 aptamers) contained the 68-mer
6
oligonucleotides
7
5’-GCGGAATTCAACAGTCCGAGCC-N30- GGGTCAATGCGTCATA-3’ (68 nt),
8
where the central N30 represented random oligonucleotides based on equal
9
incorporation of A, G, C, and T at each position. Primers P1, P2 and bio-P2
the
following
sequence:
P2)
M AN U
SC
with
RI PT
1
10
(biotin-labeled
11
(P1:5’-GCGGAATTCAACAGTCCGAGCC-3’,
12
recognition
13
specifies the BamHI recognition site).
14
At the beginning of in vitro selection, 10 µg of ManLAM was coated on a 96-well
15
plate and incubated at 37 °C for 2 hours. The wells were then washed five times and
16
subsequently blocked with 200 µl of salmon sperm DNA (100 µg/ml) at 37 °C for 1
17
hour. SsDNAs were mixed with yeast transfer RNA (5 µM) and incubated at 37 °C for
18
30 minutes. After washing, 50 µl of double-distilled H2O was added and heated at
19
99 °C for 10 minutes. The supernatant was used as the template for asymmetric
20
polymerase chain reaction amplification (PCR), and then the ssDNA products were
21
used in the next round of selection, with the ManLAM used decreased gradually to
22
0.5 µg to improve the selection efficacy. We applied the counter selection step to
P2:
used
underline
for specifies
PCR the
5’-GCGGGATCCTATGACGCATTGACCC-3’,
EcoRI
underline
AC C
EP
TE D
site;
were
7
ACCEPTED MANUSCRIPT improve aptamer specificity by the use of BSA and healthy donor serum from the 5th
2
to the 12th round of selection, following the positive selection by ManLAM. SsDNAs
3
from the 13th round of selection were amplified by PCR to obtain double-stranded
4
DNAs. Double-stranded DNAs were digested with BamHI and EcoRI and subcloned
5
into a pUC19 vector, which was used to transform E. coli DH5α bacteria. Selected
6
clones were sequenced. The identified single-clone aptamers were named as T1-T36.
7
The binding affinities of individual 36 aptamers were assessed as follows: 1 µg
8
ManLAM was coated into microplate. After blocking with 200 µl salmon sperm DNA
9
(100 µg/ml) at 37 °C for 1 hour, 0.3 µM biotin-labeled ssDNA aptamer was added and
10
incubated at 37 °C for 2 hours. Horseradish peroxidase (HRP)-conjugated streptavidin
11
(1:1000) was added and incubated for 30 minutes at 37 °C. After adding substrate and
12
stop buffer, absorbance was determined at 450 nm by a microplate reader. To evaluate
13
the binding specificity of aptamer, 20 g/mL of ManLAM was coated onto wells of
14
microplates, and biotin-labeled aptamer and soluble ManLAM was added to each well.
15
The absorbance at 450 nm was determined following by adding HRP-conjugated
16
streptavidin and substrate. The apparent Kd and Ki values were determined by
17
nonlinear regression for on-site binding using GraphPad Prism version 5.0 (GraphPad
18
Software, Inc.).
SC
M AN U
TE D
EP
AC C
19
RI PT
1
20
Detection of ManLAM antigen in serum or sputum samples with an
21
aptamer-based sandwich ELONA
22
The ELONA was performed according to our previous publications.19, 20 The mouse 8
ACCEPTED MANUSCRIPT anti-ManLAM pAb (1:3000 dilution) was used to coat a 96-well microplate at 4 °C
2
overnight. The human serum or sputum (sputum samples were pretreated with a half
3
volume of 3% NaOH, and then neutralized with an equal volume of 0.8 M HCl)
4
samples were added into the anti-ManLAM pAb-coated wells and then incubated at
5
37 °C for 2 hours. The biotin-labeled aptamer T9 (300 nM) was then added and
6
incubated at 37 °C for 2 hours. Horseradish Peroxidase (HRP)-conjugated streptavidin
7
(1:2000 dilution) was then added and incubated for 30 min at 37 °C. Color
8
development
9
tetramethylbenzidine (TMB) chromogen-substrate (Sigma). After adding stop buffer
10
(2 M H2SO4), the absorbance values at 450 nm (OD450) were determined using an
11
ELISA microplate reader.
achieved
adding
100
µl/well
of
ready-to-use
TE D
12
by
M AN U
was
SC
RI PT
1
Comparison between the aptamer-based ELONA and clinical T-SPOT.TB for TB
14
diagnosis
15
Fresh human blood samples from 100 TB patients collected from Wuhan Medical
16
treatment center were used in both the aptamer-based ELONA and clinical
17
T-SPOT.TB assay on the same day. The aptamer-based ELONA assay was performed
18
as mentioned above. The clinical T-SPOT.TB assay (Oxford Immunotec, Oxford,
19
United Kingdom) was performed according to the instruction manual. The
20
interpretation of positive, negative, and indeterminate outcomes followed the criteria
21
recommended by the manufacturer.
AC C
EP
13
22 9
ACCEPTED MANUSCRIPT Confocal microscopy
2
To observe the binding abilities between aptamer T9 and different M.tb strains, each
3
Rhodamine B (BD Diagnostics, Sparks, Maryland, USA)-lableled heat-inactivated
4
M.tb (at 65 °C for 30 minutes) bacterium (1 × 104 CFUs) was coated onto slides at
5
37 °C for 30 minutes. After washing with PBS, samples were incubated with
6
FAM-labeled T9 aptamer (5 µM) at 37 °C for 2 hours. After extensive washing with
7
PBS, samples were observed using confocal microscopy.
SC
Flow cytometry analysis
M AN U
8 9
RI PT
1
To assess the binding specificity of T9 aptamer, 1 × 107 CFUs of M.tb Beijing
11
genotype strain, M.tb H37Rv, BCG, and M. smegmatis (M.smeg) were fixed with 70%
12
ethanol for 10 minutes and then incubated with FAM-labeled T9 (5 µM) at 37 °C for 1
13
hour and then analyzed using a BD Accuri C6 flow cytometer (BD Biosciences).
14
TE D
10
Preparation of mouse anti-ManLAM polyclonal antibody (pAb)
16
BALB/c mice (7 week old) were obtained from the Wuhan University Center for
17
Animal Experiment/A3-Lab (Wuhan, China). Purified ManLAM (10 µg) emulsified
18
with Freund’s incomplete adjuvant was injected at five or six different sites
19
subcutaneously in the dorsal region and the footpad of the mice every week for four
20
weeks. One week after the final injection, blood was collected via retroorbital sinus
21
under anesthesia. Sera were isolated by centrifugation and stored at -80°C. The pAb
22
titer was evaluated by ELISA in ManLAM-coated microtiter plates, with serial
AC C
EP
15
10
ACCEPTED MANUSCRIPT dilutions of the mice serum as the primary antibody and HRP-conjugated goat
2
anti-mouse IgG as the secondary antibody. Serum collected before immunization was
3
used as a negative control. The maximum antibody dilution that fulfilled the criteria
4
(OD450positvie/OD450negative > 2.1) was considered as the antibody titer.
RI PT
1
5
Detection of the ManLAM antigen in serum samples with an indirect ELISA
7
using anti-ManLAM
8
The microplates were coated with ManLAM or serum samples and incubated
9
overnight at 4 °C. After blocking with 200 µl of salmon sperm DNA (100 µg/ml) at
10
37 °C for 1 hour, mouse anti-ManLAM pAb (1:3000 dilution) was added and
11
incubated at 37 °C for 2 hours. The absorbance at 450 nm was determined as
12
described above.
M AN U
TE D
13
SC
6
Detection of ManLAM antigen in serum or sputum samples with an
15
aptamer-based indirect ELONA
16
The serum samples from 100 aPTB patients and 64 HDs were coated onto microplates
17
(overnight at 4 °C), and detected with biotin-labeled T9 aptamer as a detection agent
18
(300 nM, 37 °C for 2 hours) and HRP-streptavidin conjugate (1:2000 dilution, 30 min
19
at 37 °C). The absorbance at 450 nm was determined as described above.
AC C
EP
14
20 21
Molecular typing for a panel of mycobacteria species
22
A real-time PCR-based molecular typing method adopted from a previous study21 was 11
ACCEPTED MANUSCRIPT used for the molecular typing using specific primers for Beijing genotype strain: BjF:
2
5’-CGAACTCGAGGCTGCCTACTAC- 3’, BjR: 5’-CTTGGCAGCTTCCTCGAT- 3’
3
and TaqMan probe (BjTm): 5’FAM-AACGCCAGAGACCAGCCGCCGGCT-BHQ
4
3’, under reaction conditions of 95°C for 4 min and 40 two-step cycles consisting of
5
95°C for 15s and 60°C for 35s.
RI PT
1
6
Determination of aptamer reactivity with a panel of clinical isolates
8
Sixty-five clinical isolated strains from three different geographic locations were
9
collected, and submitted to aptamer ELONA assay. Five clinically isolated Samonella
10
strains and PBS were used as negative control. Primary isolation, cultivation, and
11
species identification were performed with standard mycobacteriological methods.22
12
All bacteria were heat inactivated (65°C for one hour) before use. The microplates
13
were coated with each kinds of bacteria (1 × 105 CFUs) and incubated overnight at
14
4 °C, and detected with biotin-labeled T9 aptamer as a detection agent. The
15
absorbance at 450 nm was determined as described above.
M AN U
TE D
EP
AC C
16
SC
7
17
Statistical analysis
18
SPSS 13.0 software was used for all statistical analysis. Mann-Whitney U test were
19
used to evaluate the statistical differences in optical density (OD) values between
20
different groups. A p value of less than 0.05 was considered significant. Receiver
21
operating characteristic (ROC) curves of the OD values for ELONA assay were
22
plotted and the area under the curves (AUC) and 95% confidence intervals (95% CIs) 12
ACCEPTED MANUSCRIPT were calculated. The optimal cutoff values were chosen as when Youden’s index (YI =
2
sensitivity + specificity - 1) was maximum. The strength of the agreement between
3
ELONA and T-SPOT.TB assays was evaluated using Cohen's kappa coefficient, with
4
values of >0.75 representing excellent agreement beyond chance, 0.40-0.75
5
representing fair to good agreement beyond chance, and
