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A novel rapid fluorescent lateral-flow immunoassay for hepatitis B virus genotyping Liuwei Song, Yingbin Wang, Linlin Fang, Yong Wu, Lin Yang, Jieyu Chen, Shengxiang Ge, Jing Zhang, Youzheng Xiong, Xiumei Deng, Xiaoping Min, Jun Zhang, Pei-Jer Chen, Quan Yuan, and Ningshao Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504832c • Publication Date (Web): 20 Apr 2015 Downloaded from http://pubs.acs.org on April 22, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Analytical Chemistry
1
Title:
2
A novel rapid fluorescent lateral-flow immunoassay for hepatitis B virus
3
genotyping
4 5
Running title:
6
Fast HBV genotyping by LFIA
7 8
Authors:
9
Liu-Wei Song1,2,†, Ying-Bin Wang1,2,†, Lin-Lin Fang1,2, Yong Wu1,2, Lin Yang1,2,
10
Jie-Yu Chen5, Sheng-Xiang Ge1,2, Jing Zhang1,2, You-Zheng Xiong1,2,3, Xiu-Mei
11
Deng5, Xiao-Ping Min1,2,3,*, Jun Zhang1,2, Pei-Jer Chen4, Quan Yuan1,2,* and
12
Ning-Shao Xia1,2
13 14 15
†
These authors contributed equally to this work.
*
Co-corresponding authors
16 17
Author affiliations:
18
1
19
Diseases, State Key Laboratory of Molecular Vaccinology and Molecular
20
Diagnostics, School of Life Sciences, Xiamen University, China
21
2
22
3
National Institute of Diagnostics and Vaccine Development in Infectious
School of Public Health, Xiamen University, Xiamen, China School of Information Science and Engineering, Computer Science 1
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23 24
Department, Xiamen University, Xiamen, China 4
National Taiwan University College of Medicine, National Taiwan University,
25
Taipei, Taiwan
26
5
Xiamen Innovax Biotech Co., Ltd, Xiamen, China
27 28
Corresponding authors:
29
Address requests for reprints to: Q Yuan or XP Min, National Institute of
30
Diagnostics and Vaccine Development in Infectious Diseases, Xiamen
31
University, China. E-mail: [email protected] or [email protected]. Fax:
32
(86)-0592-2181258.
33 34
Funding
35
This work was supported by the National Scientific and Technological
36
Major Project (2012ZX10002005-001-001), the Key Research Item of Science
37
and Technology of Fujian Province (2014Y0073), and the Fujian province
38
science and technology project (2012Y4011).
39 40
Conflict of interest:
41
Jie-Yu Chen and Xiu-Mei Deng are employees of the Xiamen Innovax Biotech
42
Co., Ltd. The other authors declare no competing interests.
43 44
Key words: Hepatitis B virus, Genotype, Lateral flow immunoassay, 2
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Analytical Chemistry
45
Fluorescent, Monoclonal antibody, Chronic hepatitis B
46 47
Nonstandard abbreviations:
48
LFIA, lateral flow immunoassay; mAb, monoclonal antibody; HBV, hepatitis B
49
virus; ALT, alanine aminotransferase; HBsAg, hepatitis B surface antigen;
50
HBeAg, hepatitis B e antigen; ULN, upper limit of normal; CI, confidence
51
interval; SD, standard deviation.
52 53
3
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Page 4 of 34
54
Abstract
55
Hepatitis B virus (HBV) genotyping plays an important role in the clinical
56
management of chronic hepatitis B (CHB) patients. However, the current
57
nucleic
58
inconvenient. Here, we developed a novel DNA-independent HBV genotyping
59
tool based on a one-step fluorescent lateral flow immunoassay (LFIA).
60
Epitope-targeting immunization and screening techniques were used to
61
develop HBV genotype-specific monoclonal antibodies (mAbs). These mAbs
62
were used to develop a multi-test LFIA with a matched scanning luminoscope
63
for HBV genotyping (named the GT-LFIA). The performance of this novel
64
assay was carefully evaluated in well-characterized clinical cohorts. The
65
GT-LFIA, which can specifically differentiate HBV genotypes A, B, C and D in a
66
pretreatment-free
67
genotype-specific mAbs. The detection limits of the GT-LFIA for HBV
68
genotypes A, B, C and D were 2.5-10.0 IU HBV surface antigen/mL,
69
respectively. Among the sera from 456 CHB patients, 439 (96.3%, 95%
70
confidence interval [CI]: 94.1–97.8%) were genotype-differentiable by the
71
GT-LFIA and 437 (99.5%, 95% CI: 98.4-99.9%) were consistent with viral
72
genome sequencing. In the 21 patients receiving nucleos(t)ide analogue
73
therapy, for end-of-treatment specimens that were HBV DNA undetectable and
74
were not applicable for DNA-dependent genotyping, the GT-LFIA presented
75
genotyping results that were consistent with those obtained in pretreatment
acid-based
techniques
single
test,
are
was
expensive,
successfully
4
ACS Paragon Plus Environment
time-consuming
developed
and
using
4
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Analytical Chemistry
76
specimens by viral genome sequencing and the GT-LFIA. In conclusion, the
77
novel GT-LFIA is a convenient, fast and reliable tool for differential HBV
78
genotyping, especially in patients with low or undetectable HBV DNA levels.
79
5
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Analytical Chemistry
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80
Page 6 of 34
Introduction
81
The hepatitis B virus (HBV) is a serious, worldwide public health problem
82
because HBV infection can cause hepatitis, liver cirrhosis (LC) and
83
hepatocellular carcinoma (HCC), resulting in more than one million deaths
84
annually1,2. HBV has been classified into 8 different genotypes (A–H) as
85
determined by whole-genome sequence heterogeneity exceeding 8%3-6. The
86
different genotypes exhibit distinct geographic distribution characteristics.
87
Genotype A is prevalent in sub-Saharan Africa, North America and Europe,
88
whereas B and C are prevalent in Asia, E is prevalent in Africa, F and H are
89
prevalent in Central and South America, and D and G are present worldwide 1,7.
90
Overall, the HBV genotypes A/D and B/C represent the predominant epidemic
91
strains in Europe/North America and Asia, respectively.
92
Evidence from clinical studies indicates that the HBV genotype
93
significantly influences the natural history, liver disease progression and
94
antiviral treatment response 8,9. The rate of chronicity of acute genotype A or D
95
infections is higher than that of genotype B or C infections 9,10. HBV genotypes
96
A and B are associated with higher rates of HBV e antigen (HBeAg)
97
seroconversion and
98
interferon-based treatment when compared with genotypes D and C,
99
respectively. Additionally, when compared to patients with genotypes A and B,
100
those with genotypes C and D have a higher risk of poor disease progression
101
(LC and HCC)
9,11
HBV
surface antigen (HBsAg)
losses
following
. Moreover, recent studies have demonstrated that the HBV 6
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Analytical Chemistry
102
genotype significantly influences the on-treatment HBsAg kinetics and
103
end-of-treatment (EOT) HBsAg levels, which are associated with long-term
104
sustained virological responses and HBsAg losses, thus suggesting that
105
genotype-specific monitoring timeframes and EOT thresholds could ameliorate
106
the response-guided treatment (RGT) of chronic hepatitis B (CHB)
107
Altogether, HBV genotyping can provide important information for the guidance
108
of CHB clinical management.
12
.
The current techniques for HBV genotyping are mainly based on nucleic
109
13
110
acid tests (NATs) for genotype-specific sequences
111
genome
112
hybridization methods (e.g., the commercial INNO-LiPATM assay) 15, restriction
113
fragment polymorphisms 16, multiplex nested polymerase chain reaction (PCR)
114
17,18
115
NAT-based techniques are expensive, time-consuming and inconvenient,
116
given the requirement for a DNA extraction procedure. In this study, we aimed
117
to develop a novel HBV DNA-independent genotyping tool based on a
118
one-step fluorescent lateral flow immunoassay (LFIA) and genotype-specific
119
monoclonal antibodies (mAbs), furthermore the new assay was fully evaluated.
sequencing
followed
by
, including direct whole
phylogenetic
, oligonucleotide microarray chips
19
analysis
and real-time PCR
14
20
,
reverse
. However,
120 121
Materials and methods
122
Development of HBV genotype-specific mAbs
123
Epitopes in the preS2 regions were selected as the targets for HBV 7
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21
124
genotype-specific mAbs as described previously
125
of the antibodies, epitope-targeting immunization and screening techniques
126
were designed and performed as shown in Fig. 1. Briefly, the sequences of E1
127
(aa 32–55 of HBV genotype A preS2), E2 (aa 33–52 of HBV genotype B
128
preS2), E3 (aa 32–55 of HBV genotype C preS2) and E4 (aa 6–27 of HBV
129
genotype D preS2) were engrafted into an HBc149 protein to construct
130
chimeric virus-like particle proteins. The chimeric virus-like particles (VLPs)
131
were produced in E. coli and purified as described previously
132
HBc-E1, HBc-E2, HBc-E3 and HBc-E4 VLPs were used as immunogens to
133
generate antibodies targeting the E1, E2, E3 and E4 epitopes, respectively, via
134
standard hybridoma technology
135
culture media from the HBV genotype A–D hybridoma cell clones were
136
evaluated with a virus capture enzyme-linked immunosorbent assay (ELISA).
137
The clones producing mAbs with differential binding to different HBV
138
genotypes were preserved for further assessment. Finally, 4 HBV genotype
139
specific mAbs 2B2, 16D12, 6H3 and 3E6 were developed. The sensitivity and
140
specificity for detecting different HBV genotypes(A, B, C and D) of the 4 mAbs
141
were evaluated by enzyme-linked immunosorbent assay(ELISA).
142
LFIA for HBV genotyping
23
. To improve the efficiency
22
. The purified
. For screening, the binding activities of
143
The LFIA test strip for HBV genotyping (GT-LFIA) is composed of a
144
backing, sample pad, conjugate pad, nitrocellulose membrane (Millipore,
145
Bedford, MA, USA) and absorbent pad (Jieyi Biotechnology, Shanghai, China). 8
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Analytical Chemistry
146
The strip was positioned as shown in Fig. 2A, wherein the ends of the
147
components overlapped, thus ensuring a continuous flow of the sample
148
solution via capillary action from the sample pad to the absorbent pad. An
149
anti-HBsAg mAb (WTS; purchased from Wantai, Beijing, China) and
150
biotinylated bovine serum albumin (BSA), both of which were conjugated to
151
Fluoro-Max Fluorescent Nanoparticles (Excite at 333 nm and emit at 613 nm,
152
Thermo Scientific, Rockford, IL, USA), were diluted in a blocking reagent and
153
adsorbed onto the conjugate pad. On the nitrocellulose membrane, the
154
genotype-specific mAbs 2B2, 16D12, 6H3 and 3E6, goat anti-HBsAg
155
polyclonal (GAS) antibodies and streptavidin were immobilized on the A, B, C,
156
D, S and CT capture lines, respectively (as shown in Fig. 2A). All the distances
157
between the two adjacent lines are 3 mm except line S and A that is 4 mm. The
158
strip was subsequently dried overnight at 37°C and kept dry at room
159
temperature until use.
160
For GT-LFIA-mediated detection, an 80-µL sample (serum or plasma) was
161
directly pipetted onto the sample pad. Subsequently, the sample re-mobilized
162
the dried conjugates and viral surface antigens, including L-HBsAg (containing
163
the preS2 region), M-HBsAg (containing the preS2 region) and S-HBsAg (not
164
containing the preS2 region), in the WTS-FP conjugate-bound sample as both
165
migrated into the nitrocellulose membrane. The L/M-HBsAg-WTS-FP immune
166
complexes in the samples were then captured by the 3E6, 6H3, 16D12 or 2B2
167
antigens according to the viral antigen genotypes as they migrated past the 9
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lines
(D,
C,
B
and
A,
respectively).
Page 10 of 34
168
capture
169
S-HBsAg-WTS-FP immune complexes and biotinylated BSA-FP were
170
captured by the GAS antibody and streptavidin, which were immobilized on the
171
S and CT lines, respectively. Excess reagents moved beyond the capture lines
172
to be entrapped in the absorbent pad. The assay could be completed within 20
173
minutes, and the fluorescent intensities of all test lines on the strip could be
174
measured using a customized luminoscope reader (Fig. 2C).
175
Sample collection
176
The performance of the GT-LFIA was evaluated in three different cohorts. For
177
the first cohort, a total of 456 serum specimens were collected from CHB
178
patients at Zhongshan Hospital (Xiamen, China) and Xijing Hospital (Xi’an,
179
China). All of these specimens were successfully subjected to HBV genome
180
sequence analysis as previously described. The second group included 21
181
CHB patients who had undergone more than 1 year of nucleos(t)ide analogue
182
therapy (12 patients received entecavir and 9 patients received telbivudine).
183
The baseline and EOT serum specimens were collected for a comparative
184
analysis. The third group was derived from a phase 3 clinical trial of a hepatitis
185
E virus (HEV) vaccine that was conducted between August 2007 and June
186
2009 in Dongtai county, Jiangsu province, China. Serum samples were
187
collected every year or every other year to assess the durability of immunity in
188
two of the eleven towns (Quindong and Anfeng) where participants had been
189
enrolled24. In 2013, we followed up 7507 participants in Anfeng; this time point 10
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Simultaneously,
the
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Analytical Chemistry
190
was 55 months after the first vaccine dose had been administered. All of the
191
serum samples were screened for HBsAg, and 324 were positive. In addition,
192
42 serum samples with low HBsAg level (less than 1,000 IU/mL) from patients
193
with cirrhosis were collected from Xijing Hospital. All the samples were stored
194
at -80℃ until genotyping using both GT-LFIA and sequencing. The study was
195
approved by the medical ethics committee of the School of Public health,
196
Xiamen University.
197
Laboratory measurements
198
The HBsAg levels were quantified with the Architect HBsAg assay (Abbott
199
Laboratories, Abbott Park, IL, USA; detection range, 0.05–250 IU/mL). The
200
serum HBV DNA levels were measured with the CobasTaqman HBV Kit
201
(Roche Diagnostics, Indianapolis, IN, USA; lower limit of quantification, 12
202
IU/mL). HBeAg and Anti-HBe were detected with an Architect assay (Abbott
203
Laboratories). Aminotransferases were measured according to the local
204
standard procedures at the time of sampling. The direct sequence genotyping
205
method was determined by using whole genome or partial sequencing as
206
described in a previous study25.
207
Statistical analysis
208
Parameters such as the HBsAg level were analyzed using descriptive
209
statistics such as means and standard deviations, and the means were
210
compared with an unpaired Student’s t-test. Differences between proportions
211
were analyzed with the chi-square test. The statistical analysis was performed 11
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Page 12 of 34
212
using SPSS (Statistical Package for the Social Sciences) ver. 17.0 software
213
(SPSS Inc., Chicago, IL, USA). All statistical analyses were based on 2-tailed
214
hypothesis tests with a significance level of p
Article
A novel rapid fluorescent lateral-flow immunoassay for hepatitis B virus genotyping Liuwei Song, Yingbin Wang, Linlin Fang, Yong Wu, Lin Yang, Jieyu Chen, Shengxiang Ge, Jing Zhang, Youzheng Xiong, Xiumei Deng, Xiaoping Min, Jun Zhang, Pei-Jer Chen, Quan Yuan, and Ningshao Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504832c • Publication Date (Web): 20 Apr 2015 Downloaded from http://pubs.acs.org on April 22, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 34
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Analytical Chemistry
1
Title:
2
A novel rapid fluorescent lateral-flow immunoassay for hepatitis B virus
3
genotyping
4 5
Running title:
6
Fast HBV genotyping by LFIA
7 8
Authors:
9
Liu-Wei Song1,2,†, Ying-Bin Wang1,2,†, Lin-Lin Fang1,2, Yong Wu1,2, Lin Yang1,2,
10
Jie-Yu Chen5, Sheng-Xiang Ge1,2, Jing Zhang1,2, You-Zheng Xiong1,2,3, Xiu-Mei
11
Deng5, Xiao-Ping Min1,2,3,*, Jun Zhang1,2, Pei-Jer Chen4, Quan Yuan1,2,* and
12
Ning-Shao Xia1,2
13 14 15
†
These authors contributed equally to this work.
*
Co-corresponding authors
16 17
Author affiliations:
18
1
19
Diseases, State Key Laboratory of Molecular Vaccinology and Molecular
20
Diagnostics, School of Life Sciences, Xiamen University, China
21
2
22
3
National Institute of Diagnostics and Vaccine Development in Infectious
School of Public Health, Xiamen University, Xiamen, China School of Information Science and Engineering, Computer Science 1
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Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
23 24
Department, Xiamen University, Xiamen, China 4
National Taiwan University College of Medicine, National Taiwan University,
25
Taipei, Taiwan
26
5
Xiamen Innovax Biotech Co., Ltd, Xiamen, China
27 28
Corresponding authors:
29
Address requests for reprints to: Q Yuan or XP Min, National Institute of
30
Diagnostics and Vaccine Development in Infectious Diseases, Xiamen
31
University, China. E-mail: [email protected] or [email protected]. Fax:
32
(86)-0592-2181258.
33 34
Funding
35
This work was supported by the National Scientific and Technological
36
Major Project (2012ZX10002005-001-001), the Key Research Item of Science
37
and Technology of Fujian Province (2014Y0073), and the Fujian province
38
science and technology project (2012Y4011).
39 40
Conflict of interest:
41
Jie-Yu Chen and Xiu-Mei Deng are employees of the Xiamen Innovax Biotech
42
Co., Ltd. The other authors declare no competing interests.
43 44
Key words: Hepatitis B virus, Genotype, Lateral flow immunoassay, 2
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Analytical Chemistry
45
Fluorescent, Monoclonal antibody, Chronic hepatitis B
46 47
Nonstandard abbreviations:
48
LFIA, lateral flow immunoassay; mAb, monoclonal antibody; HBV, hepatitis B
49
virus; ALT, alanine aminotransferase; HBsAg, hepatitis B surface antigen;
50
HBeAg, hepatitis B e antigen; ULN, upper limit of normal; CI, confidence
51
interval; SD, standard deviation.
52 53
3
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Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 34
54
Abstract
55
Hepatitis B virus (HBV) genotyping plays an important role in the clinical
56
management of chronic hepatitis B (CHB) patients. However, the current
57
nucleic
58
inconvenient. Here, we developed a novel DNA-independent HBV genotyping
59
tool based on a one-step fluorescent lateral flow immunoassay (LFIA).
60
Epitope-targeting immunization and screening techniques were used to
61
develop HBV genotype-specific monoclonal antibodies (mAbs). These mAbs
62
were used to develop a multi-test LFIA with a matched scanning luminoscope
63
for HBV genotyping (named the GT-LFIA). The performance of this novel
64
assay was carefully evaluated in well-characterized clinical cohorts. The
65
GT-LFIA, which can specifically differentiate HBV genotypes A, B, C and D in a
66
pretreatment-free
67
genotype-specific mAbs. The detection limits of the GT-LFIA for HBV
68
genotypes A, B, C and D were 2.5-10.0 IU HBV surface antigen/mL,
69
respectively. Among the sera from 456 CHB patients, 439 (96.3%, 95%
70
confidence interval [CI]: 94.1–97.8%) were genotype-differentiable by the
71
GT-LFIA and 437 (99.5%, 95% CI: 98.4-99.9%) were consistent with viral
72
genome sequencing. In the 21 patients receiving nucleos(t)ide analogue
73
therapy, for end-of-treatment specimens that were HBV DNA undetectable and
74
were not applicable for DNA-dependent genotyping, the GT-LFIA presented
75
genotyping results that were consistent with those obtained in pretreatment
acid-based
techniques
single
test,
are
was
expensive,
successfully
4
ACS Paragon Plus Environment
time-consuming
developed
and
using
4
Page 5 of 34
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Analytical Chemistry
76
specimens by viral genome sequencing and the GT-LFIA. In conclusion, the
77
novel GT-LFIA is a convenient, fast and reliable tool for differential HBV
78
genotyping, especially in patients with low or undetectable HBV DNA levels.
79
5
ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
80
Page 6 of 34
Introduction
81
The hepatitis B virus (HBV) is a serious, worldwide public health problem
82
because HBV infection can cause hepatitis, liver cirrhosis (LC) and
83
hepatocellular carcinoma (HCC), resulting in more than one million deaths
84
annually1,2. HBV has been classified into 8 different genotypes (A–H) as
85
determined by whole-genome sequence heterogeneity exceeding 8%3-6. The
86
different genotypes exhibit distinct geographic distribution characteristics.
87
Genotype A is prevalent in sub-Saharan Africa, North America and Europe,
88
whereas B and C are prevalent in Asia, E is prevalent in Africa, F and H are
89
prevalent in Central and South America, and D and G are present worldwide 1,7.
90
Overall, the HBV genotypes A/D and B/C represent the predominant epidemic
91
strains in Europe/North America and Asia, respectively.
92
Evidence from clinical studies indicates that the HBV genotype
93
significantly influences the natural history, liver disease progression and
94
antiviral treatment response 8,9. The rate of chronicity of acute genotype A or D
95
infections is higher than that of genotype B or C infections 9,10. HBV genotypes
96
A and B are associated with higher rates of HBV e antigen (HBeAg)
97
seroconversion and
98
interferon-based treatment when compared with genotypes D and C,
99
respectively. Additionally, when compared to patients with genotypes A and B,
100
those with genotypes C and D have a higher risk of poor disease progression
101
(LC and HCC)
9,11
HBV
surface antigen (HBsAg)
losses
following
. Moreover, recent studies have demonstrated that the HBV 6
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Analytical Chemistry
102
genotype significantly influences the on-treatment HBsAg kinetics and
103
end-of-treatment (EOT) HBsAg levels, which are associated with long-term
104
sustained virological responses and HBsAg losses, thus suggesting that
105
genotype-specific monitoring timeframes and EOT thresholds could ameliorate
106
the response-guided treatment (RGT) of chronic hepatitis B (CHB)
107
Altogether, HBV genotyping can provide important information for the guidance
108
of CHB clinical management.
12
.
The current techniques for HBV genotyping are mainly based on nucleic
109
13
110
acid tests (NATs) for genotype-specific sequences
111
genome
112
hybridization methods (e.g., the commercial INNO-LiPATM assay) 15, restriction
113
fragment polymorphisms 16, multiplex nested polymerase chain reaction (PCR)
114
17,18
115
NAT-based techniques are expensive, time-consuming and inconvenient,
116
given the requirement for a DNA extraction procedure. In this study, we aimed
117
to develop a novel HBV DNA-independent genotyping tool based on a
118
one-step fluorescent lateral flow immunoassay (LFIA) and genotype-specific
119
monoclonal antibodies (mAbs), furthermore the new assay was fully evaluated.
sequencing
followed
by
, including direct whole
phylogenetic
, oligonucleotide microarray chips
19
analysis
and real-time PCR
14
20
,
reverse
. However,
120 121
Materials and methods
122
Development of HBV genotype-specific mAbs
123
Epitopes in the preS2 regions were selected as the targets for HBV 7
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Page 8 of 34
21
124
genotype-specific mAbs as described previously
125
of the antibodies, epitope-targeting immunization and screening techniques
126
were designed and performed as shown in Fig. 1. Briefly, the sequences of E1
127
(aa 32–55 of HBV genotype A preS2), E2 (aa 33–52 of HBV genotype B
128
preS2), E3 (aa 32–55 of HBV genotype C preS2) and E4 (aa 6–27 of HBV
129
genotype D preS2) were engrafted into an HBc149 protein to construct
130
chimeric virus-like particle proteins. The chimeric virus-like particles (VLPs)
131
were produced in E. coli and purified as described previously
132
HBc-E1, HBc-E2, HBc-E3 and HBc-E4 VLPs were used as immunogens to
133
generate antibodies targeting the E1, E2, E3 and E4 epitopes, respectively, via
134
standard hybridoma technology
135
culture media from the HBV genotype A–D hybridoma cell clones were
136
evaluated with a virus capture enzyme-linked immunosorbent assay (ELISA).
137
The clones producing mAbs with differential binding to different HBV
138
genotypes were preserved for further assessment. Finally, 4 HBV genotype
139
specific mAbs 2B2, 16D12, 6H3 and 3E6 were developed. The sensitivity and
140
specificity for detecting different HBV genotypes(A, B, C and D) of the 4 mAbs
141
were evaluated by enzyme-linked immunosorbent assay(ELISA).
142
LFIA for HBV genotyping
23
. To improve the efficiency
22
. The purified
. For screening, the binding activities of
143
The LFIA test strip for HBV genotyping (GT-LFIA) is composed of a
144
backing, sample pad, conjugate pad, nitrocellulose membrane (Millipore,
145
Bedford, MA, USA) and absorbent pad (Jieyi Biotechnology, Shanghai, China). 8
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Analytical Chemistry
146
The strip was positioned as shown in Fig. 2A, wherein the ends of the
147
components overlapped, thus ensuring a continuous flow of the sample
148
solution via capillary action from the sample pad to the absorbent pad. An
149
anti-HBsAg mAb (WTS; purchased from Wantai, Beijing, China) and
150
biotinylated bovine serum albumin (BSA), both of which were conjugated to
151
Fluoro-Max Fluorescent Nanoparticles (Excite at 333 nm and emit at 613 nm,
152
Thermo Scientific, Rockford, IL, USA), were diluted in a blocking reagent and
153
adsorbed onto the conjugate pad. On the nitrocellulose membrane, the
154
genotype-specific mAbs 2B2, 16D12, 6H3 and 3E6, goat anti-HBsAg
155
polyclonal (GAS) antibodies and streptavidin were immobilized on the A, B, C,
156
D, S and CT capture lines, respectively (as shown in Fig. 2A). All the distances
157
between the two adjacent lines are 3 mm except line S and A that is 4 mm. The
158
strip was subsequently dried overnight at 37°C and kept dry at room
159
temperature until use.
160
For GT-LFIA-mediated detection, an 80-µL sample (serum or plasma) was
161
directly pipetted onto the sample pad. Subsequently, the sample re-mobilized
162
the dried conjugates and viral surface antigens, including L-HBsAg (containing
163
the preS2 region), M-HBsAg (containing the preS2 region) and S-HBsAg (not
164
containing the preS2 region), in the WTS-FP conjugate-bound sample as both
165
migrated into the nitrocellulose membrane. The L/M-HBsAg-WTS-FP immune
166
complexes in the samples were then captured by the 3E6, 6H3, 16D12 or 2B2
167
antigens according to the viral antigen genotypes as they migrated past the 9
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lines
(D,
C,
B
and
A,
respectively).
Page 10 of 34
168
capture
169
S-HBsAg-WTS-FP immune complexes and biotinylated BSA-FP were
170
captured by the GAS antibody and streptavidin, which were immobilized on the
171
S and CT lines, respectively. Excess reagents moved beyond the capture lines
172
to be entrapped in the absorbent pad. The assay could be completed within 20
173
minutes, and the fluorescent intensities of all test lines on the strip could be
174
measured using a customized luminoscope reader (Fig. 2C).
175
Sample collection
176
The performance of the GT-LFIA was evaluated in three different cohorts. For
177
the first cohort, a total of 456 serum specimens were collected from CHB
178
patients at Zhongshan Hospital (Xiamen, China) and Xijing Hospital (Xi’an,
179
China). All of these specimens were successfully subjected to HBV genome
180
sequence analysis as previously described. The second group included 21
181
CHB patients who had undergone more than 1 year of nucleos(t)ide analogue
182
therapy (12 patients received entecavir and 9 patients received telbivudine).
183
The baseline and EOT serum specimens were collected for a comparative
184
analysis. The third group was derived from a phase 3 clinical trial of a hepatitis
185
E virus (HEV) vaccine that was conducted between August 2007 and June
186
2009 in Dongtai county, Jiangsu province, China. Serum samples were
187
collected every year or every other year to assess the durability of immunity in
188
two of the eleven towns (Quindong and Anfeng) where participants had been
189
enrolled24. In 2013, we followed up 7507 participants in Anfeng; this time point 10
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Simultaneously,
the
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Analytical Chemistry
190
was 55 months after the first vaccine dose had been administered. All of the
191
serum samples were screened for HBsAg, and 324 were positive. In addition,
192
42 serum samples with low HBsAg level (less than 1,000 IU/mL) from patients
193
with cirrhosis were collected from Xijing Hospital. All the samples were stored
194
at -80℃ until genotyping using both GT-LFIA and sequencing. The study was
195
approved by the medical ethics committee of the School of Public health,
196
Xiamen University.
197
Laboratory measurements
198
The HBsAg levels were quantified with the Architect HBsAg assay (Abbott
199
Laboratories, Abbott Park, IL, USA; detection range, 0.05–250 IU/mL). The
200
serum HBV DNA levels were measured with the CobasTaqman HBV Kit
201
(Roche Diagnostics, Indianapolis, IN, USA; lower limit of quantification, 12
202
IU/mL). HBeAg and Anti-HBe were detected with an Architect assay (Abbott
203
Laboratories). Aminotransferases were measured according to the local
204
standard procedures at the time of sampling. The direct sequence genotyping
205
method was determined by using whole genome or partial sequencing as
206
described in a previous study25.
207
Statistical analysis
208
Parameters such as the HBsAg level were analyzed using descriptive
209
statistics such as means and standard deviations, and the means were
210
compared with an unpaired Student’s t-test. Differences between proportions
211
were analyzed with the chi-square test. The statistical analysis was performed 11
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Page 12 of 34
212
using SPSS (Statistical Package for the Social Sciences) ver. 17.0 software
213
(SPSS Inc., Chicago, IL, USA). All statistical analyses were based on 2-tailed
214
hypothesis tests with a significance level of p
