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An optimized double-antibody sandwich ELISA for quantitative detection of WSSV in artificially infected crayfish
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An optimized double-antibody sandwich ELISA for quantitative detection of WSSV in artificially infected crayfish Xiaoqian Tanga,b, Qianrong Lianga, Lushan Liua, Xiuzhen Shenga, Jing Xinga,b, Wenbin Zhana,b,
⁎
a
Laboratory of Pathology and Immunology of Aquatic Animals, KLM, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, No.1 Wenhai Road, Aoshanwei Town, Jimo, Qingdao 266071, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: DAS-ELISA WSSV Quantitative detection Crayfish Viral propagation
Developing a rapid, accurate and quantitative method for detecting white spot syndrome virus (WSSV) is extremely urgent and critical for reducing the risk of white spot disease outbreaks. In the present work, an optimized double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for quantitative detection of WSSV. The method employed rabbit polyclonal antibodies against WSSV as the capture antibody and previously produced anti-WSSV monoclonal antibodies as the detector antibody. A standard curve of the log concentration of WSSV versus OD value was established, which was linear in the concentration range of 120–7680 ng/mL, and the linear regression equation was y = 0.166x − 0.151. Viral proteins in different tissues of crayfish (Procambarus clarkia) post artificial infection with WSSV were quantitatively measured using the DAS-ELISA. WSSV proliferated quickly within 60 h post infection and gradually slowed down afterwards. According to the linear regression relationship, the viral proteins in hemolymph, gut and gonad were firstly able to be quantified at 24 h post infection with the concentrations of 186, 158 and 128 ng/mL, respectively. These three tissues also contained higher viral proteins than the gill, heart, hepatopancreas and muscle during the entire infection period. The viral protein concentration in gut reached the highest level of 6220 ng/mL at 72 h post infection. Real time quantitative PCR was also used to detect the dynamic change of viral copies in crayfish hemolymph post WSSV infection, with similar results for both assays. The developed DAS-ELISA could detect WSSV propagation from initial to moribund stage in infected crayfish and demonstrated potential application for diagnosis of WSSV.
1. Introduction White spot syndrome, caused by the white spot syndrome virus (WSSV), is one of the most serious epizootic diseases in the shrimp farming industry throughout the world (Chou et al., 1995; Flegel, 1997). WSSV belongs to the genus Whispovirus in a new virus family Nimaviridae (Mayo, 2002), which is an enveloped virus with a doublestranded circular DNA genome of about 300 kb (van Hunlten et al., 2001; Yang et al., 2001) and contains approximately 181 putative open reading frames, encoding more than 39 structural proteins (SánchezPaz, 2010). The virus could infect shrimps and other freshwater and marine crustaceans (Lo et al., 1996a; Wang et al., 1998). Many methods have been developed for detecting WSSV, including PCR (Lo et al., 1996b; Kim et al., 1998), in situ PCR (Jian et al., 2005), in situ hybridization (Wongteerasupaya et al., 1996; Nunan and Lightner, 1997), immune methods (Huang et al., 1995; Nadala and Loh, 2000; Zhan et al., 1999; Wang and Zhan, 2006) and loop-mediated isothermal
⁎
amplification assay (Kono et al., 2004), etc. However, all of these assays are qualitative and cannot determine the exact amount of virus. Recent studies showed that fluorescent quantitative PCR could provide a sensitive method for quantifying the number of DNA templates (Durand and Lightner, 2002; Sritunyalucksana et al., 2006), which was widely proved to be rapid, accurate, and available to detect WSSV in laboratory facilities. The qPCR determines the viral load by detecting the copies of a specific gene segment, however, it does not necessarily reflects the amount of packaged mature viral particles that might better reflect the infecting potential and risk of outbreak. In contrast, the double antibody ELISA generally adopts a polyclonal antibody that could recognize multiple epitopes as the capturer and specific monoclonal antibodies as the detector to quantify the antigen at protein level (Mecham, 2006; Niu et al., 2013; Chang et al., 2016), which provided an accurate and sensitive method for detecting viral pathogens and could be further applied in WSSV detection for shrimps. So far there have been no effective measures to control WSSV, thus it is of great
Corresponding author at: Laboratory of Pathology and Immunology of Aquatic Animals, KLM, Ocean University of China,5 Yushan Road, Qingdao 266003, PR China. E-mail address: [email protected] (W. Zhan).
http://dx.doi.org/10.1016/j.jviromet.2017.10.020 Received 24 July 2017; Received in revised form 20 October 2017; Accepted 20 October 2017 0166-0934/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Tang, X., Journal of Virological Methods (2017), http://dx.doi.org/10.1016/j.jviromet.2017.10.020
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significance to monitor the infection status and evaluate infection severity of cultured shrimp for preventing the spread and outbreak of white syndrome disease. Therefore, in the present work, an optimized double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed and utilized to systematically investigate the temporal and spatial dynamic WSSV proliferation in the infected crayfish.
were harvested 7–10 days after inoculation, then the MAbs were purified from the mice ascites fluids by the caprylic acid/ammonium sulfate precipitation method (Sheng et al., 2012). The purity of the purified MAbs was checked by SDS-PAGE and the titer (2E6 and 2A3 mixed in a 1:1 ratio) was also determined by indirect ELISA as above.
2. Materials and methods
Microtiter plates were coated with 100 μL of optimal dilution of capture antibody (rabbit anti-WSSV polyclonal antibodies) in 0.01 M carbonate-bicarbonate coating buffer (pH 9.6) for 1 h 37 °C, and stored at 4 °C overnight, then the unbounded antibodies were washed off using 200 μL PBST for three times and 150 μL blocking buffer was added to all the wells for 1 h at 37 °C. After washing as above, wells were incubated with 100 μL serial 2-fold dilutions of purified WSSV (30,720 ng/ mL–7.5 ng/mL) in triplicate at 37 °C for 1 h and then washed as before. Then, the wells were incubated with 100 μL optimal dilution of detective antibodies (MAbs 2E6 and 2A3 mixed in a 1:1 ratio) at 37 °C for 1 h. After washing as above, the wells were then incubated with 100 μL AP-conjugated goat anti-mouse Ig diluted 1:3000 in PBS at 37 °C for 1 h. After washing, 100 μL of 0.1% (w/v) pNPP diluted in pNPP buffer was added to each well of the plate. The reaction was allowed to proceed for 30 min at 37 °C and stopped with 50 μL per well of 2 M NaOH. The plate was read at 405 nm in the microplate reader. PBS instead of the purified WSSV was set as negative control. To confirm the optimal dilution for capture and detective antibody, a checkerboard titration was previously performed. All the ELISA steps were performed as described above. The standard curve was obtained by plotting mean absorbance values against the logarithm of purified WSSV concentrations and fitted to a four-parameter logistic equation using Origin 8.0 (OriginLab, USA) software package.
2.4. Establishment of standard curve for the DAS-ELISA
2.1. WSSV virion purification Moribund shrimp (Fenneropenaeus chinensis) with symptomatic white spots were collected from one shrimp farm in Shandong Province, China. After confirmation of WSSV infection by nested PCR (Lo et al., 1996b), the gills of the shrimp were used for WSSV purification according to the previous method (Wang and Zhan, 2006). Briefly, the homogenate of WSSV-infected gills was centrifuged at 2000g for 10 min at 4 °C, and the supernatant fluid was added to a discontinuous sucrose gradient of 35%, 50% and 60% (w/v) and ultracentrifuged (Hitachi CP 100MX) at 80, 000 g for 2 h at 4 °C. The virus band was collected by puncturing the tube with a syringe needle and then mixed with 5 times TNE buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA-Na2, pH 7.4) and repelleted at 80, 000 g for 1.5 h at 4 °C. The resulting pellet was dissolved in 0.01 M phosphate buffered saline (0.01 M PBS, pH 7.4). The concentration of purified virus was determined by Bradford assay. To check for quality and quantity, virus samples were negatively stained and examined under a transmission electron microscope (JEOL JEM1010). 2.2. Preparation of anti-WSSV polyclonal antibody Anti-WSSV polyclonal antibodies were prepared according to the method described by Zhang et al. (2010) with modifications. Briefly, an aliquot equivalent to 400 μg of purified WSSV was mixed with the same volume of Freund’s complete adjuvant and injected subcutaneously in multiple spots on the back of a 2.5 kg female New Zealand rabbit. After 2 weeks, the first booster immunization was given to the rabbit with the same dosage of purified WSSV in Freund's incomplete adjuvant (Sigma, USA). Another two booster injections were given with 200 μg WSSV without adjuvant at one-week interval. Rabbit serum was collected 7 days after the final injection and followed with centrifugation, the serum supernatants were stored at −80 °C for later use. Indirect ELISA was conducted to confirm specificity of the polyclonal antibody and determine the binding titers. For titer detection, 100 μL of the purified WSSV solution containing 3 μg of purified WSSV was coated on each well of the microtiter plate at 4 °C overnight, then followed with three washes by PBST (PBS containing 0.05% Tween-20, Solarbio, USA), 150 μL blocking buffer (4% bovine serum albumin in PBS) was added. After blocking, 100 μL of serially diluted polyclonal antibodies (from 1:400 to 1:4 × 105) or PBS were incubated at 37 °C for 1 h. Then, the AP-conjugated goat anti-mouse Ig (Sigma, USA) diluted 1:3000 in PBS at 37 °C for 1 h. After washing, 100 μL of 0.1% (w/v) pNPP (Sigma, USA) diluted in pNPP buffer (1% diethanolamine, 0.5 mM MgCl2, pH 9.8) was added to each well of the plate. The reaction was allowed to proceed for 30 min at 37 °C and stopped with 50 μL per well of 2 M NaOH. The plate was read at 405 nm in a microplate reader (Molecular Devices).
Y = {(A − D)/[1 + (x/C)B} + D
(1)
where A is the Y value corresponding to the asymptote at the low values of the X-axis and D is the Y value corresponding to the asymptote at high values of the X-axis. The coefficient C is the X-value corresponding to the midpoint between A and D. The coefficient B describes how rapidly the curve makes its transition from the asymptote in the center of the curve. The linear fit was based on the equation Y = A + B*log(X) where X is the concentration of purified WSSV. The curve fitting is based on the Lavenberg-Marquardt method (Kumuda and White, 1998). Then the total WSSV concentrations of the samples could be determined according to the model. 2.5. Validation of WSSV DAS-ELISA The calibration model was evaluated using standard curves from three validation runs. The relative error (RE) and recovery were analyzed between the back-calculated concentration and nominal concentration of each WSSV dilution. RE = [(observed concentration/nominal concentration) − 1] × 100% (2) Recovery = (observed concentration/nominal concentration) × 100% (3) The putative validated range of the assay, between the upper limit of quantitation (ULOQ) and lower limit of quantitation (LLOQ), was determined using data from sextuplicate of WSSV serial dilutions from 30,720 to 7.5 ng/mL. The mean%RE for each dilution level was determined and the two-sided 90% confidence intervals (CI%RE) for the mean were constructed using Microsoft Excel software:
2.3. Preparation of anti-WSSV monoclonal antibodies (MAbs) Two previously produced hybridoma cell lines, 2E6 and 2A3, which produce monoclonal antibodies reactive towards viral envelope and capsid of WSSV respectively (Wang and Zhan, 2006), were employed for ascites fluid production. Pristane-primed BALB/c mice were injected intraperitoneally with the activated hybridomas, and the ascites fluids
90% CI%RE = ū ± 1.645 (δ/n1/2)
(4)
where ū = mean%RE, δ = standard deviation of ū, and n = number of 2
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data points for ū. The ULOQ and LLOQ were defined as the two-sided 90% CI%RE for which mean%RE was −25% and +25%, respectively (Findlay et al., 2000). The repeatability (intra-assay reproducibility) and intermediate precision (inter- assay reproducibility) of the assay were assessed using serially diluted purified WSSV samples, with the concentrations of stock WSSV solution determined to be 30,720 ng/mL by Bradford assay. The stock solution was serially diluted in PBS to achieve the following concentrations: 7680, 3840, 1920, 960, 480, 240 and 120 ng/mL and assayed in sextuplicate during four separate runs of the WSSV sandwich ELISA. The precision (%CV) was calculated for each sample for each run and among all runs.
Table 1 Checkerboard titration of the capture and detector antibodies. Dilution of detector antibody (1:X)
50 100 200 400 800 1600 3200 6400 Negative control
2.6. Quantification of WSSV protein concentration in crayfish during infection using the DAS-ELISA
Dilution of capture antibody (1:X)
200
400
800
1600
3200
6400
12800
25600
0.494 0.475 0.469 0.387 0.372 0.347 0.327 0.301 0.086
0.488 0.471 0.470 0.382 0.367 0.339 0.316 0.295 0.093
0.489 0.464 0.445 0.384 0.356 0.318 0.299 0.299 0.096
0.478 0.466 0.457 0.373 0.349 0.313 0.292 0.280 0.093
0.496 0.454 0.449 0.362 0.345 0.303 0.287 0.268 0.090
0.470 0.447 0.442 0.351 0.334 0.296 0.279 0.267 0.090
0.438 0.424 0.416 0.320 0.306 0.286 0.268 0.251 0.093
0.406 0.393 0.385 0.312 0.295 0.267 0.250 0.240 0.106
The underlined value means the OD405 value obtained under the optimal dilutions of capture antibody and detective antibody.
The experimental crayfish (Procambarus Clarkia) with an average weight of 15 g were purchased from a local aquatic product market in Qingdao, Shandong Province, China, which were confirmed WSSV-free by nested PCR assay (Lo et al., 1996b). The crayfish were maintained at 25 °C in fresh water with continuous aeration for one week, then 200 crayfishes were randomly selected and averagely divided into two groups. Each crayfish from one group was intramuscularly injected with 50 μL WSSV inoculum containing 10−1 ng WSSV, the crayfish injected with equal volume of PBS was performed as control. At 0, 24, 36, 48, 60, 72, 84 and 96 h post infection (h p.i.), eight crayfish were randomly selected both from the WSSV-infected group and control group, and the hemolymph, gill, hepatopancreas, heart, gonad, gut and muscle were sampled and mixed with two volumes of homogenization buffer (PBS containing 0.5 M EDTA), then homogenized at 4 °C with 18 strokes of a Dounce homogenizer. In order to inhibit the endogenous alkaline phosphatase activity (Zhan et al., 2004), the homogenates were incubated at 37 °C for 1 h, and then the WSSV concentrations were detected using the DAS-ELISA as described above. Meanwhile, proportions of hemolymph were also sampled at different time points post WSSV infection and used for detection of viral copies by real time qPCR later.
monoclonal antibodies were determined to be 1:4 × 105 and 1:6400, respectively (Tables A. 1 and A. 2 in Supplementary material). Then several dilutions of anti-WSSV MAbs and rabbit anti-WSSV antibodies were used in a checkerboard titration to determine the working dilution for each reagent. The antibodies display similarly strong absorbance values up to a dilution of 1/6400 for mouse and 1/200 for rabbit (Table 1). A dilution of 1/3200 was selected for the rabbit antisera thereby ensuring a slight excess of capture antibody on the plates, and a dilution of 1/100 anti-WSSV MAbs was selected to maximize the detection of captured virions, while OD405 value of 0.454 obtained using the optimized dilutions of two antibodies was underlined in Table 1. 3.2. The standard curve After the purified WSSV suspensions with serial dilution from 7.5 to 30,720 ng/mL was assayed via the optimized DAS-ELISA, a sigmoidal standard curve was generated, which was fitted well (r = 0.99) to a four-parameter logistic equation (Fig. 1A). The four-parameter logistic equation is expressed as: Y = {(A1 − A2)/[1 + (x/x0)p]} + A2, in which A1 = 0.1442 ± 0.0065, A2 = 0.5530 ± 0.0196, x0 = 2.9652 ± 0.0589, p = 5.5973 ± 0.4941. The minimum detection limit was 120 ng/mL with a detection range of 120–30,720 ng/mL. The mean OD405 values were at least two times greater than the negative control. Based on the sigmoidal standard curve, a linear fit equation was obtained with a concentration range of 120–7680 ng/mL with R2 = 0.992. The linear equation was expressed as: Y = 0.166X − 0.151 (Fig. 1B). The recovery of the optimized DAS-ELISA was between 0.84–1.10, and the relative error (RE) was between −15.17 to 10.42 (Table 2). Intra-assay reproducibility was determined with coefficient of variation (CV) to be in a range of 0.66–7.11% (Table 3), and the inter-assay reproducibility was in a range of 2.11–7.32% (Table 4).
2.7. Quantification of WSSV copies in crayfish hemolymph by real time qPCR In order to confirm the reliability of DAS-ELISA, the dynamic change of WSSV copies in the hemolymph sampled at different time points post WSSV infection was detected as previously described (Yuan et al., 2007). Briefly, series dilutions of WSSV recombinant plasmid (copy number known) were prepared as standards for quantification. Then total genomic DNA in sampled hemolymph was extracted using DNA extraction kit (Takara, Japan) following the manufacturer’s instruction. An equal quantity of DNA (50 ng) of each sampling time was added into the SYBR Green Premix with the specific primers (F:5′-AAACCTCCGCATTCCTGTGA-3′, R:5′-TCCGCATCTTCTTCCTTCAT-3′, Tm: 57 °C) for amplification. Quantitation of the amount of WSSV copies in hemolymph was accomplished using the standard curve.
3.3. Dynamic changes of viral protein load in WSSV-infected crayfish The optimized DAS-ELISA was employed to investigate the dynamic changes of WSSV concentrations in different tissues of crayfishes after WSSV infection. According to values calculated from the linear fit equation, the earliest time to detect WSSV-positive signal was at 24 h in hemolymph, gut and gonad with the concentrations of 186, 158 and 128 ng/mL, respectively. At 36 h post infection, the WSSV-positive signals could be detected in all the 7 sampled tissues, among them, the viral concentrations in gut and gonad were significantly higher than the other tissues, which mounted up to 2166 and 1012 ng/mL, respectively. During the whole infection process, the viral proliferation in gut, hemolymph and gonad were obviously higher than the other tissues, which reached their peak levels with 6220, 3715 and 2681 ng/mL, respectively. Since 60 h post infection, the viral concentrations in
3. Results 3.1. Optimization of capture and detector antibodies determined by checkerboard titration method Abundant intact WSSV virions with a high purity were observed by transmission electron microscope (Fig. A. 1 in Supplementary material), which was used as the standard sample. The purified anti-WSSV MAbs (2E6 and 2A3) was analyzed by SDS-PAGE, which showed two clear bands with the expected molecular weight masses of heavy (about 55 kDa) and light (about 25 kDa) chains (Fig. A. 2 in Supplementary material). By indirect ELISA, the binding titers of polyclonal and 3
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Fig. 1. The standard curves for WSSV DAS-ELISA.
Table 2 Recovery and relative error (RE) of WSSV DAS-ELISA. Nominal concentration (ng/ mL)
Observed concentration (ng/mL)
Recovery
RE
7680.00 3840.00 1920.00 960.00 480.00 240.00 120.00
8226.93 3956.57 1907.36 1060.00 407.17 214.59 117.48
1.07 1.03 0.99 1.10 0.85 0.89 0.98
7.12 3.04 −0.66 10.42 −15.17 −10.59 −2.10
Nominal concentration: The WSSV concentration determined by the Bradford assay; Observed concentration: The WSSV concentration calculated by the DAS-ELISA.
Table 3 Intra-assay reproducibility of DAS-ELISA. Concentration of WSSV (ng/mL)
Plate A (%CV)
Plate B (%CV)
Plate C (%CV)
Plate D (%CV)
7680 3840 1920 960 480 240 120 Negative control
4.17 0.66 2.22 5.28 6.74 6.85 5.78 1.13
3.72 4.21 2.35 4.17 1.57 3.29 2.55 1.73
5.69 6.01 4.12 2.24 5.08 3.93 4.56 2.59
6.22 7.11 6.46 4.56 6.05 2.34 3.23 2.38
Fig. 2. The variation of WSSV concentrations in different tissues of crayfishes during WSSV infection.
different tissues maintained relatively stable, and then all of the infected crayfishes eventually died off at 96 h post infection (Fig. 2). 3.4. Dynamic change of WSSV DNA copies in WSSV-infected crayfish hemolymph The number of WSSV copies in hemolymph samples was calculated according to the standard curve generated from samples of purified cloned-WSSV plasmid tested by qPCR. The results showed that WSSV copies in the hemolymph could be detected as early as 6 h p.i. with 101 per ng DNA, and it maintained at a low level till 18 hp.i., but significantly increased since 24 hp.i. (p < 0.05), during period of 24–48 hp.i., the copy number significantly increased from about 103 to 105, and then the viral load increased slightly after 48 hp.i. (Fig. 3).
Each dilution sample was tested in sextuplicate in each plate.
Table 4 Inter-assay reproducibility of DAS-ELISA. Concentration of WSSV (ng/mL)
7680 3840 1920 960 480 240 120 Negative control
OD405 value of Plate A–D Mean ± SD
Inter-assay reproducibility (%CV)
0.486 0.455 0.427 0.343 0.289 0.245 0.204 0.095
6.58 5.27 7.32 5.93 4.17 4.96 4.48 2.11
± ± ± ± ± ± ± ±
0.032 0.024 0.030 0.020 0.012 0.012 0.009 0.002
4. Discussion Double antibody sandwich ELISA has higher sensitivity and specificity than indirect ELISA (Hutchings and Ferris, 2006; Luo et al., 2012; Li et al., 2014), and can accurately quantify antigens with easy experimental operation (Fæste and Plassen, 2008; Tang et al., 2010; Haaf et al., 2017). In the present work, an optimized DAS-ELISA technique was established for WSSV detection at protein level, which used the rabbit antisera against WSSV and mixed MAbs against WSSV as the 4
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to inhibit this non-specific reactions during practical tests, endogenous alkaline phosphatases were inactivated with 0.5 M EDTA according to our previous procedure (Zhan et al., 2004). Freshwater crayfish is sensitive to the WSSV and has become an excellent animal model to study this virus (Huang et al., 2001). In order to confirm that the developed DAS-ELISA could efficiently detect WSSV virions in the viral infected shrimp, here Procambarus Clarkia was used as an artificially infected model. Our results showed that WSSV proliferated quickly before within 36 hp.i., then obviously slowed down from 48 to 96 h p.i. and the virus protein load remained relatively stable afterwards. In addition, during the whole infection process, the positive signal of WSSV was earliest detected at 24 h in hemolymph, gonad and gut, and the highest WSSV loads were also found in these tissues, which suggested that hemolymph, gonad and gut are the most susceptible to WSSV. Similar proliferation processes were also found in the WSSV-infected Litopenaeus vannamei and Fenneropenaeus chinensis by competitive PCR and real time qPCR (Tang and Lightner, 2000; Sun et al., 2013; Zhong et al., 2013). In this work, the hemolymph was chosen for the WSSV copy detection by qPCR to confirm the reliability of DAS-ELISA, and the qPCR results agreed well with the DAS-ELISA results from 24 hp.i. to 96 hp.i. Compared with DAS-ELISA, qPCR could detect the viral copies within 18 hp.i., suggesting that there was no or very few packaged mature virions in the hemolymph during this period. These results suggested that no matter which detecting target molecule, DNA or protein, similar results could be obtained, which also indicated that the DAS-ELISA developed in this study could be applied for the WSSV detection. In conclusion, compared with real time qPCR, the DAS-ELISA had a higher detection limit may not allow for the very early detection in infected animals, but could determine the amount of packaged mature virions and might better reflect the risk of outbreak. Also, the WSSVpositive signal was able to be detected from the initial to moribund stage in viral infected crayfish by the DAS-ELISA, which has a potential value in evaluating the viral infection state and prevention of WSSV outbreak.
Fig. 3. Dynamic state of WSSV copies in infected crayfish hemolymph.
capture and detection antibodies, respectively. The MAbs used were previously proved to be specific to WSSV, which guaranteed the specificity of developed DAS-ELISA. In this method, a sigmoidal and a linear standard curves were both generated with the valid detection scopes of 7.5–30,720 ng/mL and 120–7680 ng/mL, respectively. The reproducibility of this method was also proved to be well by examining the CV values. All these results suggested that the DAS-ELISA developed has a good sensitivity, specificity and producibility, which could be employed for qualifying the WSSV in a convenient and simple way. The characters of antibodies used in the DAS-ELISA would directly influence the specificity and sensitivity of the detection. Previous studies have shown that the antibodies produced using the elaborately purified virions as immunogen could increase DAS-ELISA sensitivity by 100-fold in detecting the bluetongue virus (Pál et al., 2005; Chand et al., 2009; Mecham, 2006). Therefore, purified natural antigen is of extremely great importance in the production of antibodies for immunological assay, which would directly influence the sensitivity, specificity and stability of the final assay (Pál et al., 2005; Chand et al., 2009). In this study, quite abundant intact and high-purity WSSV virions were prepared for the production of rabbit antisera as capture antibodies. Similarly, many sandwich ELISA assays developed so far often adopted the polyclonal antibodies as the capturer (Mecham, 2006; Niu et al., 2013; Chang et al., 2016). It was believed that using the polyclonal antibodies instead of monoclonal antibody as the capture antibodies could efficiently increase the sensitivity of the sandwich ELISA by nearly 10-fold (Mecham and Wilson, 2004; Mecham, 2006), which was probably that the polyclonal antibodies could recognize more epitopes of antigen than the MAbs. As for the detection antibodies, the mixed MAbs against the WSSV were adopted (recognize WSSV virions both on the envelope and the capsid), which not only guaranteed the specificity, but also increased the sensitivity of the detection assay. Finally, a sigmoidal and a linear standard curves were generated with the minimum detection limits of 7.5 and 120 ng/mL, respectively. Although, the minimum detection limit of linear standard curve was a little higher, which is more convenient and simpler for calculating, as well as could satisfy common measurement. Moreover, by comparing the results of DAS-ELISA checkboard titration with indirect ELISA, the OD values of DAS-ELISA were higher than the indirect ELISA when the mixed MAbs were used at the same dilution, which suggested that the optimized DAS-ELISA developed in the present work had higher sensitivity than the indirect ELISA. In addition, our previous studies had showed that endogenous alkaline phosphatases commonly exist in shrimp tissues (Zhan et al., 2003; Zhan et al., 2004), which would usually lead to the false-positive results when using the shrimp tissue samples as the antigens without any pretreatment. Thus, in order
Acknowledgments This research was financially supported by Qingdao National Laboratory for Marine Science and Technology (QNLM2016ORP0307) and the Taishan Scholar Program of Shandong Province. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet.2017.10.020. References 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. Chang, Y.H., Xing, J., Tang, X.Q., Sheng, X.Z., Zhan, W.B., 2016. Haemocyanin content of shrimp (Fenneropenaeus chinensis) associated with white spot syndrome virus and Vibrio harveyi infection process. Fish Shellfish Immunol. 48, 185–189. Chou, H.Y., Huang, C.Y., Wang, C.H., Lo, C.F., 1995. Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Dis. Aquat. Org. 23, 165–173. Durand, S.V., Lightner, D.V., 2002. Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp. J. Fish Dis. 25, 381–389. Fæste, C., Plassen, C., 2008. Quantitative sandwich ELISA for the determination of fish in foods. J. Immunol. Methods 329, 45–55. Findlay, J.W.A., Smith, W.C., Lee, J.W., Nordblom, G.D., Das, I., DeSilva, B.S., Khan, M.N., Bowsher, R.R., 2000. Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective. J. Pharm. Biomed. Anal. 21, 1249–1273. Flegel, T.W., 1997. Major viral diseases of the black tiger prawn (Penaeus monodon) in Thailand. World J. Microbiol. Biotechnol. 13, 433–442. Haaf, T.A., Kohl, J., Pscherer, S., Hamann, H.P., Eskens, H.U., Bastian, M., Gattenlöhner, S., Tur, M.K., 2017. Development of a monoclonal sandwich ELISA for direct
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detection of bluetongue virus 8 in infected animals. J. Virol. Methods 243, 172–176. Huang, J., Yu, J., Wang, X.H., Song, X.L., Ma, C.S., Zhao, F.Z., Yang, C.H., 1995. Survey on the pathogen and route of transmission of baculoviral hypodermal and hematopoietic necrosis in shrimp by ELISA of monoclonal antibody. Mar. Fish. Res. 16, 40–45. Huang, C.H., Zhang, L.R., Zhang, J.H., Xiao, L., Wu, Q., Chen, D., Li, J.K., 2001. Purification and characterization of White Spot Syndrome Virus (WSSV) produced in an alternate host: crayfish, Cambarus clarkii. Virus Res. 76, 115–125. Hutchings, G.H., Ferris, N.P., 2006. Indirect sandwich ELISA for antigen detection of African swine fever virus: comparison of polyclonal and monoclonal antibodies. J. Virol. Methods 131, 213–217. Jian, X.F., Lu, L., Chen, Y.G., Chan, S.M., He, J.G., 2005. Comparison of a novel in situ polymerase chain reaction (ISPCR) method to other methods for white spot syndrome virus (WSSV) detection in Penaeus vannamei. Dis. Aquat. Org. 67, 171–176. Kim, C.K., Kim, P.K., Sohn, S.G., Sim, D.S., Park, M.A., Heo, M.S., Lee, T.H., Lee, J.D., Jun, H.K., Jang, K.L., 1998. Development of a polymerase chain reaction (PCR) procedure for the detection of baculovirus associated with white spot syndrome (WSBV) in Penaeid shrimp. J. Fish Dis. 21, 11–17. Kono, T., Savan, R., Sakai, M., Itami, T., 2004. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification. J. Virol. Methods 115, 59–65. Kumuda, C.D., White, C.W., 1998. Detection of thioredoxin in human serum and biological samples using a sensitive sandwich ELISA with digoxigenin-labeled antibody. J. Immunol. Methods 211, 9–20. Li, J.K., Huang, R.R., Xia, K., Liu, L., 2014. Double antibodies sandwich enzyme-linked immunosorbent assay for the detection of Alicyclobacillus acidoterrestris in apple juice concentrate. Food Control 40, 172–176. Lo, C.F., Ho, C.H., Peng, S.E., Chen, C.H., Hsu, H.C., Chiu, Y.L., Chang, C.F., Liu, K.F., Su, M.S., Wang, C.H., Kou, G.H., 1996a. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis. Aquat. Org. 27, 215–225. Lo, C.F., Leu, J.H., Ho, C.H., Chen, C.H., Peng, S.E., Chen, Y.T., Chou, C.M., Yeh, P.Y., Huang, C.J., Chou, H.Y., Wang, C.H., Kou, G.H., 1996b. Detection of baculovirus associated with white spot syndrome (WSBV) in Penaeid shrimps using polymerase chain reaction. Dis. Aquat. Org. 25, 133–141. Luo, Y.Z., Terkawi, M.A., Jia, H.L., Aboge, G.O., Goo, Y.K., Cao, S.N., Li, Y., Yu, L.Z., Ooka, H., Kamyingkird, K., Masatani, T., Zhang, S.F., Nishikawa, Y., Igarashi, I., Xuan, X.N., 2012. A double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model. Exp. Parasitol. 130, 178–182. Mayo, M.A., 2002. A summary of taxonomic changes recently approved by ICTV. Arch. Virol. 147, 1655. Mecham, J.O., Wilson, W.C., 2004. Antigen capture competitive enzyme-linked immunosorbent assay using baculovirus-expressed antigens for diagnosis of bluetongue virus and epizootic hemorrhagic disease virus. J. Clin. Microbiol. 42, 518–523. Mecham, J.O., 2006. Detection and titration of bluetongue virus in Culicoides insects cell culture by an antigen-capture enzyme-linked immunosorbent assay. J. Virol. methods 135, 269–271. Nadala Jr., E.C.B., Loh, P.C., 2000. Dot-blot nitrocellulose enzyme immunoassays for the detection of white-spot virus and yellow-head virus of Penaeid shrimp. J. Virol. Methods 84, 175–179. Niu, H.M., Huang, X.M., Han, K.K., Liu, Y.Z., Zhao, D.M., Zhang, J.F., Liu, F., Li, T.T., Zhou, X.B., Li, X.R., Li, Y., 2013. Development of double antibody sandwich ELISA for detection of duck or goose flavivirus. J. Integr. Agricul. 12, 1638–1643. Nunan, L.M., Lightner, D.V., 1997. Development of a non-radioactive gene probe by PCR
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An optimized double-antibody sandwich ELISA for quantitative detection of WSSV in artificially infected crayfish Xiaoqian Tanga,b, Qianrong Lianga, Lushan Liua, Xiuzhen Shenga, Jing Xinga,b, Wenbin Zhana,b,
⁎
a
Laboratory of Pathology and Immunology of Aquatic Animals, KLM, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, No.1 Wenhai Road, Aoshanwei Town, Jimo, Qingdao 266071, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: DAS-ELISA WSSV Quantitative detection Crayfish Viral propagation
Developing a rapid, accurate and quantitative method for detecting white spot syndrome virus (WSSV) is extremely urgent and critical for reducing the risk of white spot disease outbreaks. In the present work, an optimized double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for quantitative detection of WSSV. The method employed rabbit polyclonal antibodies against WSSV as the capture antibody and previously produced anti-WSSV monoclonal antibodies as the detector antibody. A standard curve of the log concentration of WSSV versus OD value was established, which was linear in the concentration range of 120–7680 ng/mL, and the linear regression equation was y = 0.166x − 0.151. Viral proteins in different tissues of crayfish (Procambarus clarkia) post artificial infection with WSSV were quantitatively measured using the DAS-ELISA. WSSV proliferated quickly within 60 h post infection and gradually slowed down afterwards. According to the linear regression relationship, the viral proteins in hemolymph, gut and gonad were firstly able to be quantified at 24 h post infection with the concentrations of 186, 158 and 128 ng/mL, respectively. These three tissues also contained higher viral proteins than the gill, heart, hepatopancreas and muscle during the entire infection period. The viral protein concentration in gut reached the highest level of 6220 ng/mL at 72 h post infection. Real time quantitative PCR was also used to detect the dynamic change of viral copies in crayfish hemolymph post WSSV infection, with similar results for both assays. The developed DAS-ELISA could detect WSSV propagation from initial to moribund stage in infected crayfish and demonstrated potential application for diagnosis of WSSV.
1. Introduction White spot syndrome, caused by the white spot syndrome virus (WSSV), is one of the most serious epizootic diseases in the shrimp farming industry throughout the world (Chou et al., 1995; Flegel, 1997). WSSV belongs to the genus Whispovirus in a new virus family Nimaviridae (Mayo, 2002), which is an enveloped virus with a doublestranded circular DNA genome of about 300 kb (van Hunlten et al., 2001; Yang et al., 2001) and contains approximately 181 putative open reading frames, encoding more than 39 structural proteins (SánchezPaz, 2010). The virus could infect shrimps and other freshwater and marine crustaceans (Lo et al., 1996a; Wang et al., 1998). Many methods have been developed for detecting WSSV, including PCR (Lo et al., 1996b; Kim et al., 1998), in situ PCR (Jian et al., 2005), in situ hybridization (Wongteerasupaya et al., 1996; Nunan and Lightner, 1997), immune methods (Huang et al., 1995; Nadala and Loh, 2000; Zhan et al., 1999; Wang and Zhan, 2006) and loop-mediated isothermal
⁎
amplification assay (Kono et al., 2004), etc. However, all of these assays are qualitative and cannot determine the exact amount of virus. Recent studies showed that fluorescent quantitative PCR could provide a sensitive method for quantifying the number of DNA templates (Durand and Lightner, 2002; Sritunyalucksana et al., 2006), which was widely proved to be rapid, accurate, and available to detect WSSV in laboratory facilities. The qPCR determines the viral load by detecting the copies of a specific gene segment, however, it does not necessarily reflects the amount of packaged mature viral particles that might better reflect the infecting potential and risk of outbreak. In contrast, the double antibody ELISA generally adopts a polyclonal antibody that could recognize multiple epitopes as the capturer and specific monoclonal antibodies as the detector to quantify the antigen at protein level (Mecham, 2006; Niu et al., 2013; Chang et al., 2016), which provided an accurate and sensitive method for detecting viral pathogens and could be further applied in WSSV detection for shrimps. So far there have been no effective measures to control WSSV, thus it is of great
Corresponding author at: Laboratory of Pathology and Immunology of Aquatic Animals, KLM, Ocean University of China,5 Yushan Road, Qingdao 266003, PR China. E-mail address: [email protected] (W. Zhan).
http://dx.doi.org/10.1016/j.jviromet.2017.10.020 Received 24 July 2017; Received in revised form 20 October 2017; Accepted 20 October 2017 0166-0934/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Tang, X., Journal of Virological Methods (2017), http://dx.doi.org/10.1016/j.jviromet.2017.10.020
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significance to monitor the infection status and evaluate infection severity of cultured shrimp for preventing the spread and outbreak of white syndrome disease. Therefore, in the present work, an optimized double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed and utilized to systematically investigate the temporal and spatial dynamic WSSV proliferation in the infected crayfish.
were harvested 7–10 days after inoculation, then the MAbs were purified from the mice ascites fluids by the caprylic acid/ammonium sulfate precipitation method (Sheng et al., 2012). The purity of the purified MAbs was checked by SDS-PAGE and the titer (2E6 and 2A3 mixed in a 1:1 ratio) was also determined by indirect ELISA as above.
2. Materials and methods
Microtiter plates were coated with 100 μL of optimal dilution of capture antibody (rabbit anti-WSSV polyclonal antibodies) in 0.01 M carbonate-bicarbonate coating buffer (pH 9.6) for 1 h 37 °C, and stored at 4 °C overnight, then the unbounded antibodies were washed off using 200 μL PBST for three times and 150 μL blocking buffer was added to all the wells for 1 h at 37 °C. After washing as above, wells were incubated with 100 μL serial 2-fold dilutions of purified WSSV (30,720 ng/ mL–7.5 ng/mL) in triplicate at 37 °C for 1 h and then washed as before. Then, the wells were incubated with 100 μL optimal dilution of detective antibodies (MAbs 2E6 and 2A3 mixed in a 1:1 ratio) at 37 °C for 1 h. After washing as above, the wells were then incubated with 100 μL AP-conjugated goat anti-mouse Ig diluted 1:3000 in PBS at 37 °C for 1 h. After washing, 100 μL of 0.1% (w/v) pNPP diluted in pNPP buffer was added to each well of the plate. The reaction was allowed to proceed for 30 min at 37 °C and stopped with 50 μL per well of 2 M NaOH. The plate was read at 405 nm in the microplate reader. PBS instead of the purified WSSV was set as negative control. To confirm the optimal dilution for capture and detective antibody, a checkerboard titration was previously performed. All the ELISA steps were performed as described above. The standard curve was obtained by plotting mean absorbance values against the logarithm of purified WSSV concentrations and fitted to a four-parameter logistic equation using Origin 8.0 (OriginLab, USA) software package.
2.4. Establishment of standard curve for the DAS-ELISA
2.1. WSSV virion purification Moribund shrimp (Fenneropenaeus chinensis) with symptomatic white spots were collected from one shrimp farm in Shandong Province, China. After confirmation of WSSV infection by nested PCR (Lo et al., 1996b), the gills of the shrimp were used for WSSV purification according to the previous method (Wang and Zhan, 2006). Briefly, the homogenate of WSSV-infected gills was centrifuged at 2000g for 10 min at 4 °C, and the supernatant fluid was added to a discontinuous sucrose gradient of 35%, 50% and 60% (w/v) and ultracentrifuged (Hitachi CP 100MX) at 80, 000 g for 2 h at 4 °C. The virus band was collected by puncturing the tube with a syringe needle and then mixed with 5 times TNE buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA-Na2, pH 7.4) and repelleted at 80, 000 g for 1.5 h at 4 °C. The resulting pellet was dissolved in 0.01 M phosphate buffered saline (0.01 M PBS, pH 7.4). The concentration of purified virus was determined by Bradford assay. To check for quality and quantity, virus samples were negatively stained and examined under a transmission electron microscope (JEOL JEM1010). 2.2. Preparation of anti-WSSV polyclonal antibody Anti-WSSV polyclonal antibodies were prepared according to the method described by Zhang et al. (2010) with modifications. Briefly, an aliquot equivalent to 400 μg of purified WSSV was mixed with the same volume of Freund’s complete adjuvant and injected subcutaneously in multiple spots on the back of a 2.5 kg female New Zealand rabbit. After 2 weeks, the first booster immunization was given to the rabbit with the same dosage of purified WSSV in Freund's incomplete adjuvant (Sigma, USA). Another two booster injections were given with 200 μg WSSV without adjuvant at one-week interval. Rabbit serum was collected 7 days after the final injection and followed with centrifugation, the serum supernatants were stored at −80 °C for later use. Indirect ELISA was conducted to confirm specificity of the polyclonal antibody and determine the binding titers. For titer detection, 100 μL of the purified WSSV solution containing 3 μg of purified WSSV was coated on each well of the microtiter plate at 4 °C overnight, then followed with three washes by PBST (PBS containing 0.05% Tween-20, Solarbio, USA), 150 μL blocking buffer (4% bovine serum albumin in PBS) was added. After blocking, 100 μL of serially diluted polyclonal antibodies (from 1:400 to 1:4 × 105) or PBS were incubated at 37 °C for 1 h. Then, the AP-conjugated goat anti-mouse Ig (Sigma, USA) diluted 1:3000 in PBS at 37 °C for 1 h. After washing, 100 μL of 0.1% (w/v) pNPP (Sigma, USA) diluted in pNPP buffer (1% diethanolamine, 0.5 mM MgCl2, pH 9.8) was added to each well of the plate. The reaction was allowed to proceed for 30 min at 37 °C and stopped with 50 μL per well of 2 M NaOH. The plate was read at 405 nm in a microplate reader (Molecular Devices).
Y = {(A − D)/[1 + (x/C)B} + D
(1)
where A is the Y value corresponding to the asymptote at the low values of the X-axis and D is the Y value corresponding to the asymptote at high values of the X-axis. The coefficient C is the X-value corresponding to the midpoint between A and D. The coefficient B describes how rapidly the curve makes its transition from the asymptote in the center of the curve. The linear fit was based on the equation Y = A + B*log(X) where X is the concentration of purified WSSV. The curve fitting is based on the Lavenberg-Marquardt method (Kumuda and White, 1998). Then the total WSSV concentrations of the samples could be determined according to the model. 2.5. Validation of WSSV DAS-ELISA The calibration model was evaluated using standard curves from three validation runs. The relative error (RE) and recovery were analyzed between the back-calculated concentration and nominal concentration of each WSSV dilution. RE = [(observed concentration/nominal concentration) − 1] × 100% (2) Recovery = (observed concentration/nominal concentration) × 100% (3) The putative validated range of the assay, between the upper limit of quantitation (ULOQ) and lower limit of quantitation (LLOQ), was determined using data from sextuplicate of WSSV serial dilutions from 30,720 to 7.5 ng/mL. The mean%RE for each dilution level was determined and the two-sided 90% confidence intervals (CI%RE) for the mean were constructed using Microsoft Excel software:
2.3. Preparation of anti-WSSV monoclonal antibodies (MAbs) Two previously produced hybridoma cell lines, 2E6 and 2A3, which produce monoclonal antibodies reactive towards viral envelope and capsid of WSSV respectively (Wang and Zhan, 2006), were employed for ascites fluid production. Pristane-primed BALB/c mice were injected intraperitoneally with the activated hybridomas, and the ascites fluids
90% CI%RE = ū ± 1.645 (δ/n1/2)
(4)
where ū = mean%RE, δ = standard deviation of ū, and n = number of 2
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data points for ū. The ULOQ and LLOQ were defined as the two-sided 90% CI%RE for which mean%RE was −25% and +25%, respectively (Findlay et al., 2000). The repeatability (intra-assay reproducibility) and intermediate precision (inter- assay reproducibility) of the assay were assessed using serially diluted purified WSSV samples, with the concentrations of stock WSSV solution determined to be 30,720 ng/mL by Bradford assay. The stock solution was serially diluted in PBS to achieve the following concentrations: 7680, 3840, 1920, 960, 480, 240 and 120 ng/mL and assayed in sextuplicate during four separate runs of the WSSV sandwich ELISA. The precision (%CV) was calculated for each sample for each run and among all runs.
Table 1 Checkerboard titration of the capture and detector antibodies. Dilution of detector antibody (1:X)
50 100 200 400 800 1600 3200 6400 Negative control
2.6. Quantification of WSSV protein concentration in crayfish during infection using the DAS-ELISA
Dilution of capture antibody (1:X)
200
400
800
1600
3200
6400
12800
25600
0.494 0.475 0.469 0.387 0.372 0.347 0.327 0.301 0.086
0.488 0.471 0.470 0.382 0.367 0.339 0.316 0.295 0.093
0.489 0.464 0.445 0.384 0.356 0.318 0.299 0.299 0.096
0.478 0.466 0.457 0.373 0.349 0.313 0.292 0.280 0.093
0.496 0.454 0.449 0.362 0.345 0.303 0.287 0.268 0.090
0.470 0.447 0.442 0.351 0.334 0.296 0.279 0.267 0.090
0.438 0.424 0.416 0.320 0.306 0.286 0.268 0.251 0.093
0.406 0.393 0.385 0.312 0.295 0.267 0.250 0.240 0.106
The underlined value means the OD405 value obtained under the optimal dilutions of capture antibody and detective antibody.
The experimental crayfish (Procambarus Clarkia) with an average weight of 15 g were purchased from a local aquatic product market in Qingdao, Shandong Province, China, which were confirmed WSSV-free by nested PCR assay (Lo et al., 1996b). The crayfish were maintained at 25 °C in fresh water with continuous aeration for one week, then 200 crayfishes were randomly selected and averagely divided into two groups. Each crayfish from one group was intramuscularly injected with 50 μL WSSV inoculum containing 10−1 ng WSSV, the crayfish injected with equal volume of PBS was performed as control. At 0, 24, 36, 48, 60, 72, 84 and 96 h post infection (h p.i.), eight crayfish were randomly selected both from the WSSV-infected group and control group, and the hemolymph, gill, hepatopancreas, heart, gonad, gut and muscle were sampled and mixed with two volumes of homogenization buffer (PBS containing 0.5 M EDTA), then homogenized at 4 °C with 18 strokes of a Dounce homogenizer. In order to inhibit the endogenous alkaline phosphatase activity (Zhan et al., 2004), the homogenates were incubated at 37 °C for 1 h, and then the WSSV concentrations were detected using the DAS-ELISA as described above. Meanwhile, proportions of hemolymph were also sampled at different time points post WSSV infection and used for detection of viral copies by real time qPCR later.
monoclonal antibodies were determined to be 1:4 × 105 and 1:6400, respectively (Tables A. 1 and A. 2 in Supplementary material). Then several dilutions of anti-WSSV MAbs and rabbit anti-WSSV antibodies were used in a checkerboard titration to determine the working dilution for each reagent. The antibodies display similarly strong absorbance values up to a dilution of 1/6400 for mouse and 1/200 for rabbit (Table 1). A dilution of 1/3200 was selected for the rabbit antisera thereby ensuring a slight excess of capture antibody on the plates, and a dilution of 1/100 anti-WSSV MAbs was selected to maximize the detection of captured virions, while OD405 value of 0.454 obtained using the optimized dilutions of two antibodies was underlined in Table 1. 3.2. The standard curve After the purified WSSV suspensions with serial dilution from 7.5 to 30,720 ng/mL was assayed via the optimized DAS-ELISA, a sigmoidal standard curve was generated, which was fitted well (r = 0.99) to a four-parameter logistic equation (Fig. 1A). The four-parameter logistic equation is expressed as: Y = {(A1 − A2)/[1 + (x/x0)p]} + A2, in which A1 = 0.1442 ± 0.0065, A2 = 0.5530 ± 0.0196, x0 = 2.9652 ± 0.0589, p = 5.5973 ± 0.4941. The minimum detection limit was 120 ng/mL with a detection range of 120–30,720 ng/mL. The mean OD405 values were at least two times greater than the negative control. Based on the sigmoidal standard curve, a linear fit equation was obtained with a concentration range of 120–7680 ng/mL with R2 = 0.992. The linear equation was expressed as: Y = 0.166X − 0.151 (Fig. 1B). The recovery of the optimized DAS-ELISA was between 0.84–1.10, and the relative error (RE) was between −15.17 to 10.42 (Table 2). Intra-assay reproducibility was determined with coefficient of variation (CV) to be in a range of 0.66–7.11% (Table 3), and the inter-assay reproducibility was in a range of 2.11–7.32% (Table 4).
2.7. Quantification of WSSV copies in crayfish hemolymph by real time qPCR In order to confirm the reliability of DAS-ELISA, the dynamic change of WSSV copies in the hemolymph sampled at different time points post WSSV infection was detected as previously described (Yuan et al., 2007). Briefly, series dilutions of WSSV recombinant plasmid (copy number known) were prepared as standards for quantification. Then total genomic DNA in sampled hemolymph was extracted using DNA extraction kit (Takara, Japan) following the manufacturer’s instruction. An equal quantity of DNA (50 ng) of each sampling time was added into the SYBR Green Premix with the specific primers (F:5′-AAACCTCCGCATTCCTGTGA-3′, R:5′-TCCGCATCTTCTTCCTTCAT-3′, Tm: 57 °C) for amplification. Quantitation of the amount of WSSV copies in hemolymph was accomplished using the standard curve.
3.3. Dynamic changes of viral protein load in WSSV-infected crayfish The optimized DAS-ELISA was employed to investigate the dynamic changes of WSSV concentrations in different tissues of crayfishes after WSSV infection. According to values calculated from the linear fit equation, the earliest time to detect WSSV-positive signal was at 24 h in hemolymph, gut and gonad with the concentrations of 186, 158 and 128 ng/mL, respectively. At 36 h post infection, the WSSV-positive signals could be detected in all the 7 sampled tissues, among them, the viral concentrations in gut and gonad were significantly higher than the other tissues, which mounted up to 2166 and 1012 ng/mL, respectively. During the whole infection process, the viral proliferation in gut, hemolymph and gonad were obviously higher than the other tissues, which reached their peak levels with 6220, 3715 and 2681 ng/mL, respectively. Since 60 h post infection, the viral concentrations in
3. Results 3.1. Optimization of capture and detector antibodies determined by checkerboard titration method Abundant intact WSSV virions with a high purity were observed by transmission electron microscope (Fig. A. 1 in Supplementary material), which was used as the standard sample. The purified anti-WSSV MAbs (2E6 and 2A3) was analyzed by SDS-PAGE, which showed two clear bands with the expected molecular weight masses of heavy (about 55 kDa) and light (about 25 kDa) chains (Fig. A. 2 in Supplementary material). By indirect ELISA, the binding titers of polyclonal and 3
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Fig. 1. The standard curves for WSSV DAS-ELISA.
Table 2 Recovery and relative error (RE) of WSSV DAS-ELISA. Nominal concentration (ng/ mL)
Observed concentration (ng/mL)
Recovery
RE
7680.00 3840.00 1920.00 960.00 480.00 240.00 120.00
8226.93 3956.57 1907.36 1060.00 407.17 214.59 117.48
1.07 1.03 0.99 1.10 0.85 0.89 0.98
7.12 3.04 −0.66 10.42 −15.17 −10.59 −2.10
Nominal concentration: The WSSV concentration determined by the Bradford assay; Observed concentration: The WSSV concentration calculated by the DAS-ELISA.
Table 3 Intra-assay reproducibility of DAS-ELISA. Concentration of WSSV (ng/mL)
Plate A (%CV)
Plate B (%CV)
Plate C (%CV)
Plate D (%CV)
7680 3840 1920 960 480 240 120 Negative control
4.17 0.66 2.22 5.28 6.74 6.85 5.78 1.13
3.72 4.21 2.35 4.17 1.57 3.29 2.55 1.73
5.69 6.01 4.12 2.24 5.08 3.93 4.56 2.59
6.22 7.11 6.46 4.56 6.05 2.34 3.23 2.38
Fig. 2. The variation of WSSV concentrations in different tissues of crayfishes during WSSV infection.
different tissues maintained relatively stable, and then all of the infected crayfishes eventually died off at 96 h post infection (Fig. 2). 3.4. Dynamic change of WSSV DNA copies in WSSV-infected crayfish hemolymph The number of WSSV copies in hemolymph samples was calculated according to the standard curve generated from samples of purified cloned-WSSV plasmid tested by qPCR. The results showed that WSSV copies in the hemolymph could be detected as early as 6 h p.i. with 101 per ng DNA, and it maintained at a low level till 18 hp.i., but significantly increased since 24 hp.i. (p < 0.05), during period of 24–48 hp.i., the copy number significantly increased from about 103 to 105, and then the viral load increased slightly after 48 hp.i. (Fig. 3).
Each dilution sample was tested in sextuplicate in each plate.
Table 4 Inter-assay reproducibility of DAS-ELISA. Concentration of WSSV (ng/mL)
7680 3840 1920 960 480 240 120 Negative control
OD405 value of Plate A–D Mean ± SD
Inter-assay reproducibility (%CV)
0.486 0.455 0.427 0.343 0.289 0.245 0.204 0.095
6.58 5.27 7.32 5.93 4.17 4.96 4.48 2.11
± ± ± ± ± ± ± ±
0.032 0.024 0.030 0.020 0.012 0.012 0.009 0.002
4. Discussion Double antibody sandwich ELISA has higher sensitivity and specificity than indirect ELISA (Hutchings and Ferris, 2006; Luo et al., 2012; Li et al., 2014), and can accurately quantify antigens with easy experimental operation (Fæste and Plassen, 2008; Tang et al., 2010; Haaf et al., 2017). In the present work, an optimized DAS-ELISA technique was established for WSSV detection at protein level, which used the rabbit antisera against WSSV and mixed MAbs against WSSV as the 4
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to inhibit this non-specific reactions during practical tests, endogenous alkaline phosphatases were inactivated with 0.5 M EDTA according to our previous procedure (Zhan et al., 2004). Freshwater crayfish is sensitive to the WSSV and has become an excellent animal model to study this virus (Huang et al., 2001). In order to confirm that the developed DAS-ELISA could efficiently detect WSSV virions in the viral infected shrimp, here Procambarus Clarkia was used as an artificially infected model. Our results showed that WSSV proliferated quickly before within 36 hp.i., then obviously slowed down from 48 to 96 h p.i. and the virus protein load remained relatively stable afterwards. In addition, during the whole infection process, the positive signal of WSSV was earliest detected at 24 h in hemolymph, gonad and gut, and the highest WSSV loads were also found in these tissues, which suggested that hemolymph, gonad and gut are the most susceptible to WSSV. Similar proliferation processes were also found in the WSSV-infected Litopenaeus vannamei and Fenneropenaeus chinensis by competitive PCR and real time qPCR (Tang and Lightner, 2000; Sun et al., 2013; Zhong et al., 2013). In this work, the hemolymph was chosen for the WSSV copy detection by qPCR to confirm the reliability of DAS-ELISA, and the qPCR results agreed well with the DAS-ELISA results from 24 hp.i. to 96 hp.i. Compared with DAS-ELISA, qPCR could detect the viral copies within 18 hp.i., suggesting that there was no or very few packaged mature virions in the hemolymph during this period. These results suggested that no matter which detecting target molecule, DNA or protein, similar results could be obtained, which also indicated that the DAS-ELISA developed in this study could be applied for the WSSV detection. In conclusion, compared with real time qPCR, the DAS-ELISA had a higher detection limit may not allow for the very early detection in infected animals, but could determine the amount of packaged mature virions and might better reflect the risk of outbreak. Also, the WSSVpositive signal was able to be detected from the initial to moribund stage in viral infected crayfish by the DAS-ELISA, which has a potential value in evaluating the viral infection state and prevention of WSSV outbreak.
Fig. 3. Dynamic state of WSSV copies in infected crayfish hemolymph.
capture and detection antibodies, respectively. The MAbs used were previously proved to be specific to WSSV, which guaranteed the specificity of developed DAS-ELISA. In this method, a sigmoidal and a linear standard curves were both generated with the valid detection scopes of 7.5–30,720 ng/mL and 120–7680 ng/mL, respectively. The reproducibility of this method was also proved to be well by examining the CV values. All these results suggested that the DAS-ELISA developed has a good sensitivity, specificity and producibility, which could be employed for qualifying the WSSV in a convenient and simple way. The characters of antibodies used in the DAS-ELISA would directly influence the specificity and sensitivity of the detection. Previous studies have shown that the antibodies produced using the elaborately purified virions as immunogen could increase DAS-ELISA sensitivity by 100-fold in detecting the bluetongue virus (Pál et al., 2005; Chand et al., 2009; Mecham, 2006). Therefore, purified natural antigen is of extremely great importance in the production of antibodies for immunological assay, which would directly influence the sensitivity, specificity and stability of the final assay (Pál et al., 2005; Chand et al., 2009). In this study, quite abundant intact and high-purity WSSV virions were prepared for the production of rabbit antisera as capture antibodies. Similarly, many sandwich ELISA assays developed so far often adopted the polyclonal antibodies as the capturer (Mecham, 2006; Niu et al., 2013; Chang et al., 2016). It was believed that using the polyclonal antibodies instead of monoclonal antibody as the capture antibodies could efficiently increase the sensitivity of the sandwich ELISA by nearly 10-fold (Mecham and Wilson, 2004; Mecham, 2006), which was probably that the polyclonal antibodies could recognize more epitopes of antigen than the MAbs. As for the detection antibodies, the mixed MAbs against the WSSV were adopted (recognize WSSV virions both on the envelope and the capsid), which not only guaranteed the specificity, but also increased the sensitivity of the detection assay. Finally, a sigmoidal and a linear standard curves were generated with the minimum detection limits of 7.5 and 120 ng/mL, respectively. Although, the minimum detection limit of linear standard curve was a little higher, which is more convenient and simpler for calculating, as well as could satisfy common measurement. Moreover, by comparing the results of DAS-ELISA checkboard titration with indirect ELISA, the OD values of DAS-ELISA were higher than the indirect ELISA when the mixed MAbs were used at the same dilution, which suggested that the optimized DAS-ELISA developed in the present work had higher sensitivity than the indirect ELISA. In addition, our previous studies had showed that endogenous alkaline phosphatases commonly exist in shrimp tissues (Zhan et al., 2003; Zhan et al., 2004), which would usually lead to the false-positive results when using the shrimp tissue samples as the antigens without any pretreatment. Thus, in order
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