Rapid Fluorescent Lateral-Flow Immunoassay for Hepatitis B Virus

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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

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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

ACS Paragon Plus Environment


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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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Fluorescent, Monoclonal antibody, Chronic hepatitis B

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Nonstandard abbreviations:

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LFIA, lateral flow immunoassay; mAb, monoclonal antibody; HBV, hepatitis B

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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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Abstract

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Hepatitis B virus (HBV) genotyping plays an important role in the clinical

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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

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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

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assay was carefully evaluated in well-characterized clinical cohorts. The

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GT-LFIA, which can specifically differentiate HBV genotypes A, B, C and D in a

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pretreatment-free

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genotype-specific mAbs. The detection limits of the GT-LFIA for HBV

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genotypes A, B, C and D were 2.5-10.0 IU HBV surface antigen/mL,

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respectively. Among the sera from 456 CHB patients, 439 (96.3%, 95%

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confidence interval [CI]: 94.1–97.8%) were genotype-differentiable by the

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GT-LFIA and 437 (99.5%, 95% CI: 98.4-99.9%) were consistent with viral

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genome sequencing. In the 21 patients receiving nucleos(t)ide analogue

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therapy, for end-of-treatment specimens that were HBV DNA undetectable and

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were not applicable for DNA-dependent genotyping, the GT-LFIA presented

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genotyping results that were consistent with those obtained in pretreatment

acid-based

techniques

single

test,

are

was

expensive,

successfully

4


time-consuming

developed

and

using

4

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specimens by viral genome sequencing and the GT-LFIA. In conclusion, the

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novel GT-LFIA is a convenient, fast and reliable tool for differential HBV

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genotyping, especially in patients with low or undetectable HBV DNA levels.

79

5



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Introduction

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The hepatitis B virus (HBV) is a serious, worldwide public health problem

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because HBV infection can cause hepatitis, liver cirrhosis (LC) and

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hepatocellular carcinoma (HCC), resulting in more than one million deaths

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annually1,2. HBV has been classified into 8 different genotypes (A–H) as

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determined by whole-genome sequence heterogeneity exceeding 8%3-6. The

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different genotypes exhibit distinct geographic distribution characteristics.

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Genotype A is prevalent in sub-Saharan Africa, North America and Europe,

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whereas B and C are prevalent in Asia, E is prevalent in Africa, F and H are

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prevalent in Central and South America, and D and G are present worldwide 1,7.

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Overall, the HBV genotypes A/D and B/C represent the predominant epidemic

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strains in Europe/North America and Asia, respectively.

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Evidence from clinical studies indicates that the HBV genotype

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significantly influences the natural history, liver disease progression and

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antiviral treatment response 8,9. The rate of chronicity of acute genotype A or D

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infections is higher than that of genotype B or C infections 9,10. HBV genotypes

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A and B are associated with higher rates of HBV e antigen (HBeAg)

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seroconversion and

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interferon-based treatment when compared with genotypes D and C,

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respectively. Additionally, when compared to patients with genotypes A and B,

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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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genotype significantly influences the on-treatment HBsAg kinetics and

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end-of-treatment (EOT) HBsAg levels, which are associated with long-term

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sustained virological responses and HBsAg losses, thus suggesting that

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genotype-specific monitoring timeframes and EOT thresholds could ameliorate

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the response-guided treatment (RGT) of chronic hepatitis B (CHB)

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Altogether, HBV genotyping can provide important information for the guidance

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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

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hybridization methods (e.g., the commercial INNO-LiPATM assay) 15, restriction

113

fragment polymorphisms 16, multiplex nested polymerase chain reaction (PCR)

114

17,18

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NAT-based techniques are expensive, time-consuming and inconvenient,

116

given the requirement for a DNA extraction procedure. In this study, we aimed

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to develop a novel HBV DNA-independent genotyping tool based on a

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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

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Development of HBV genotype-specific mAbs

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Epitopes in the preS2 regions were selected as the targets for HBV 7



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21

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genotype-specific mAbs as described previously

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of the antibodies, epitope-targeting immunization and screening techniques

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were designed and performed as shown in Fig. 1. Briefly, the sequences of E1

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(aa 32–55 of HBV genotype A preS2), E2 (aa 33–52 of HBV genotype B

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preS2), E3 (aa 32–55 of HBV genotype C preS2) and E4 (aa 6–27 of HBV

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genotype D preS2) were engrafted into an HBc149 protein to construct

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chimeric virus-like particle proteins. The chimeric virus-like particles (VLPs)

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were produced in E. coli and purified as described previously

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HBc-E1, HBc-E2, HBc-E3 and HBc-E4 VLPs were used as immunogens to

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generate antibodies targeting the E1, E2, E3 and E4 epitopes, respectively, via

134

standard hybridoma technology

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culture media from the HBV genotype A–D hybridoma cell clones were

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evaluated with a virus capture enzyme-linked immunosorbent assay (ELISA).

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The clones producing mAbs with differential binding to different HBV

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genotypes were preserved for further assessment. Finally, 4 HBV genotype

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specific mAbs 2B2, 16D12, 6H3 and 3E6 were developed. The sensitivity and

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specificity for detecting different HBV genotypes(A, B, C and D) of the 4 mAbs

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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

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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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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

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adsorbed onto the conjugate pad. On the nitrocellulose membrane, the

154

genotype-specific mAbs 2B2, 16D12, 6H3 and 3E6, goat anti-HBsAg

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polyclonal (GAS) antibodies and streptavidin were immobilized on the A, B, C,

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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.

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For GT-LFIA-mediated detection, an 80-µL sample (serum or plasma) was

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directly pipetted onto the sample pad. Subsequently, the sample re-mobilized

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the dried conjugates and viral surface antigens, including L-HBsAg (containing

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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).

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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

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measured using a customized luminoscope reader (Fig. 2C).

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Sample collection

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The performance of the GT-LFIA was evaluated in three different cohorts. For

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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

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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).

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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

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E virus (HEV) vaccine that was conducted between August 2007 and June

186

2009 in Dongtai county, Jiangsu province, China. Serum samples were

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collected every year or every other year to assess the durability of immunity in

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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


Simultaneously,

the

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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,

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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

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method was determined by using whole genome or partial sequencing as

206

described in a previous study25.

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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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using SPSS (Statistical Package for the Social Sciences) ver. 17.0 software

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(SPSS Inc., Chicago, IL, USA). All statistical analyses were based on 2-tailed

214

hypothesis tests with a significance level of p

Rapid Fluorescent Lateral-Flow Immunoassay for Hepatitis B Virus

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