Development of a simple and rapid immunochromatographic strip test for detecting duck plague virus antibodies based on gI protein

Journal Pre-proof Development of a simple and rapid immunochromatographic strip test for detecting duck plague virus antibodies based on gI protein Xiaofeng Zhou, Mingshu Wang, Anchun Cheng, Qiao Yang, Ying Wu, Renyong Jia, Mafeng Liu, Dekang Zhu, Shun Chen, Shaqiu Zhang, Xin-Xin Zhao, Juan Huang, Sai Mao, Xumin Ou, Qun Gao, Yunya Liu, Yanling Yu, Ling Zhang, Bin Tian, Leichang Pan, Mujeeb Ur Rehman, Xiaoyue Chen

PII:

S0166-0934(19)30387-8

DOI:

https://doi.org/10.1016/j.jviromet.2019.113803

Reference:

VIRMET 113803

To appear in:

Journal of Virological Methods

Received Date:

26 August 2019

Revised Date:

16 December 2019

Accepted Date:

16 December 2019

Please cite this article as: Zhou X, Wang M, Cheng A, Yang Q, Ying W, Jia R, Liu M, Zhu D, Chen S, Zhang S, Zhao X-Xin, Huang J, Mao S, Xumin O, Gao Q, Liu Y, Yanling Y, Zhang L, Tian B, Pan L, Rehman MU, Chen X, Development of a simple and rapid immunochromatographic strip test for detecting duck plague virus antibodies based on gI protein, Journal of Virological Methods (2019), doi: https://doi.org/10.1016/j.jviromet.2019.113803

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Development of a simple and rapid immunochromatographic strip test for detecting duck plague virus antibodies based on gI protein Xiaofeng Zhou1,2,3¶, Mingshu Wang1,2,3¶, Anchun Cheng1,2,3*, Qiao Yang1,2,3, Ying Wu1,2,3, Renyong Jia1,2,3, Mafeng Liu1,2,3, Dekang Zhu2,3, Shun Chen1,2,3, Shaqiu Zhang1,2,3,Xin-Xin Zhao1,2,3,Juan Huang1,2,3, Sai Mao1,2,3,Xumin Ou1,2,3, Qun Gao1,2,3,Yunya Liu1,2,3, Yanling Yu1,2,3, Ling Zhang1,2,3 , Bin Tian1,3,Leichang Pan1,3, Mujeeb Ur Rehman1,3, Xiaoyue Chen1,2,3

1

Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu

2

Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural

University, Wenjiang, Chengdu City, Sichuan, 611130, P.R. China

Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University,

Wenjiang, Chengdu City, Sichuan, 611130, P.R. China

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City, Sichuan, 611130, P.R. China

¶These authors contributed equally to this work as first authors.

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Page number: 11

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Figure number: 6 Table Number: 2

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*Corresponding authors: Anchun Cheng

Highlights

We expressed numerous DPV gI protein by E. coli expression system and

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obtained purified protein. We established a colloidal gold test strip method capable of detecting anti-

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DPV antibodies.

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We compared this new method with neutralization test and ELISA to evaluate it’s effect.

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We demonstrated that this mothod was suitable for detect anti-DPV 1

antibodies.

ABSTRACT A colloidal gold strip (CGS) for detecting antibodies to duck plague virus(DPV) was developed. Colloidal gold-labeled DPV gI protein and goat anti-rabbit IgG were dispensed on a conjugate pad as tracers. The recombinant DPV gI protein and rabbit IgG were used as capture reagents at the test line and control line, respectively. The

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detection limit of this assay was 1:256. Additionally, the CGS did not react with antisera from other common duck diseases, only reacting with anti-DPV serum and yielding a

specific and strong red signal. 123 serum samples were tested by CGS and enzymelinked immunosorbent assay (ELISA), and the results showed good agreement. The

clinical testing and large-scale detection.

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CGS test results can be observed in 15 min with the naked eye, should be suitable for

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Keywords: Duck plague virus; DPV gI protein; Colloidal gold strip (CGS)

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Duck plague is an acute, infectious and fatal disease to ducks, geese, swans and other wild geese. This disease is characterized by widespread epidemics that are rapidly spread and have high morbidity, decreased egg production in domestic and wild waterfowl (Chang et al., 2011; Deng et al., 2008; Guiping et al.; Liu et al., 2017; Walker et al., 1969; Xuefeng et al., 2008; Yang et al., 2014; Yuan et al., 2005), causing huge economic losses to the waterfowl industry. At present, methods for diagnosing duck plague include polymerase chain reaction (PCR) (Cheng et al., 2004; Guo et al., 2009; Lin et al., 2013; Qi et al., 2009; Wu et al.,

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2011; Zou et al., 2010), loop-mediated isothermal amplification (LAMP) (Jun et al., 2009), ELISA (Wen et al., 2010; Xue-Feng et al., 2007), neutralization test (NT) and agar gel diffusion experiments, etc. However, these diagnosis methods have their own

shortcomings; PCR relies on specific equipment. NT is the gold standard assay for DPV

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antibody detection (Toro and Kaleta, 1986), but it requires the use of live virus and up to 1 week to obtain results, meaning that it is easy for the virus to spread and cause

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environmental pollution while performing the test. ELISA takes at least 3.5h. The CGS is a novel immunolabeling technique based on the specific binding characteristics of an

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antigen-antibody. The results could be detected by the naked eye, guaranteeing clinical convenience (Se-HwanPaek et al., 1999) and making it suitable for large quantities of

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serum samples. DPV-gI protein is an envelope glycoprotein encoded by the DPV gene US7. It is an antigenic determinant on the surface of the virus and has certain immunogenicity, which makes it suitable as a target antigen for serological tests. In this

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study, we used the recombinant DPV gI protein to develop an CGS test for the simple, fast, sensitive and specific detection of duck plague serum antibodies. We compared

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our new method with the NT and ELISA to evaluate its effect. The pET-32a(+)-gI recombinant expression plasmid was transformed into E. coli

BL21(DE3). A single colony was cultured in Luria Bertani (LB) containing 50 μg/ml ampicillin overnight at 37°C to obtain a seed culture. The 2 ml seed culture was then expanded in 200 ml LB medium. After incubating for 2 h at 37°C, the cells reached logarithmic phase (at OD600 of 0.5-0.6), and isopropyl-β-D-thiogalactogyranoside 3

(IPTG, Takara) was added (final concentration 0.2 mM) to the cultures, followed by continued incubation at 37°C for an additional 4 h. After induction, the cells were harvested by centrifugation at 5000 rpm/min for 30 min. The harvested bacterial cell pellet was resuspended in PBS and then ultrasonically disrupted on ice. The disrupted cells were then centrifuged at 10000 rpm/min for 10 min to obtain the inclusion body precipitate. The recombinant protein was analyzed by SDS-PAGE (Figure 1a). There was abundant gI fusion protein expressed with a molecular weight of approximately 65 kDa. Duck anti-DPV antibody as first antibody, horseradish peroxidase-labeled goat

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anti-duck immunoglobulin G (IgG) (KPL; Amersham) as second antibody, for western blotting (WB). Clear and specific band was detected using DAB solution (Tiangen biotech Co., Ltd. PA110) (Figure 1b). Suggesting that the purified fusion gI protein was suitable as the capture for the CGS.

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The recombinant His-tagged protein was purified by nickel affinity

chromatography according to the manufacturer’s protocol (Bio-Rad). The target protein

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was dialyzed in urea buffer after which the urea concentration was gradually reduced to remove the urea and renature the protein. The recombinant protein was then dialyzed

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overnight in 10 mmol/L PBS at 4 °C.

To determine the optimum pH of colloidal gold-labeled gI, six eppendorf tubes

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were supplemented with 500 μl 20 nM colloidal gold solution. Then, 1, 2, 3, 4, 5, and 6 μl of 0.2 mol/l potassium carbonate (K2CO3) was added to each tubes and mixed evenly. Next, 10 µg recombinant gI protein was added , and the mixture was allowed

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to rest for 30 min. Then the mixture was centrifuged at 12,000 r/min for 30 min at 4 °C. The samples were observed to determine whether a precipitate was present and whether

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the precipitate could be evenly resuspended. Prepare colloidal gold-gI and determine the optimal concentration of colloidal

gold-gI, 10ml of a 20 nM colloidal gold solution was added to small beakers and adjusted to the optimum pH and then continuously stirred for 5 min. 100ug of recombinant gI protein was added and stirring was continued for 30 min. Then, 1.3ml of 10% (w/v) BSA was added to each of the solution, and the solution was stirred for 4

10 min. After stirring, the colloidal gold solution was allowed to rest for 30 min. The colloidal gold-labeled solution was divided into 8 parts on average and then centrifuged at 12000 r/min for 30 min, the resulting precipitate was resuspended with resuspension buffer (20 mmol/L Tris-HCl and 1% BSA), obtained final concentration from 20ug/ml to 100ug/ml. As shown in Figure 2, clear red bands were obtained when the colloidal gold-labeled gI concentration reached 100 µg/ml. The same procedure was used to obtain the colloidal gold-labeled goat anti-rabbit IgG. The fusion gI protein was diluted with PBS from 3 mg/ml to 0.5 mg/ml. There

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were clear red bands at the test line at gI protein concentrations of 3 mg/ml, 2.5 mg/ml, and 2 mg/ml. As shown in Figure 3. A continuous increase in the antigen concentration did not improve the CGS effect. Thus, 2 mg/ml gI protein was selected as the capture concentration at the test line.

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As shown in Figure 4. membrane, sample pad, conjugate pad, and absorption pad were sequentially adhered to the PCV bottom plate. The control line was close to the

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absorption pad, the test line was close to the conjugate pad, and the adjacent parts overlapped by approximately 2 mm. The CGS was assembled, cut into 4 mm wide strips

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and sealed in a dry environment.

The DPV standard positive serum was sequentially diluted with PB buffer (1:2,

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1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512) to determine the detection limit of the CGS. At the highest dilution (1:256) (Figure 5), two clear red bands developed at the test and control lines. Compared the sensitivity with ELISA and NT, 11 serum

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samples were detected respectively. As shown in table 1. The CGS is more sensitive than NT and can be used to detect low amounts of DPV antiserum antibodies.

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In total, 123 serum samples from healthy ducks, DPV-CHV and DPV deletion

strain infected ducks and attenuated vaccine immunized ducks were simultaneously detected by the CGS and gI-ELISA. As shown in Table 2. There were 39 positive sera (31.7%) detected by CGS; the percentage of positive sera was comparable to 31 positive sera (25.2%) detected by the gI-ELISA. The consistency of the percentages of positive sera were analyzed using McNemar’s test, and the kappa statistic was used to mearsure 5

the strength of agreement among the results between the two methods with R(version 3.3.3) software. The coincident rate between CGS and ELISA is 83.73%. These data showed that there were no significant differences in the compliance rate between the two methods. The specificity of the strip was evaluated by using other duck susceptible pathogens, inculding Riemerella anatipestifer (RA), Tembusu virus, duck hepatitis virus type I(DHV-1) and duck hepatitis virus type 3(DHV-3), duck Pasteurella, and duck Salmonella. All of the other samples showed negative results, only the anti-DPV

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serum yielded a specific and strong signal(Figure 6). The CGS assembled in this study were stored at 4 °C. Test strips were taken out

every two months until the 12th month to detect DPV-positive and -negative sera (3 parts). Serum (3 parts) tests were repeated three times. The same results were also

of the known negative samples were negative.

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obtained at different months. All of the known positive samples were positive, and all

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The ability of duck plague to establish a latent infection and its high levels of transmission make it difficult to monitor and control (Burgess, Ossa, and Yuill, 1979).

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Rapid diagnosis is an important measure for controlling duck plague allow scientists to take effective control measures. Diagnostic methods are mainly based on pathogen and

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antibody. Studies have shown that DPV establishes an asymptomatic carrier state in waterfowl, it can only be detected during the intermittent shedding period. It is difficult to directly detect duck plague pathogens. CGS are widely used in different methods of

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testing based on a certain recombinant protein. In this study, we developed a colloidal gold strip capable of detecting duck plague serum antibodies. The sensitivity of the

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CGS developed in this study reached 1:256; compared with the gold standard assay, NT, the sensitivity of the CGS was obviously higher than NT. The CGS also displayed high consistency rates with ELISA. This high consistency with ELISA further supports the clinical use of the test strip. In contrast to other detection methods, the CGS does not require special equipment or professional operators. The CGS can be used on-site, and its results can be observed with the naked eye within 15 min. 6

At present, there are no specific drug treatments for duck plague. Thus, it is very important to carry out vaccine prevention. Attenuated or naturally apathogenic DPV strains are used in live vaccines to protect ducks from duck plague (Lian et al., 2010; Liu et al., 2007). Combined with other detection methods to monitor DPV-specific antibodies is key to evaluating the immune response of DPV vaccines and developing a reasonable immunization program. In conclusion, we have successfully developed an CGS that is simple and sensitive and quickly detects anti-DPV serum antibodies. Conflict of interest

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The authors declare that they have no conflict of interest.

Acknowledgments

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This work was supported by the National Key Research and Development Program of China (2017YFD0500800), the China Agricultural Research System (CARS-42-17), the Sichuan Veterinary Medicine and Drug Innovation Group of China Agricultural Research System (CARS-SVDIP).

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W.G. and Ming, W., 2004. The Preliminary Application of PCR in Research of Clinical

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Guo, Y., Cheng, A., Wang, M., Shen, C., Jia, R., Chen, S. and Na, Z., 2009. Development of TaqMan® MGB fluorescent real-time PCR assay for the detection of anatid herpesvirus 1. Virology Journal 6, 71-71.

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isothermal amplification. Research in Veterinary Science 87, 53-58. Lian, B., Xu, C., Cheng, A., Wang, M., Zhu, D., Luo, Q., Jia, R., Bi, F., Chen, Z. and Zhou, Y., 2010. Identification and characterization of duck plague virus glycoprotein C gene and gene product. Virology Journal 7, 349. Lin, M., Jia, R., Wang, M., Gao, X., Zhu, D. and Chen…, S., 2013. The transcription analysis of duck enteritis virus UL49.5 gene using real-time quantitative reverse transcription PCR.

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Walker, J.W., Pfow, C.J., Newcomb, S.S., Urban, W.D., Nadler, H.E. and Locke, L.N., 1969. Status of duck virus enteritis (duck plague) in the United States. Proc Annu Meet U S Anim Health Assoc 73, 254-279. Wen, Y., Cheng, A., Wang, M., Han, G., Shen, C., Liu, S., Xiang, J., Jia, R., Zhu, D. and Chen, X., 2010. A Thymidine Kinase recombinant protein-based ELISA for detecting antibodies to Duck Plague Virus. Virology Journal 7, 77-77.

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Quantitative analysis of virulent duck enteritis virus loads in experimentally infected

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genomic sequence analysis between a standard challenge strain and a vaccine strain of duck enteritis virus in China. Virus Genes 48, 296-303.

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Zou, Q., Sun, K., Cheng, A., Wang, M. and Chen, X., 2010. Detection of anatid herpesvirus 1 gC gene by TaqManTM fluorescent quantitative real-time PCR with specific primers and probe. Virology Journal 7, 37-37.

Figure caption

(b)

(a)

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KDa 180 130

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70 55

40

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Fig. 1. SDS-PAGE and WB of the recombinant gI protein. (a) SDS-PAGE of the

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recombinant gI protein. Lane M, molecular mass marker (in KDa); lane 1, pET32a-gI incubated without IPTG; lane2, pET32a-gI after induction; lane3, the

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supernatant of induced E.coli pET32a-gI; lane 4, the precipitate of induced E.coli pET32a-gI; lane 5, purified recombinant gI protein. The molecular weight of the protein is 65 KDa. (b)WB of recombinant gI protein. Lane 1, pET32a-gI

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incubated without IPTG; lane 2, pET32a-gI after induction; Lane M, molecular

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mass markers (in KDa).

20 ug/ml

40 ug/ml

60 ug/ml

80 ug/ml

100 ug/ml

Negative

Fig. 2. Optimization of the amount of colloidal gold-labeled gI protein. Different amounts of colloidal gold-labeled gI protein were obtained. The last strip was a negative control. The data show that the test strips have a good effect when the 11

colloidal gold labeled gI protein concentration reached 100 µg/ml.

Fig. 3. Optimization of the recombinant protein concentration coating the T line. Different concentrations of gI protein coating the test line. There were two clear

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red bands at the 2 mg/ml concentration.

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Fig. 4. The schematic structure of the CGS. The purified recombinant gI protein and rabbit IgG were immobilized at the T line and C line, respectively. Colloidal gold-

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labeled gI protein and goat anti-rabbit IgG were sprayed onto the conjugated pad.

1:8

1:16

1:32

1:64

1:128

1:256

1:512

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Fig. 5. The sensitivity of the CGS. Positive anti-DPV serum was 2-fold gradient diluted with PB buffer and tested. The highest dilution was 1:1256.

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DPV

RA

Pasteurella

Salmonella

Tembusu

DHV-1

DHV-3

Fig. 6. The specificity of the colloidal gold strip. Antiserum form DPV, Rlemerella anatipestifer (RA), Pasteurella, Salmonella, Tembusu virus, DHV-1 and DHV-3

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were tested by the CGS. Similar result patterns were reproduced in the repeat

1:6400

experiments. Table 1 The sensitivity of CGS, ELISA and NT. 2

3

4

5

6

ELISA

0

1:400

1:400

1:800

1:400

1:800

CGS

0

1:128

1:64

1:128

1:64

1:64

NT

0

2-2

2-4

2-4.375

2-3.33

2-3.16

8

9

10

11

0

1:1600

1:1600

1:1600

1:256

1:64

1:256

1:64

1:64

2-3.16

2-4.33

2-4.33

2-4.5

2-3.87

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Serums no.

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Notes: The serum include Duck anti-DPV antibody(CHv), Duck anti-DPV gE deletion strain antibody and Duck anti-DPV LORF5 deletion strain antibody. From this

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table we observed that CGS is less sensitive than ELISA but more sensitive than NT. Table 2

Agreement between the CGS and ELISA. The CGS

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ELISA

Total Negative

Positive

25

6

31

Negative

14

78

92

Total

39

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123

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Positive

Table 3 serum

healthy

DPV-△gI

DPV-△gE

attenuated vaccine DPV-CHV infected 13

samples

ducks

infected ducks infected ducks

immunized ducks

ducks

75

15

3

10

20

Notes: Serum samples for ELISA and CGS. Here are 90 negative serum samples

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and 33 positive serum samples for ELISA and CGS.

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