Development of double-antibody sandwich ELISA for rapidly quantitative detection of antigen concentration in inactivated SCRV vaccine

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Development of double-antibody sandwich ELISA for rapidly quantitative detection of antigen concentration in inactivated SCRV vaccine Yinjie Niua,1, Peng Zhangb,1, Luyao Wanga,b, Ningqiu Lia, Qiang Lina, Lihui Liua, Hongru Lianga, Zhibin Huanga, Xiaozhe Fua,∗ a

Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Provinces, Guangzhou, 510380, China b College of Fisheries and Life Science, Dalian Ocean University, Dalian, 116023, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Siniperca chuatsi rhabdovirus(SCRV) DAS-ELISA Quantitative detection SCRV-QY inactivated vaccines

In recent years, Siniperca chuatsi rhabdovirus (SCRV) caused serious threats and huge economic losses in Siniperca chuatsi aquaculture industry. Vaccination is the most efficacious and cost-effective strategy to control this viral disease. However, SCRV-QY vaccine quality control is vital for successful prevention. Herein, we generated a pair of high affinity antibodies against SCRV-QY virus and established a double-antibody sandwich enzymelinked immunosorbent assay (DAS-ELISA) for detecting the SCRV-QY antigen amounts. In this assay, monoclonal antibody 4H8 was selected as capture antibody and 4E12 labeled HRP for detector antibody. A standard curve was generated using the SCRV concentration versus OD value with the linear range of concentration of 78.125~5000 ng/ml. The antigen content of 3 batches SCRV-QY inactivated vaccines were quantitatively detected by using the DAS-ELISA. The results showed that antigen contents of SCRV-QY inactivated vaccines were positively correlated with the viral titers. In conclusion, this DAS-ELISA was an accurate, quick and efficacious method for detecting antigen concentration of inactivated SCRV-QY vaccines.

1. Introduction Siniperca chuatsi rhabdovirus (SCRV) disease is mainly caused by SCRV, which belongs to the genus Perhabdovirus in the family Rhabdoviridae (Assenberg et al., 2010). In 1999, Zhang et al. observed the virions in the tissues of diseased Siniperca chuatsi (Lin et al., 2017; Tao et al., 2007). The skin, gill, liver, and gut of infected fish were spot bleeding (Lin et al., 2017). SCRV is enveloped and bullet-shaped, and its genome is an unsegmented, negative sense (-) single-stranded RNA molecule with the length of approximately 11 kb, which encodes five viral proteins:nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase protein (large protein, L) (Fu et al., 2017; Liu et al., 2014). The G glycoprotein at the virion surface is the major antigenic proteins and involved in viral tropism, viral entry, pathogenicity, and host selection (Blondel et al., 2015). In recent years, the outbreak of SCRV disease had caused huge economic losses and serious threats to Siniperca chuatsi aquaculture. And, the prevention of SCRV disease was mainly relied on the vaccination. Inactivated vaccines cultured in cells are widely used due to its

stable prevention, efficacy, simple operation and low cost. The antigen content of the inactivated vaccine is the key for vaccine quality control. But for the inactivated virus vaccine, the virus titers could not accurately detect the antigen concentration. A simple and effective detecting method is urgently established. The double antibody sandwich enzymelinked immunosorbent assays (DAS-ELISA) is a simple, sensitive and specific method for detecting of the antigen amounts. DAS-ELISA for detecting rabies virus antigen has been established (Martine ChabaudRiou et al., 2017; Morgeaux et al., 2017; Wang et al., 2018). In this study, we prepared two monoclonal antibodies (4H8 and 4E12) against the SCRV-QY virus, and established a convenient and time-saving DAS-ELISA for detecting the SCRV antigen contents of inactivated vaccine using the capture antibody 4H8 and detection antibody 4E12. 2. Materials and methods 2.1. Cells and virus The CPB cells from Chinese perch (also called mandarin fish) brain

∗

Corresponding author. 1 XingYu Road, Pearl River Fishery Research Institute, Chinese Academy Fishery Sciences, Guangzhou, 510380, China. E-mail address: [email protected] (X. Fu). 1 Yinjie Niu and Peng Zhang contributed equally to this work. https://doi.org/10.1016/j.aquaculture.2019.734671 Received 26 September 2019; Received in revised form 8 October 2019; Accepted 3 November 2019 Available online 05 November 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Yinjie Niu, et al., Aquaculture, https://doi.org/10.1016/j.aquaculture.2019.734671

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ascites was purified by affinity chromatography. Briefly, the protein A agarose was loaded into chromatographic column, washed in turn by 10 times column volume of deionized water and 1% NaAc. Ascites through 0.22 μm membrane was added into the chromatographic column. The crude protein were rinsed by 30 mL 1% NaAc, the pH of antibody eluent was adjusted to neutral by using 3.5% HAc, and then concentrated by a 10 kDa molecular weight cut off (Millipore). The purified antibody was finally achieved by dialysis using 10L PBS for 24 h, and evaluated by SDS-PAGE. The sensitivity of the purified antibodies was assessed by indirect ELISA with the purified antibodies diluting in 4% SMP by serial 2-fold.

were established and stored in our laboratory (Fu et al., 2015). The SCRV-QY strain was isolated and stored in our laboratory (Fu et al., 2017). The virus was propagated in CPB cells, and the viral titer was measured by Reed-Muench as described previously (Lin et al., 2017). 2.2. Virus purification The cells infected with SCRV-QY virus were harvested and cell debris was removed by centrifugation at 4000×g for 15 min at 4 °C. The supernatant was centrifuged at 100000 g for 2 h. The SCRV-QY virus pellet was resuspended in PBS and centrifuged through a discontinuous sucrose gradient (30, 40, 50, 60%, w/v) at 80000 g for 1.5 h. The distinct virus bands were aspirated, diluted in PBS, and pelleted at 100000 g for 2 h. The SCRV-QY virus pellet was resuspended in TE buffer (10 mM Tris–Cl, 1 mM EDTA, pH 7.4) and stored at −20 °C. The purified SCRV-QY viruses were analyzed by electron microscope and SDS-PAGE.

2.5. Indirect immunofluorescence assay (IFA) and Western Blotting The reaction between antibody and SCRV-QY virus was evaluated by IFA and Western Blotting. Briefly, CPB cells were infected with SCRV-QY strain (MOI = 0.01), and fixed in 4% paraformaldehyde at 12 h post-infection. The fixed cells were incubated with the 4H8 and 4E12 antibodies for 1 h, respectively, then FITC-conjugated goat antimouse IgG was added to detect the bound antibodies. The infected cell samples were separated by SDS-PAGE, and transferred onto a nitrocellulose membrane by semi-dry apparatus (BioRad). After blocking, blotted membranes were incubated with the 4H8 and 4E12 antibodies and washed three times, respectively, then incubated with the secondary antibody diluted in PBS with 1:5000 at room temperature for 1 h. The bands were visualized using DAB (ComWin Bio, Beijing, China) and SuperSignal®West Pico Trial Kit (Thermo, Rockford, USA) according to the manufacturer's instructions.

2.3. Preparation of monoclonal antibodies Five 6~8-week-old SPF BALB/C mices were initially immunized subcutaneously with 50 μg/mice antigen (purified SCRV-QY viruses) emulsified with complete Freund's adjuvant (Sigma, Missouri, USA). Subsequently, the mice were immunized with 50 μg/mice immunogen emulsified with Freund's incomplete adjuvant three times at 2-week intervals. Antiserum was collected from the immunized mice at 7 days after the fourth immunization. Antiserum titers was monitored by indirect-ELISA. Two weeks after the final immunization, the mice were injected intraperitoneally with 50 μg/mice antigen in sterile PBS, and cell fusion was conducted on three days post-infection. Single splenocyte suspensions from the adequate mice were fused with mouse myeloma cell SP2/0 using polyethylene glycol 3350 (PEG3350, Sigma-Aldrich). The hybridomas were cultured into 96-well culture plates (Corning Costar, Bodenhein, Germany) for 10 days, the cell supernatants were screened by indirect ELISA. Briefly, the 96-well microtiter plates were coated with 100 μL purified SCRV-QY viruses (4 μg/mL) overnight at 4 °C. After washing three times with PBS containing 0.05% Tween-20 (PBST), the plates were blocked with 250 μL/ well 8% skimmed milk proteins (SMP) for 1 h at 37 °C. The cell supernatants were added into each well and incubated for 1 h at 37 °C. After washing three times with PBST, the plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10000, Jackson, America) for 1 h at 37 °C. Following incubation, the plates were washed three times and inoculated with a chromogen reagent (3,3′,5,5′-tetramethylbenzidine; Sigma –Aldrich, St. Louis, MO, USA). The absorbance was measured at 450 nm. The positive clones were subcloned at least twice by limiting dilution and expanding culture. The subtype of the mAbs was confirmed using mouse monoclonal antibody isotype reagents (Sigma-Aldrich). The reaction with SCRV of selected mAbs was detected by indirect ELISA as the above described. The epitope of 4H8 and 4E12 were identified by indirect competitive ELISA. Briefly, the concentration of 4H12 with Biotin (1:100) were determined by indirect ELISA. The 96-well microtiter plates were coated with 100 μL purified SCRV-QY viruses (5 μg/mL) overnight at 4 °C. The primary antibodies were 50 μL 4H12-Bio + 50 μL pending next antibody, the 50 μL 4H12-Bio+50 μL SP2/0 supernant was used as positive control and the 50 μL 4H12-Bio+50 μL 4H12 was served as negative control. The second antibody was HRP-conjugated goat antimouse IgG. The OD450 was similar to positive control, which indicated that 4H12 and the pending next antibody possessed different epitope.

2.6. Development of DAS-ELISA The optimal concentrations of capture mAb (2 μg/mL and 4 μg/mL) and detection mAb (1:100, 1:200, 1:400 and 1:800) were determined by checkerboard titration. The procedure in detail of DAS-ELISA was performed as described previously (Ming et al., 2019). In order to obtain standard curve of the DAS-ELISA, microtiter plates were coated with 100 μl (2 μg/mL) capture antibody 4H8 for overnight at 4 °C. After washing and blocking, the 100 μL serial 2-fold dilutions of purified SCRV-QY (5000 ng/mL~78.125 ng/mL) were added into the wells and incubated for 1 h. The conjugated HRP-avidin 4E12 was added after washing. The OD was measured at 450 nm. The standard curve was calculated with the plotting OD450 against the purified SCRV concentration by using scatter plot in Excel. 2.7. Duplicability test Four batches of the inactivated SCRV-QY vaccine were detected by DAS-ELISA, and the variable coefficient of DAS-ELISA was established based on the detected data. Variation coefficient less than 10% represented good repeatability. 2.8. Application of DAS-ELISA in inactivated SCRV vaccine Three batches of SCRV-QY viruses were harvested and measured by Reed-Muench, and inactivated with 1‰ formaldehyde for 12 h or 2‰ BPL for 72 h, respectively. The antigen contents of different batches inactivated SCRV-QY vaccine were detected by the DAS-ELISA. 3. Results 3.1. Virus purification

2.4. The ascites preparation, purification, and sensitivity The large number of bullet-shaped virus was observed by electron microscope, and many bands with molecular weight 35 kDa–66.2 kDa of viral proteins were detected in purified viruses (Fig. 1a and b).

To induce the ascites fluids, BALB/c mice were inoculated intraperitoneally with the two hybridoma cell lines, 4H8 and 4E12. The 2

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with R2 = 0.9896 (Fig. 3). Variation coefficient 9.8% showed that the DAS-ELISA has good repeatability. 3.5. Application of DAS-ELISA in inactivated SCRV vaccine In order to assess the efficacy of DAS-ELISA, three batches of different SCRV viral titer (109.8, 1010.25, and 1010.67) were inactivated and tested by DAS-ELISA. The antigen concentration (351.62, 446.35, 481 ng/mL with formaldehyde, and 203.50, 342.09, 384.01 ng/mL with BPL, respectively) was in parallel with the viral titer. The results showed that the antigen contents of three batches inactivated vaccines were positive correlated with virus TCID50, and DAS-ELISA can detect the slight changes in the antigen amounts of the different SCRV virus TCID50 (Table 5).

Fig. 1. The identification of purified SCRV virions. a: The electron microscopy of purified SCRV viruses; b: SDS-PAGE analysis of purified viruses, M: marker, virus: purified virus.

Table 1 The reaction with purified SCRV viruses of different hybridoma cell lines. Strains

6F10

5H4

4H8

4E12

OD(SCRV)

0.6527

0.8087

1.5974

2.4326

4. Discussion Rhabdoviruses have a broad spectrum host range including plants, insects, fish, mammals, and reptiles (Kuzmin et al., 2009). They caused huge economic losses in humans, livestock and aquaculture. SCRV can cause serious disease outbreaks and threat for Siniperca chuatsi aquaculture (Basak et al., 2007; Yoder et al., 2019; Zhao et al., 2019). Vaccination was the main way of preventing SCRV. SCRV vaccines in different R&D stages are now being developed by research institutes and enterprises in China. Vaccine quality control is vital to the SCRV vaccine production. Double antibody sandwich ELISA was higher sensitivity and specificity than indirect ELISA, which can accurately quantify antigens with simple operation (Hutchings and Ferris, 2006; Li et al., 2014; Luo et al., 2012; Ten Haaf et al., 2017). In this study, we established an optimized DAS-ELISA method based on the capture antibody 4H8 and detector antibody 4E12 for testing SCRV vaccine antigen content. The characteristics of antibodies directly determine the specificity and sensitivity of the DAS-ELISA. Purified natural antigens play an important role in the production antibodies for immunological assay (Chand et al., 2009; Pál et al., 2005). The sensitivity of DAS-ELISA for detecting bluetongue virus increased 100-fold by using antibodies against purified virions as immunogen (Chand et al., 2009; Hans Bunschoten et al., 1989; Kramer et al., 2005). The rabies virus G protein can induce neutralizing antibodies. However, the production of neutralizing antibodies largely depends on the three-dimensional structure of G protein. The soluble recombinant rabies virus G protein is less immunogenic than its purified virions (AT Piza et al., 2002; Dietzschold et al., 1983; Gamoh et al., 1996). In this study, we produced antibodies by using purified SCRV-QY virions as immunogen. Antibodies 4H8 and 4E12 had high affinity for purified SCRV-QY virions. And the size of the SCRV-QY antigen recognized by 4H8 and 4E12 is approximately 70 kDa. It have been reported that the SCRV G protein band size of 66 kDa was detected by mAb (Chen et al., 2012). Therefore, we speculated that 4H8 and 4E12 was targeted the native G protein from SCRV virus. These results showed that antibodies 4H8 and 4E12 can identify SCRV-QY virus with specificity and high affinity. We selected the 4H8 and 4E12for increasing the sensitivity and specificity of the DAS-ELISA. In this detection method, the linear curve was generated with the valid detection ranges of 78.125~5000 ng/mL. The detection spectrum of DAS-ELISA was a narrower range. However, this method is simple, convenient and time-saving for quantifying the SCRV antigen amounts. The OD values of DAS-ELISA

3.2. The characterization of SCRV-QY mAbs 4 hybridoma cell lines against purified SCRV-QY virus (named 6F10, 5H4, 4H8 and 4E12) were achieved after limiting dilutions. The isotype of 6F10, 5H4, 4H8 and 4E12 were identified as IgG1. According to ELISA results, 6F10, 5H4, 4H8 and 4E12 can recognize the purified SCRV-QY viruses, and 4H8 and 4E12 had high affinity with the natural SCRV-QY virus (Table 1). In order to establish a DAS-ELISA method for accurately detecting the SCRV-QY antigen amounts, we selected 4H8 and 4E12 with high affinity for the SCRV-QY strain antigen (Table 1). Meanwhile, the OD450 of 4H8+4E12(Bio) was 1.2327, it indicated that 4H8 and 4E12 have different epitopes (Table 2). 3.3. The sensitivity and reactivity of purified antibody 4H8 and 4E12 contained k light chains, and the concentration of 4H8 and 4E12 were 4 mg/mL and 6 mg/mL (Fig. 2a). The sensitivity of ELISA results showed that 15.625 ng/mL of the two purified 4H8 and 4E12 have a strong signal with purified SCRV QY particles (OD450 = 0.4629 and OD450 = 0.576) (Table 3). WB results revealed that 4H8 and 4E12 reacted with the SCRV-QY strain. And the size of the SCRV antigen recognized by 4H8 and 4E12 is approximately 70 kDa (Fig. 2b). According to IFA results, 4H8 and 4E12 reacted with the SCRV-QY infected CPB cells, but not uninfected CPB cells (Fig. 2c). These results showed that 4H8 and 4E12 can recognize the SCRV-QY virus with specificity and high affinity. 3.4. Establishment of DAS-ELISA 4H8 and 4E12 were selected as the optimal capture and detector antibody according to the above ELISA results. The results of checkerboard titration showed that the optimal concentration of the capture antibody was 2 μg/mL (100 μL), and the detector antibody (HRPavidin) was 1:400 (15 μg/mL) (Table 4). The linear standard curve was obtained with the detection range of 78.125~5000 ng/mL. The linear equation was Y = 1792.1x-260.16 Table 2 The identification epitope of antibodies. Coated(Virus)

5 μg/mL

Primary antibody OD(Values)

4H8+4E12(Bio) 1.2327

5H4+4E12(Bio) 1.2836

3

4E12+4E12(Bio) 0.5873

4E12(Bio)+sp2/0 supernatants 1.095

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Fig. 2. The purity and characterization of the monoclonal antibodies 4H8 and 4E12. a: The SDS-PAGE analysis of purified antibodies 4H8 and 4E12; b: The 4H8 and 4E12 reacted with SCRV viruses; c: The monoclonal antibodies 4H8 and 4E12 recognized SCRV infected CPB cells by immunofluorescence. Table 3 The reaction and sensitivity with purified SCRV viruses of 4H8 and 4E12. SCRV virus (ng/mL)

1000 μg/mL

500 μg/mL

250 μg/mL

125 μg/mL

62.5 μg/mL

31.25 μg/μg/mL

15.625 μg/mL

Con

4E12 4H8

2.8958 2.5951

2.9638 2.5282

2.6686 2.3508

2.5802 2.0791

1.9918 1.4554

1.2007 0.867

0.576 0.4629

0.0375 0.0179

Table 4 The result of checkerboard titration. Detector Capture antibody

4E12(1:100) 4E12(1:200) 4E12(1:400) 4E12(1:800)

4H8 4 μg/mL(coated)

2 μg/mL(coated)

SCRV(μg/ mL)

OD(values)

SCRV(μg/ mL)

OD(values)

5 0 5 0 5 0 5 0

3.1366 0.6528 4.4708 0.3836 4.4204 0.2414 2.2375 0.1359

5 0 5 0 5 0 5 0

3.2269 0.5611 3.2159 0.3487 2.7288 0.1651 0.6284 0.1271

Fig. 3. The linear standard curve of DAS-ELISA with the detection range of 78.125~5000 ng/mL. The linear equation:Y = 1792.1x-260.16 with R2 = 0.9896.

decreased when the SCRV sample is further diluted, indicating that the method could detect the slight changes in the antigen amounts of different SCRV virus titer. Meanwhile, this method may detect the antigen contents of different batches vaccines in the industry. Those results suggested the DAS-ELISA satisfied our common measurements. In conclusion, we obtained two mAbs (4H8 and 4E12) against SCRV virus with high affinity, which may have potential applications in the study of pathogenic mechanism. At the same time, we established the DAS-ELISA using 4H8 for capture antibody and 4E12 for detector antibody. It has demonstrated that the DAS-ELISA can be better suited to

accurately quantify antigen concentration of inactivated SCRV vaccine, which has a potential role in monitoring vaccine quality and the SCRV vaccine production.

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Table 5 Antigen quantification of different batches of SCRV vaccine. SCRV(TCID50/ml)

9.8

A(10 ) B(1010.25) C(1010.67)

Antigen concentration(ng/mL) Formaldehyde

BPL

351.62 446.35 481

203.50 342.09 384.04

Compliance with ethical standards This study does not contain any studies with human participants performed by any of the authors. All experimental procedures were performed with institutional guidelines for the care and use of laboratory animals in Sun Yat-sen University, Guangzhou, China, and conformed to the National Institutes of Health Guide for Care and Use of laboratory Animals (Publication No. 85-23, revised 1985). Notes The authors declare no competing financial interest. Declaration of competing interest The authors declare that have no conflict of interest. Acknowledgements This study was financially supported by Chinese Academy of Fishery Sciences Fundamental Research Projects, China (2019ZD0702); the special funds for Marine Fishery Science & Technology Promotion Project of Guangdong Provinces, China (the study on the key technology of ISKNV and SCRV inactivated combined vaccine development and large-scale preparation); Special Funds for Economic Development of Marine Economy of Guangdong Province, China (GDME-2018C007); China-ASEAN Maritime Cooperation Fund, China; Guangdong Provincial Special Fund For Modern Agriculture Industry Technology Innovation Teams, China (2019KJ140); Pearl River Science & Technology NovaProgram of Guang zhou City, China (no. 201710010087). References Assenberg, R., Delmas, O., Morin, B., Graham, S.C., De Lamballerie, X., Laubert, C., Coutard, B., Grimes, J.M., Neyts, J., Owens, R.J., Brandt, B.W., Gorbalenya, A., Tucker, P., Stuart, D.I., Canard, B., Bourhy, H., 2010. Genomics and structure/ function studies of Rhabdoviridae proteins involved in replication and transcription. Antivir. Res. 87, 149–161. AT Piza, K.P., Lusa, G.M., Caporale, G.M.M., Terreran, M.T., Machado, L.A., Zanetti, C., 2002. Effect of the contents and form of rabies glycoprotein on the potency of rabies vaccination in cattle. Mem. Inst. Oswaldo Cruz 97, 265–268. Basak, S., Mondal, A., Polley, S., Mukhopadhyay, S., Chattopadhyay, D., 2007. Reviewing Chandipura: a vesiculovirus in human epidemics. Biosci. Rep. 27, 275–298. Blondel, D., Maarifi, G., Nisole, S., Chelbi-Alix, M.K., 2015. Resistance to Rhabdoviridae infection and subversion of antiviral responses. Viruses 7, 3675–3702. Chand, K., Biswas, S.K., De, A., Sing, B., Mondal, B., 2009. A polyclonal antibody-based sandwich ELISA for the detection of bluetongue virus in cell culture and blood of sheep infected experimentally. J. Virol Methods 160, 189–192. Chen, Z.Y., Lei, X.Y., Zhang, Q.Y., 2012. The antiviral defense mechanisms in Mandarin

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