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Development and application of antibody microarray for lymphocystis disease virus detection in fish
Journal of Virological Methods 189 (2013) 243–249
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Development and application of antibody microarray for lymphocystis disease virus detection in fish Xiuzhen Sheng, Xiaoli Xu, Wenbin Zhan ∗ Laboratory of Pathology and Immunology of Aquatic Animals, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China
a b s t r a c t Article history: Received 19 June 2012 Received in revised form 1 February 2013 Accepted 27 February 2013 Available online 7 March 2013 Keywords: Lymphocystis disease virus (LCDV) Antibody microarray Agarose gel Virus detection
Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease affecting marine and freshwater fish worldwide. Here an antibody microarray was developed and employed to detect LCDV in fish. Rabbit anti-LCDV serum was arrayed on agarose gel-modified slides as capture antibody, and Cy3conjugated anti-LCDV monoclonal antibody (MAbs) was added as detection antibody. The signals were imaged with a laser chip scanner and analyzed by corresponding software. To improve the sensitivity, different substrate binders (poly-l-lysine, MPTS, aldehyde, APES and agarose gel modified slides, and commercially available amino-modified slides), markers (fluorescein isothiocyanate, Cy3, horseradish peroxidase, biotin or colloidal gold) conjugated to anti-LCDV Mabs, and storage time of the antibody were assessed. The results showed that the antibody microarrays based on agarose gel-modified slides gave a lower detection limit of 0.55 g/ml of LCDV when Cy3 and HRP conjugated anti-LCDV MAbs were used as detection antibody; and the lowest detectable LCDV protein concentration was 0.0686 g/ml when streptavidin–biotin conjugated to anti-LCDV MAbs served as detection antibody. The developed antibody microarray proved to have a high specificity for LCDV detection and a shelf-life of more than 8 months at −20 ◦ C. Furthermore, the LCDV detection results of the microarray in fish gills or fins (n = 50) presented a concordance rate of 100% with enzyme-linked immunosorbent assay (ELISA) and 98% with immunofluorescence assay technique (IFAT). These results revealed that the developed antibody microarray could serve as an effective tool for diagnostic and epidemiological studies of LCDV in fish. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease (LCD) affecting approximately 140 species of marine and freshwater fish worldwide (Lang, 1994; Plumb, 1993). Fish infected with LCDV exhibit characteristic external symptoms of wart-like nodules on the body skin, fins, mouth and gills, causing a decrease in commercial value (Tidona and Darai, 1997). In addition, diseased fish are more susceptible to secondary infection by other microorganisms, resulting in high mortalities and great economic losses for aquaculture worldwide (Chinchar, 2002; Iwamoto et al., 2002). Since there are currently no available treatments for LCD, rapid and accurate detection of LCDV is crucial in prevention and control of lymphocystis disease. Diagnosis of LCD is generally based on typical skin lesion observation. At present, the techniques used frequently for LCDV detection include polymerase chain reaction (PCR), real-time PCR, virus isolation and neutralization using cell lines, and several immunological techniques based on anti-LCDV antibody
∗ Corresponding author. Tel.: +86 532 82032284; fax: +86 532 82032284. E-mail address: [email protected] (W. Zhan). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.02.015
(Garcíarosado et al., 2002; Kitamura et al., 2006; Cano et al., 2006, 2007). PCR and real-time PCR are of specific, sensitive, rapid and cost-effective; however, their performances require highly trained personnel and specialized equipments. The isolation of viral pathogens in cell cultures has been regarded as the “gold standard” for their convenience and accuracy, but this approach is often slow and requires considerable technical expertise. Immunological techniques (ELISA, immunofluorescence assay technique, Western-blot) based on anti-LCDV antibody are sensitive and specific, but time-consuming with complicated operation. Antibody microarray-based analysis is a cost-effective approach that yields reproducible results and can allow replicate analyses in a single assay run. As a next-generation tool, antibody microarrays are increasing in popularity, for they offer unparalleled throughput, minimal reagent consumption and sensitive detection of multiple targets simultaneously (Angenendt, 2005); and test results could be read by naked eyes when combined with immunoenzyme techniques or immunogold-sliver staining. However the accuracy conferred by this method is linked with the quality of antibodies employed closely, and there are issues relating to cross-reactivity and antibody availability, both of which are crucial for sensitive and specific strain typing particularly (Mahesh et al., 2008). Given the listed benefits, antibody microarrays have been and are being
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developed that have growing potential for clinical, biothreat, and point-of-care applications (Huelseweh et al., 2006; Lian et al., 2010). For disease diagnosis in aquaculture, the antibody microarray for white spot syndrome virus (WSSV) detection in shrimp have been developed (Xu et al., 2011), however for fish viral detection there are, to our knowledge, no reports of antibody microarray diagnostics so far. This paper described the development and application of a low-density antibody microarray for LCDV detection in fish tissue extracts, as well as improvement of antibody microarray sensitivity. With the prepared antibody-microarray-integrated system, the entire testing procedures could be completed in less than 2 h, and the multiple samples from diverse sources could be tested in a single assay without the need for labeling. As a supplementary technique for detection of LCDV in fish, the developed antibody microarray is convenient, accurate, sensitive and time-saving, and suitable for diagnostic and epidemiological detection to LCDV. 2. Materials and methods 2.1. Preparation of samples The LCDV-free flounders (Paralichthys olivaceus, 15 ± 1 cm in length) tested by PCR (Zhan et al., 2010) were obtained from a fish farm located in Qingdao of Shandong province, China; LCDVinfected flounders (20 ± 2 cm in length) with various-sized nodules on body surface were from a fish farm in Hebei province. Common sea bass (Lateolabrax japonicus, 23 ± 2 cm) and sting fish (Sebastes schlegeli, 20 ± 2 cm) were from a fish farm in Shandong province. All these fish were shipped in chill containers, and then anaesthetized with tricaine (0.065 mg/ml) (MS-222, Sigma–Aldrich, St. Louis, US). The tissues (gill, fins, skin, lymphocystis nodules and internal organs) were taken and frozen at −80 ◦ C. Sampled fish fin or gill (target organs of LCDV) homogenates were used for LCDV detection. The fish gills or fins (∼1 g) were washed with TNE buffer (50 mmol/l Tris, 100 mmol/l NaCl, 1 mmol/l EDTA, pH 7.4) for 5 min at a ratio of 10% (w/v), homogenized and suspended in TNE buffer at 4 ◦ C. The suspension was subjected to rapid freeze–thaw (3×), ultrasonication, and centrifuged at 500 × g and then 1800 × g for 20 min at 4 ◦ C, respectively. The homogenates of gills and fins from the same fish were pooled and stored at −80 ◦ C until use. Purified LCDV solution was prepared from lymphocystis nodule homogenate using the method developed previously (Cheng et al., 2006) and resuspended with 0.01 mol/l PBS (pH 7.4), and the viral protein concentration was measured 0.878 mg/ml. Graded reagents and MilliQ grade water were used throughout the experiment. All studies were conducted in accordance with institutional, national and international standards on animal welfare. 2.2. Antibody production and purification Rabbit anti-LCDV antibody (as capture antibody of the microarray) and rabbit anti-mouse Ig antibody (as positive control) were prepared by immunizing adult New Zealand rabbits with purified LCDV and mouse Ig in conventional methods respectively (Huang et al., 2005). One week after the last injection, blood was taken from the rabbit and the rabbit antiserum was purified by the Ampure PA kit (GE Healthcare, Fairfield, US) following the manufacturer’s protocol. The reactivity of LCDV with rabbit antiserum was determined by immunofluorescence assay technique (IFAT) using pre-immune serum as control (Cheng et al., 2006). Four anti-LCDV MAbs, 3G3, 2B6, 1D7 and 2D11 developed previously (Cheng et al., 2006), were produced in ascites by injecting the hybridoma clone into the peritoneal cavity of Balb/c mice, and purified in the same way above-mentioned. These MAbs were then
labeled with Cy3 (GE Healthcare, Fairfield, US) according to the manufacturers’ instructions and used as detection antibody after mixed in equal proportion. 2.3. Antibody microarray fabrication for LCDV detection 2.3.1. Optimal concentration of capture antibody Agarose gel modified slides were prepared following the methods of Xu et al. (2011). Rabbit anti-LCDV antibody was diluted at the concentration range of 0.5 g/ml to 1.5 mg/ml, and spotted on agarose gel-modified slides as capture antibody in 3 × 3 matrix respectively. The slides were put in humid chambers at 37 ◦ C for 2 h to complete the immobilization of antibodies and then washed 3 times with PBST (PBS containing 0.5% Tween-20) for 5 min each. Slide surface was blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at 37 ◦ C. After 3 washes, LCDV diluent at 0.1 mg/ml was incubated with the capture antibody, and the antigen–antibody complex formed was identified by Cy3-conjugated anti-LCDV antibody. Detection results were measured by laser chipscanner at 532 nm and quantified with software EcoscanCHS. The optimal concentration of capture antibody was confirmed when the relative signal value of the microarray tended to be stable. 2.3.2. Integration of antibody microarray The capture antibody, 0.5 mg/ml rabbit anti-LCDV antibody in printing buffer (PBS containing 50% glycerol) was spotted onto the agarose gel-modified slide with a spot volume of 20–50 nl. The microarray contained eight groups deposited in 4 × 4 matrixes and 2 × 4 arrangement on one slide. In each matrix, the printing buffer as negative control and the anti-mouse Ig antibody as positive control were spotted to form four replicates, respectively, and the capture antibody was spotted in eight replicates. To perform multiplexed assays on the same slide, the microarrays were compartmentalized into eight subarrays by Super PAP Pen or silicone gasket. After arraying, the slides were incubated in humid chambers at 37 ◦ C for immobilization of antibodies and then blocked by 3% BSA. The antibody microarrays were washed and allowed to dry, and stored at 4 ◦ C or −20 ◦ C until use. 2.4. LCDV detection Detection samples, 40 l per matrix, were added on the antibody microarray. The slides were put in a humid chamber at 37 ◦ C for 15–30 min and then washed 3 times, followed by incubation with Cy3-conjugated anti-LCDV antibody at 37 ◦ C for 30 min. The microarrays were washed again and imaged by laser chip scanner, and fluorescence signals were quantified with software EcoscanCHS. The fluorescence intensity of negative control was used as the background value, and the mean fluorescence intensity value of the eight replicates of capture antibody in one matrix was determined as the relative signal value after subtracting the corresponding background value. 2.5. Detection limit of antibody microarray Purified LCDV (0.878 mg/ml) was diluted 1:50–6400 with PBS, and added to the integrated antibody microarrays in 40 l per matrix. The detection limit of the antibody microarray was defined as the lowest LCDV concentration that could be detected reliably and positively and also a measure of sensitivity. For improvement of detection limit, the supports of microarray, markers labeled to anti-LCDV MAbs, and shelf life of antibody microarray, were assessed. In addition, the detection limit of the antibody microarray was compared with that of indirect sandwich enzyme-linked immunosorbent assay (ELISA) (Luo et al., 2009). The 96-well EIA plates (Corning, New York, USA) were coated with 50 l rabbit
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anti-LCDV antibody (0.1 mg/ml), and anti-LCDV MAbs served as the primary antibody and goat anti-mouse Ig serum conjugated with AP (Sigma–Aldrich, St. Louis, US) served as the second antibody. The dilution buffer was used as negative control. The experiment was repeated three times. The antibody microarray sensitivity was also determined by analyzing serial dilutions of LCDV suspension (homogenate of lymphocystis nodules) corresponding to median tissue culture infectious dose (TCID50). The flounder gill (FG) cell line was incubated in MEM supplemented with 10% FBS at 20 ◦ C (Tong et al., 1997). The TCID50 of LCDV suspensions was tested by FG cells (Xing et al., 2006), and the antibody microarray sensitivity expressed as TCID50 was then obtained by conversion. 2.5.1. Detection limit based on different supports Commercially available amino-modified slides were purchased from CEL CO (USA). Poly-l-lysine, MPTS, aldehyde, APES and agarose gel modified slides for microarrays were prepared according to the references (Masanori, 1991; Kusnezow et al., 2003; Rubina et al., 2003; Panagiota et al., 2007; Xu et al., 2011). The capture antibody was spotted on the six types of slides in an array format and then detection limit was tested using Cy3-conjugated anti-LCDV antibody as detection antibody, respectively. 2.5.2. Detection limit based on different markers Purified anti-LCDV MAbs, 3G3, 2B6, 1D7 and 2D11 were mixed in a volume ratio 1:1:1:1 and labeled with horseradish peroxidase (HRP) (Galaxybio, Beijing, China), fluorescein isothiocyanate (FITC) and biotin (Sigma–Aldrich, St. Louis, US) according to the manufacturers’ instructions, respectively. Conjugation of anti-LCDV MAbs to colloidal gold was prepared following the method of Wang and Zhan (2006). The labeled anti-LCDV MAbs mixture was used as detection antibody. Integrated antibody microarray based on agarose gel-modified slides was incubated with different LCDV diluents. After three washes the slides were exposed to the detection antibody labeled with different markers. The biotin labeled anti-LCDV MAbs were coincubated with Cy3 or HRP conjugated streptavidin simultaneously on the antibody microarray. Following three washes, the signals were visualized with the following methods. The fluorescence intensity of FITC and Cy3 were imaged with professional laser scanner at 494 nm and 532 nm, respectively, and analyzed by corresponding software. The signals of HRP were visualized with 3,3 ,5,5 -tetramethylbenzidine (TMB) (Galaxybio, Beijing, China) and the reaction was stopped by 2 M sulfuric acid. The signals of colloidal gold were visualized by soaking the slides in silver enhancement reagent (Sigma–Aldrich, St. Louis, US) for 10 min in the dark and fixed with sodium thiosulfate solution for 3 min at room temperature.
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The antibody microarrays prepared as mentioned previously, were stored at 4 ◦ C and −20 ◦ C. Slides and antibody microarray were tested every 15 days with different LCDV diluents, using HRP and Cy3 conjugated anti-LCDV MAbs as detection antibody. Agarose gel-modified slide and antibody microarray prepared freshly were used as controls. The shelf life of antibody microarrays was determined according to the relative signal value tested over a 1-year period. 2.8. Validation of antibody microarray for LCDV detection The accuracy of integrated microarray was compared to that of the indirect ELISA and IFAT by detecting LCDV in 50 samples of asymptomatic cultured flounder (P. olivaceus), common sea bass (L. japonicus) and sting fish (S. schlegeli) from different farms of Shandong province, China. The purified LCDV solution was used as positive control and the samples from healthy individuals served as negative control. LCDV detection by indirect ELISA was performed as described by Zhan et al. (2004). For ELISA, the purified LCDV solution was used as positive control and the supernatants of fish gills or fins were all pretreated with 0.5 M EDTA for 1 h. The dilution buffer and the samples from healthy individuals served as background and negative control, respectively, and mixture of four anti-LCDV MAbs served as primary antibody. OD values over three times higher than the negative control were evaluated positive. LCDV detection with IFAT was carried out following the method of Cheng et al. (2006). Cryosections (about 7 m thick) were prepared from detected fish fins or gills. Mixture of four anti-LCDV MAbs served as primary antibody and FITC-conjugated goat antimouse Ig serum (Sigma–Aldrich, St. Louis, US) diluted at 1:256 was used as the second antibody. Results were imaged by fluorescence microscope (Olympus, Tokyo, Japan). 3. Results 3.1. Optimum concentration of capture antibody Reactivity of the rabbit anti-LCDV antibody was tested by IFAT and a strong positive signal was found, but no positive signal was detected with pre-immune serum. Rabbit anti-LCDV antibody in various concentrations was spotted onto agarose gel-modified slides, and results indicated that the relative signal value of the microarrays continued to go up as the concentration of capture antibody increased (Fig. 1a and b), and reached saturation at 0.5 mg/ml. Thus 0.5 mg/ml of rabbit anti-LCDV antibody was confirmed as the appropriate concentration of capture antibody for the microarray. 3.2. Detection limit of antibody microarray based on different supports and markers
2.6. Specificity of antibody microarray To determine specificity of antibody microarray for LCDV detection, gill homogenates of LCDV-infected P. olivaceus, infectious hematopoietic necrosis virus (IHNV)-infected P. olivaceus, turbot reddish body iridovirus (TRBIV)-infected Scophthalmus maximus (Qingdao, China) and healthy P. olivaceus were incubated with the antibody microarray in different matrixes, respectively. Purified LCDV was used as positive control. 2.7. Stability of agarose gel-modified slides and antibody microarray Measuring their efficacy in immobilizing antibody tested the shelf life of agarose gel-modified slides. Agarose gel-modified slides were stored at 4 ◦ C, −20 ◦ C and room temperature (RT) before use.
Different supports were used to prepare the antibody microarrays and Cy3-conjugated anti-LCDV antibody was used as detection antibody to test detection limits. The results revealed that the relative signal value increased with increasing LCDV concentration, and the lowest detectable LCDV protein concentration of the antibody microarrays was 0.55 g/ml, which was performed by the microarray based on agarose gel-modified slides and aldehyde slides. The antibody microarray based on APES-modified slides, amino-modified slides and mercapto slides could allow detection of LCDV at concentration of 1.1 g/ml, while the microarray based on poly-l-lysine modified slide gave a detection limit of 1.46 g/ml (Fig. 2). In order to enhance detection sensitivity, anti-LCDV MAbs conjugated to different markers were used as detection antibodies. As shown in Table 1, Cy3 and HRP conjugated anti-LCDV MAbs gave
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Fig. 1. Relationship between capture antibody concentration and relative signal value. Rabbit anti-LCDV serum as capture antibody was immobilized on agarose gel-modified at a concentration of 1–1500 g/ml with a spot volume of 20–50 nl. The microarray was covered by LCDV diluent and then incubated with Cy3-conjugated anti-LCDV MAbs. The results were imaged by a laser scanner and analyzed by Ecoscan CHS software. (a) The relative signal value enhanced with the increasing concentration of capture antibody and reached saturation at 0.5 mg/ml. (b) The images of capture antibody at various protein concentrations used for the immobilization.
(IGSS) and Cy3-conjugated streptavidin-biotin detection system was 0.88 g/ml, 1.1 g/ml and 0.14 g/ml, respectively (Fig. 3). TCID50 of LCDV suspensions was 24.10 /40 l tested by FG cells which means 40 l LCDV suspensions of 17 times dilution is 1 TCID50 . When LCDV suspension was up to 29 -fold dilution, it was detected positive by antibody microarray with HRP-conjugated streptavidin–biotin detection system. Therefore the sensitivity of antibody microarray was 2−4.9 TCID50 /40 l for LCDV by conversion.
3.3. Specificity of antibody microarray Fig. 2. Detection limits based on different supports. Antibody microarrays based on six types of supports were incubated with LCDV diluents and the detection limits were tested. As low as 0.55 g/ml of LCDV could be detected by the antibody microarray based on agarose gel-modified slides (I) and aldehyde slides (V), whereas 1.1 g/ml of LCDV were detected by the antibody microarray based on amine slide (II), APES modified slide (III) and mercapto slide (IV), and 1.46 g/ml of LCDV by the antibody microarray based on poly-l-lysine modified slide (XI).
Cross-reactivity studies using gill homogenates of LCDVinfected P. olivaceus, IHNV-infected P. olivaceus, TRBIV-infected S. maximus and healthy P. olivaceus showed that positive signals were observed in the positive control and LCDV-infected P. olivaceus, but not in IHNV, TRBIV and healthy samples.
Table 1 Detection limit with different markers conjugated to anti-LCDV MAbs.
3.4. Shelf-life stability of agarose gel-modified slides
Markers
Detection limit of LCDV (g/ml)
FITC HRP IGSS Cy3 HRP–avidin–biotin Cy3–avidin–biotin
0.88 0.55 1.10 0.55 0.0686 0.14
a detection limit of 0.55 g/ml; while indirect sandwich ELISA was 0.28 g/ml (data not shown). Streptavidin–biotin detection system was applied to amplify the detection signal and HRP-conjugated streptavidin gave the lowest detection limit of 0.0686 g/ml. The detection limit produced by FITC, immunogold-sliver staining
We tested the stability of the agarose gel-modified slides stored under dry condition over 1 year at different temperatures (Fig. 4). The results illustrated that agarose gel-modified slides have a good capability of immobilizing protein after 1-year storage compared to the slides modified freshly, and the detection limit was still 0.55 g/ml of LCDV by using HRP-conjugated anti-LCDV MAbs as detection antibody. However, when Cy3-conjugated anti-LCDV MAbs were used as detection antibody, the background value increased with prolonged storage resulting in the decline of relative signal value, which was more obvious for the supports stored at −20 ◦ C, and as a result the microarray based on the agarose gelmodified slides exhibited a higher detection limit of 1.46 g/ml after 4-month storage at −20 ◦ C.
Fig. 3. Detection limit of antibody microarray with Cy3 (a), HRP (b) and colloidal gold (c) conjugated to anti-LCDV MAbs as detection antibody. LCDV diluents were incubated with the antibody microarrays and the antibody–antigen complexes were identified by detection antibody with different markers. The left column of (a)–(c) was positive control, and then the viral protein concentrations were 4.39, 2.2, 1.1, 0.88 and 0.55 g/ml in order for (a) and (b), and 8.78, 4.39, 2.2, 1.46 and 1.1 g/ml in order for (c).
Relative signal value
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Relative signal value, stored at RT Relative signal value, stored at 4ºC
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that 8/50 samples were detected LCDV-positive, suggesting 8 samples were infected by LCDV. Partial analysis results of antibody microarray were present in Fig. 6, showing samples III, IV, V, VI, VII and VIII were LCDV-positive and samples I and II were LCDVnegative. Additionally 8/50 samples were confirmed positive by ELISA, which had 100% agreement with the antibody microarray and the correlation coefficient (r) was 0.9853 (Fig. 6b). Only 7 samples were validated positive by IFAT and had 98% agreement (data not shown), which was also in good concordance with antibody microarray.
Relative signal value, stored at 20ºC
4. Discussion
Fig. 4. Agarose gel-modified slides stability. Agarose gel-modified slides stored under dry condition for a 1-year period still have a good capability of immobilizing protein compared to modified slides freshly with HRP conjugated to anti-LCDV MAbs as detection antibody.
3.5. Stability of antibody microarray For stability of the antibody microarray stored at 4 ◦ C and −20 ◦ C, the shelf life of capture antibody spotted on the agarose gelmodified slides was tested every 15 days. When Cy3-conjugated anti-LCDV MAbs were used as detection antibody, the relative signal value of microarrays decreased gradually after 4-month storage due to the background value increasing, especially for the microarrays stored at −20 ◦ C, though the fluorescence intensities of antibody spots were still saturated. The detection limit was 1.1 g/ml after 4-month storage. When HRP-conjugated MAbs were used as detection antibody, no obvious decrease in relative signal value was seen until 8-month storage at −20 ◦ C (Fig. 5), and the detection limit then declined to 1.1 g/ml 1 year later compared to the antibody microarray prepared freshly (0.55 g/ml). The antibody microarrays stored at 4 ◦ C were less stable and the detection limit declined to 1.1 g/ml after 6-month storage. Fig. 5 showed the relative amount of LCDV detection limit as a function of storage time at −20 ◦ C. These data indicated that capture antibody coupled onto agarose gel-modified slides and stored at −20 ◦ C was stable for at least 8 months when HRP conjugated to anti-LCDV MAbs were used as detection antibody. 3.6. Concordance comparisons of antibody microarray, ELISA and IFAT Positive samples were detected in the antibody microarray by exhibiting obvious fluorescence intensities. The results showed
Fig. 5. Variation of antibody microarrays sensitivity under −20 ◦ C storage with HRP conjugated to anti-LCDV MAbs as detection antibody for up to 1 year. The detection limit was 0.55 g/ml after 8 months of storage and 1.1 g/ml 1 year later, a comparable amount relative to antibody microarray prepared freshly.
LCDV infections pose a serious threat to the aquaculture industry and are responsible for significant economic losses worldwide (Lang, 1994; Plumb, 1993). Although it is reported that there is self-healing phenomenon in LCDV-infected fish causing tumor-like lesions to disappear when exposed to warmer water temperatures, virus particles seem to persist in asymptomatic carriers until animals become stressed resulting in a recrudescence of clinical symptoms. LCDV was still detectable in fish skin, caudal fin and eyeballs up to four weeks after all pathognomonic external signs of LCD have resolved (Cano et al., 2006). This suggests that the recovered individuals may still be infective to naive fish in the same pond. This has been suggested after detection of LCDV in the caudal fin of asymptomatic gilthead sea bream (Sparus aurata), showing reappearance of LCDV symptoms when animals are maintained under stressful conditions (Cano et al., 2007). Therefore it is important to establish rapid, convenient, and reliable methods for the tracking and monitoring of LCDV exposure to prevent further transmission and outbreaks of lymphocystis disease. Antibody microarray technology has provided new molecular diagnostic tools with a high-throughput capacity that enables a multitude of tests to be performed simultaneously (Park et al., 2005). Nowadays antibody microarray has been used in clinical practice for detection of hepatitis virus, biomarkers or surface antigen profiler of colorectal cancer, or cardiovascular risk markers (Teresa et al., 2006; Ozgur et al., 2007; Xu et al., 2007; Zhou et al., 2010). In this study, rabbit anti-LCDV antibody (0.5 mg/ml) was selected as capture antibody and the antibody microarray was developed for LCDV diagnosis, which exhibited reasonable sensitivity and specificity. Anti-LCDV MAbs were used as capture antibody in pilot research, however, purified rabbit anti-LCDV antibody, which had the same titer as anti-LCDV MAbs, could improve the microarray performance dramatically. A likely explanation is that polyclonal antibody enhances the antigen capture by binding multiple sites on the analyte (Lian et al., 2010). Agarose gel-modified slides are chosen as the supports of antibody microarray because they provide three-dimensional surface capable of immobilizing protein in high efficiency (Xu et al., 2011). In this paper, antibody microarray based on agarose gel modified slides also gave rise to a higher sensitivity. To achieve the best sensitivity of microarray on agarose gel-modified slides, different markers including colloidal gold, FITC, Cy3, HRP and two additional streptavidin conjugates (streptavidin–Cy3 and streptavidin–HRP) were examined. Fluorescent dyes are becoming more and more popular and are the universal choice for labeling and detection of molecules in microarray applications currently, and FITC is a common fluorescent probe; also Cy3, HRP and immunogold assay are also used widely in microarray technology (James et al., 2004; Yu et al., 2004; Jiang et al., 2008). In the paper, anti-LCDV MAbs conjugated to different markers were used as detection antibodies, and HRP and Cy3 conjugated anti-LCDV MAbs gave a higher sensitivity of 0.55 g/ml. Jiang et al. (2008) compared sensitivities of fluorescent and colorimetric detection methods-based protein microarray
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Fig. 6. LCDV detection in the fish gills or fins tissue homogenates. Fish gill tissue homogenates were incubated with antibody microarray and then with Cy3-conjugated antiLCDV MAbs as detection antibody. Indirect ELISA measured the same samples with dilution buffer used as a negative control. (a) Partial LCDV detection results by antibody microarray were shown, revealing LCDV-positive in III, IV, V, VI, VII and VIII samples. (b) Correlation of antibody microarray results vs. ELISA. The correlation coefficient (r) was 0.9853.
for serodiagnosis of TORCH infections, and the detection limits of IgM antibody on the microarrays were 0.48 and 0.24 g/ml, which was in accordance with this research. The use of fluorescence dye Cy3 can avoid interruption of endogenous enzyme clearly, yet the measurement yielded by HRP-conjugated MAbs can be visualized with naked eye, which is more practical in on-site detection of fish disease. Fluorescent or HRP conjugates of streptavidin are often used as amplifiers to detect biomolecules (Liu et al., 2005; Ye et al., 2005; Lucarelli et al., 2006), because biomolecules can be labeled with biotin easily, and the biotin–streptavidin interaction has been established as a representative system for amplifying signal after the hybridization (Vijaya et al., 2007). Vijaya et al. (2007) presented applications of various streptavidin–fluorophore conjugates including streptavidin–Cy3, streptavidin–Cy5, streptavidin–Alexa Fluor 555 and streptavidin–phycoerythrin, and chose streptavidin–Cy3 conjugate to enhance the detection limit of the microarray finally. In this research, the best sensitivity was produced by streptavidin–HRP. Also, it was found that the co-incubation method, covering the slides with biotin-conjugated anti-LCDV MAbs and Cy3-conjugated streptavidin simultaneously after incubation with LCDV diluent or the detected samples, gave the same fluorescent or staining signal as obtained with the conventional method. The co-incubation could simplify the operating procedures and shorten time-consuming operating procedures, which not only reduced labor costs, but also reduced sample-handing errors potentially (Park et al., 2010). The shelf life stability of antibody microarray based on agarose gel-modified slides over time is also an important practical consideration in facilitating the manufacture of sensitive and reproducible assays (Du et al., 2003; Seshi et al., 2004). In this study, the shelf life of antibody microarray was stable for at least 8 months at −20 ◦ C when detected with HRP-conjugated anti-LCDV MAbs. However, when detected with Cy3-conjugated anti-LCDV MAbs, the relative signal value decreased 4 months later as the background value increased with this phenomenon more evident when microarray was stored at −20 ◦ C. The shelf life stability of the support showed that the background value of agarose-gel modified slides stored at −20 ◦ C increased greatly after 4-month storage when detected with Cy3-conjugated anti-LCDV MAbs. It seemed that the agarose gel surface had changed after long-term frozen storage, and the dye Cy3, a small molecule, was easy to be adsorbed and hard to be washed away from the surface; however, storing at −20 ◦ C had the advantage of protecting the antibody activity on the microarray. Therefore, HRP-conjugated anti-LCDV MAbs as a detection antibody using the HRP-TMB Chromogenic system was recommended because it was both more economical and convenient. In conclusion, an antibody microarray for LCDV detection with high specificity and sensitivity was developed. The
lowest detectable protein concentration of LCDV by the antibody microarrays was 0.0686 g/ml (sample volume was 40 l/array), which was more sensitive than that of indirect sandwich ELISA (0.28 g/ml) or indirect dot–blot immunoenzymatic assay (0.5 g/ml) (Garcíarosado et al., 2002). The sensitivity was expressed as 2−4.9 TCID50 /40 l for LCDV. Furthermore, scaling down permitted low consumption of both samples and reagents, reducing the amounts of biohazardous waste as well as assay costs (Park et al., 2010). The diagnostic results of antibody microarray showed 100% agreement with conventional ELISA, and 98% agreement with IFAT which might be due to the low sensitivity of IFAT or sampling location differences. The developed antibody microarray can be utilized to detect LCDV in multiple samples simultaneously, making it suitable for aquatic animal health inspection diagnostic testing, for meeting import and export requirements of quarantine of aquatic animals, and for conducting diagnostic and epidemiological studies and surveys of LCDV infections in aquaculture and wild populations. However, considering sensitivity of immunological methods is confined by viral extraction, suspected positive samples tested with the antibody microarray may show negative results. It is necessary that the tiny infection samples should be further confirmed by cell culture or real-time PCR as complement. Acknowledgements This study was supported by National Natural Science Foundation of China (Grants. 31172429 and 31072232), and the National Science and Technology Supporting Program (Grant 2012BAD17B01). Prof. E. Scott Weber, from University of California Davis, is acknowledged for his kind assistance in perfecting the text. References Angenendt, P., 2005. Progress in protein and antibody microarray technology. Drug Discov. Today 10, 503–511. Cano, I., Alonso, M.C., Garcia-Rosado, E., Saint-Jean, S.R., Castro, D., Borrego, J.J., 2006. Detection of lymphocystis disease virus (LCDV) in asymptomatic cultured gilt-head seabream (Sparus aurata L.) using an immunoblot technique. Vet. Microbiol. 113, 137–141. Cano, I., Ferro, P., Alonso, M.C., Bergmann, S.M., Romer-Oberdorfer, A., GarciaRosado, E., Castro, D., Borrego, J.J., 2007. Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species. J. Appl. Microbiol. 102, 32–40. Cheng, S.F., Zhan, W.B., Xing, J., Sheng, X.Z., 2006. Development and characterization of monoclonal antibody to the lymphocystis disease virus of Japanese flounder Paralichthys olivaceus isolated from China. J. Virol. Methods 135, 173–180. Chinchar, V.G., 2002. Ranaviruses (family Iridovirudae): emerging coldblooded killers. Arch. Virol. 147, 447–470. Du, W.D., Xu, Z.S., Ma, X.L., Song, L.H., Schneider, E.M., 2003. Biochip as a potential platform of serological interferon ␣2b antibody assay. J. Biotechnol. 106, 87–100. Garcíarosado, E., Castro, D., Cano, I., Pérezprieto, S.I., Borrego, J.J., 2002. Serological techniques for detection of lymphocystis virus in fish. Aquat. Living Resour. 15, 179–185.
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Development and application of antibody microarray for lymphocystis disease virus detection in fish Xiuzhen Sheng, Xiaoli Xu, Wenbin Zhan ∗ Laboratory of Pathology and Immunology of Aquatic Animals, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China
a b s t r a c t Article history: Received 19 June 2012 Received in revised form 1 February 2013 Accepted 27 February 2013 Available online 7 March 2013 Keywords: Lymphocystis disease virus (LCDV) Antibody microarray Agarose gel Virus detection
Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease affecting marine and freshwater fish worldwide. Here an antibody microarray was developed and employed to detect LCDV in fish. Rabbit anti-LCDV serum was arrayed on agarose gel-modified slides as capture antibody, and Cy3conjugated anti-LCDV monoclonal antibody (MAbs) was added as detection antibody. The signals were imaged with a laser chip scanner and analyzed by corresponding software. To improve the sensitivity, different substrate binders (poly-l-lysine, MPTS, aldehyde, APES and agarose gel modified slides, and commercially available amino-modified slides), markers (fluorescein isothiocyanate, Cy3, horseradish peroxidase, biotin or colloidal gold) conjugated to anti-LCDV Mabs, and storage time of the antibody were assessed. The results showed that the antibody microarrays based on agarose gel-modified slides gave a lower detection limit of 0.55 g/ml of LCDV when Cy3 and HRP conjugated anti-LCDV MAbs were used as detection antibody; and the lowest detectable LCDV protein concentration was 0.0686 g/ml when streptavidin–biotin conjugated to anti-LCDV MAbs served as detection antibody. The developed antibody microarray proved to have a high specificity for LCDV detection and a shelf-life of more than 8 months at −20 ◦ C. Furthermore, the LCDV detection results of the microarray in fish gills or fins (n = 50) presented a concordance rate of 100% with enzyme-linked immunosorbent assay (ELISA) and 98% with immunofluorescence assay technique (IFAT). These results revealed that the developed antibody microarray could serve as an effective tool for diagnostic and epidemiological studies of LCDV in fish. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease (LCD) affecting approximately 140 species of marine and freshwater fish worldwide (Lang, 1994; Plumb, 1993). Fish infected with LCDV exhibit characteristic external symptoms of wart-like nodules on the body skin, fins, mouth and gills, causing a decrease in commercial value (Tidona and Darai, 1997). In addition, diseased fish are more susceptible to secondary infection by other microorganisms, resulting in high mortalities and great economic losses for aquaculture worldwide (Chinchar, 2002; Iwamoto et al., 2002). Since there are currently no available treatments for LCD, rapid and accurate detection of LCDV is crucial in prevention and control of lymphocystis disease. Diagnosis of LCD is generally based on typical skin lesion observation. At present, the techniques used frequently for LCDV detection include polymerase chain reaction (PCR), real-time PCR, virus isolation and neutralization using cell lines, and several immunological techniques based on anti-LCDV antibody
∗ Corresponding author. Tel.: +86 532 82032284; fax: +86 532 82032284. E-mail address: [email protected] (W. Zhan). 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.02.015
(Garcíarosado et al., 2002; Kitamura et al., 2006; Cano et al., 2006, 2007). PCR and real-time PCR are of specific, sensitive, rapid and cost-effective; however, their performances require highly trained personnel and specialized equipments. The isolation of viral pathogens in cell cultures has been regarded as the “gold standard” for their convenience and accuracy, but this approach is often slow and requires considerable technical expertise. Immunological techniques (ELISA, immunofluorescence assay technique, Western-blot) based on anti-LCDV antibody are sensitive and specific, but time-consuming with complicated operation. Antibody microarray-based analysis is a cost-effective approach that yields reproducible results and can allow replicate analyses in a single assay run. As a next-generation tool, antibody microarrays are increasing in popularity, for they offer unparalleled throughput, minimal reagent consumption and sensitive detection of multiple targets simultaneously (Angenendt, 2005); and test results could be read by naked eyes when combined with immunoenzyme techniques or immunogold-sliver staining. However the accuracy conferred by this method is linked with the quality of antibodies employed closely, and there are issues relating to cross-reactivity and antibody availability, both of which are crucial for sensitive and specific strain typing particularly (Mahesh et al., 2008). Given the listed benefits, antibody microarrays have been and are being
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developed that have growing potential for clinical, biothreat, and point-of-care applications (Huelseweh et al., 2006; Lian et al., 2010). For disease diagnosis in aquaculture, the antibody microarray for white spot syndrome virus (WSSV) detection in shrimp have been developed (Xu et al., 2011), however for fish viral detection there are, to our knowledge, no reports of antibody microarray diagnostics so far. This paper described the development and application of a low-density antibody microarray for LCDV detection in fish tissue extracts, as well as improvement of antibody microarray sensitivity. With the prepared antibody-microarray-integrated system, the entire testing procedures could be completed in less than 2 h, and the multiple samples from diverse sources could be tested in a single assay without the need for labeling. As a supplementary technique for detection of LCDV in fish, the developed antibody microarray is convenient, accurate, sensitive and time-saving, and suitable for diagnostic and epidemiological detection to LCDV. 2. Materials and methods 2.1. Preparation of samples The LCDV-free flounders (Paralichthys olivaceus, 15 ± 1 cm in length) tested by PCR (Zhan et al., 2010) were obtained from a fish farm located in Qingdao of Shandong province, China; LCDVinfected flounders (20 ± 2 cm in length) with various-sized nodules on body surface were from a fish farm in Hebei province. Common sea bass (Lateolabrax japonicus, 23 ± 2 cm) and sting fish (Sebastes schlegeli, 20 ± 2 cm) were from a fish farm in Shandong province. All these fish were shipped in chill containers, and then anaesthetized with tricaine (0.065 mg/ml) (MS-222, Sigma–Aldrich, St. Louis, US). The tissues (gill, fins, skin, lymphocystis nodules and internal organs) were taken and frozen at −80 ◦ C. Sampled fish fin or gill (target organs of LCDV) homogenates were used for LCDV detection. The fish gills or fins (∼1 g) were washed with TNE buffer (50 mmol/l Tris, 100 mmol/l NaCl, 1 mmol/l EDTA, pH 7.4) for 5 min at a ratio of 10% (w/v), homogenized and suspended in TNE buffer at 4 ◦ C. The suspension was subjected to rapid freeze–thaw (3×), ultrasonication, and centrifuged at 500 × g and then 1800 × g for 20 min at 4 ◦ C, respectively. The homogenates of gills and fins from the same fish were pooled and stored at −80 ◦ C until use. Purified LCDV solution was prepared from lymphocystis nodule homogenate using the method developed previously (Cheng et al., 2006) and resuspended with 0.01 mol/l PBS (pH 7.4), and the viral protein concentration was measured 0.878 mg/ml. Graded reagents and MilliQ grade water were used throughout the experiment. All studies were conducted in accordance with institutional, national and international standards on animal welfare. 2.2. Antibody production and purification Rabbit anti-LCDV antibody (as capture antibody of the microarray) and rabbit anti-mouse Ig antibody (as positive control) were prepared by immunizing adult New Zealand rabbits with purified LCDV and mouse Ig in conventional methods respectively (Huang et al., 2005). One week after the last injection, blood was taken from the rabbit and the rabbit antiserum was purified by the Ampure PA kit (GE Healthcare, Fairfield, US) following the manufacturer’s protocol. The reactivity of LCDV with rabbit antiserum was determined by immunofluorescence assay technique (IFAT) using pre-immune serum as control (Cheng et al., 2006). Four anti-LCDV MAbs, 3G3, 2B6, 1D7 and 2D11 developed previously (Cheng et al., 2006), were produced in ascites by injecting the hybridoma clone into the peritoneal cavity of Balb/c mice, and purified in the same way above-mentioned. These MAbs were then
labeled with Cy3 (GE Healthcare, Fairfield, US) according to the manufacturers’ instructions and used as detection antibody after mixed in equal proportion. 2.3. Antibody microarray fabrication for LCDV detection 2.3.1. Optimal concentration of capture antibody Agarose gel modified slides were prepared following the methods of Xu et al. (2011). Rabbit anti-LCDV antibody was diluted at the concentration range of 0.5 g/ml to 1.5 mg/ml, and spotted on agarose gel-modified slides as capture antibody in 3 × 3 matrix respectively. The slides were put in humid chambers at 37 ◦ C for 2 h to complete the immobilization of antibodies and then washed 3 times with PBST (PBS containing 0.5% Tween-20) for 5 min each. Slide surface was blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at 37 ◦ C. After 3 washes, LCDV diluent at 0.1 mg/ml was incubated with the capture antibody, and the antigen–antibody complex formed was identified by Cy3-conjugated anti-LCDV antibody. Detection results were measured by laser chipscanner at 532 nm and quantified with software EcoscanCHS. The optimal concentration of capture antibody was confirmed when the relative signal value of the microarray tended to be stable. 2.3.2. Integration of antibody microarray The capture antibody, 0.5 mg/ml rabbit anti-LCDV antibody in printing buffer (PBS containing 50% glycerol) was spotted onto the agarose gel-modified slide with a spot volume of 20–50 nl. The microarray contained eight groups deposited in 4 × 4 matrixes and 2 × 4 arrangement on one slide. In each matrix, the printing buffer as negative control and the anti-mouse Ig antibody as positive control were spotted to form four replicates, respectively, and the capture antibody was spotted in eight replicates. To perform multiplexed assays on the same slide, the microarrays were compartmentalized into eight subarrays by Super PAP Pen or silicone gasket. After arraying, the slides were incubated in humid chambers at 37 ◦ C for immobilization of antibodies and then blocked by 3% BSA. The antibody microarrays were washed and allowed to dry, and stored at 4 ◦ C or −20 ◦ C until use. 2.4. LCDV detection Detection samples, 40 l per matrix, were added on the antibody microarray. The slides were put in a humid chamber at 37 ◦ C for 15–30 min and then washed 3 times, followed by incubation with Cy3-conjugated anti-LCDV antibody at 37 ◦ C for 30 min. The microarrays were washed again and imaged by laser chip scanner, and fluorescence signals were quantified with software EcoscanCHS. The fluorescence intensity of negative control was used as the background value, and the mean fluorescence intensity value of the eight replicates of capture antibody in one matrix was determined as the relative signal value after subtracting the corresponding background value. 2.5. Detection limit of antibody microarray Purified LCDV (0.878 mg/ml) was diluted 1:50–6400 with PBS, and added to the integrated antibody microarrays in 40 l per matrix. The detection limit of the antibody microarray was defined as the lowest LCDV concentration that could be detected reliably and positively and also a measure of sensitivity. For improvement of detection limit, the supports of microarray, markers labeled to anti-LCDV MAbs, and shelf life of antibody microarray, were assessed. In addition, the detection limit of the antibody microarray was compared with that of indirect sandwich enzyme-linked immunosorbent assay (ELISA) (Luo et al., 2009). The 96-well EIA plates (Corning, New York, USA) were coated with 50 l rabbit
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anti-LCDV antibody (0.1 mg/ml), and anti-LCDV MAbs served as the primary antibody and goat anti-mouse Ig serum conjugated with AP (Sigma–Aldrich, St. Louis, US) served as the second antibody. The dilution buffer was used as negative control. The experiment was repeated three times. The antibody microarray sensitivity was also determined by analyzing serial dilutions of LCDV suspension (homogenate of lymphocystis nodules) corresponding to median tissue culture infectious dose (TCID50). The flounder gill (FG) cell line was incubated in MEM supplemented with 10% FBS at 20 ◦ C (Tong et al., 1997). The TCID50 of LCDV suspensions was tested by FG cells (Xing et al., 2006), and the antibody microarray sensitivity expressed as TCID50 was then obtained by conversion. 2.5.1. Detection limit based on different supports Commercially available amino-modified slides were purchased from CEL CO (USA). Poly-l-lysine, MPTS, aldehyde, APES and agarose gel modified slides for microarrays were prepared according to the references (Masanori, 1991; Kusnezow et al., 2003; Rubina et al., 2003; Panagiota et al., 2007; Xu et al., 2011). The capture antibody was spotted on the six types of slides in an array format and then detection limit was tested using Cy3-conjugated anti-LCDV antibody as detection antibody, respectively. 2.5.2. Detection limit based on different markers Purified anti-LCDV MAbs, 3G3, 2B6, 1D7 and 2D11 were mixed in a volume ratio 1:1:1:1 and labeled with horseradish peroxidase (HRP) (Galaxybio, Beijing, China), fluorescein isothiocyanate (FITC) and biotin (Sigma–Aldrich, St. Louis, US) according to the manufacturers’ instructions, respectively. Conjugation of anti-LCDV MAbs to colloidal gold was prepared following the method of Wang and Zhan (2006). The labeled anti-LCDV MAbs mixture was used as detection antibody. Integrated antibody microarray based on agarose gel-modified slides was incubated with different LCDV diluents. After three washes the slides were exposed to the detection antibody labeled with different markers. The biotin labeled anti-LCDV MAbs were coincubated with Cy3 or HRP conjugated streptavidin simultaneously on the antibody microarray. Following three washes, the signals were visualized with the following methods. The fluorescence intensity of FITC and Cy3 were imaged with professional laser scanner at 494 nm and 532 nm, respectively, and analyzed by corresponding software. The signals of HRP were visualized with 3,3 ,5,5 -tetramethylbenzidine (TMB) (Galaxybio, Beijing, China) and the reaction was stopped by 2 M sulfuric acid. The signals of colloidal gold were visualized by soaking the slides in silver enhancement reagent (Sigma–Aldrich, St. Louis, US) for 10 min in the dark and fixed with sodium thiosulfate solution for 3 min at room temperature.
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The antibody microarrays prepared as mentioned previously, were stored at 4 ◦ C and −20 ◦ C. Slides and antibody microarray were tested every 15 days with different LCDV diluents, using HRP and Cy3 conjugated anti-LCDV MAbs as detection antibody. Agarose gel-modified slide and antibody microarray prepared freshly were used as controls. The shelf life of antibody microarrays was determined according to the relative signal value tested over a 1-year period. 2.8. Validation of antibody microarray for LCDV detection The accuracy of integrated microarray was compared to that of the indirect ELISA and IFAT by detecting LCDV in 50 samples of asymptomatic cultured flounder (P. olivaceus), common sea bass (L. japonicus) and sting fish (S. schlegeli) from different farms of Shandong province, China. The purified LCDV solution was used as positive control and the samples from healthy individuals served as negative control. LCDV detection by indirect ELISA was performed as described by Zhan et al. (2004). For ELISA, the purified LCDV solution was used as positive control and the supernatants of fish gills or fins were all pretreated with 0.5 M EDTA for 1 h. The dilution buffer and the samples from healthy individuals served as background and negative control, respectively, and mixture of four anti-LCDV MAbs served as primary antibody. OD values over three times higher than the negative control were evaluated positive. LCDV detection with IFAT was carried out following the method of Cheng et al. (2006). Cryosections (about 7 m thick) were prepared from detected fish fins or gills. Mixture of four anti-LCDV MAbs served as primary antibody and FITC-conjugated goat antimouse Ig serum (Sigma–Aldrich, St. Louis, US) diluted at 1:256 was used as the second antibody. Results were imaged by fluorescence microscope (Olympus, Tokyo, Japan). 3. Results 3.1. Optimum concentration of capture antibody Reactivity of the rabbit anti-LCDV antibody was tested by IFAT and a strong positive signal was found, but no positive signal was detected with pre-immune serum. Rabbit anti-LCDV antibody in various concentrations was spotted onto agarose gel-modified slides, and results indicated that the relative signal value of the microarrays continued to go up as the concentration of capture antibody increased (Fig. 1a and b), and reached saturation at 0.5 mg/ml. Thus 0.5 mg/ml of rabbit anti-LCDV antibody was confirmed as the appropriate concentration of capture antibody for the microarray. 3.2. Detection limit of antibody microarray based on different supports and markers
2.6. Specificity of antibody microarray To determine specificity of antibody microarray for LCDV detection, gill homogenates of LCDV-infected P. olivaceus, infectious hematopoietic necrosis virus (IHNV)-infected P. olivaceus, turbot reddish body iridovirus (TRBIV)-infected Scophthalmus maximus (Qingdao, China) and healthy P. olivaceus were incubated with the antibody microarray in different matrixes, respectively. Purified LCDV was used as positive control. 2.7. Stability of agarose gel-modified slides and antibody microarray Measuring their efficacy in immobilizing antibody tested the shelf life of agarose gel-modified slides. Agarose gel-modified slides were stored at 4 ◦ C, −20 ◦ C and room temperature (RT) before use.
Different supports were used to prepare the antibody microarrays and Cy3-conjugated anti-LCDV antibody was used as detection antibody to test detection limits. The results revealed that the relative signal value increased with increasing LCDV concentration, and the lowest detectable LCDV protein concentration of the antibody microarrays was 0.55 g/ml, which was performed by the microarray based on agarose gel-modified slides and aldehyde slides. The antibody microarray based on APES-modified slides, amino-modified slides and mercapto slides could allow detection of LCDV at concentration of 1.1 g/ml, while the microarray based on poly-l-lysine modified slide gave a detection limit of 1.46 g/ml (Fig. 2). In order to enhance detection sensitivity, anti-LCDV MAbs conjugated to different markers were used as detection antibodies. As shown in Table 1, Cy3 and HRP conjugated anti-LCDV MAbs gave
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Fig. 1. Relationship between capture antibody concentration and relative signal value. Rabbit anti-LCDV serum as capture antibody was immobilized on agarose gel-modified at a concentration of 1–1500 g/ml with a spot volume of 20–50 nl. The microarray was covered by LCDV diluent and then incubated with Cy3-conjugated anti-LCDV MAbs. The results were imaged by a laser scanner and analyzed by Ecoscan CHS software. (a) The relative signal value enhanced with the increasing concentration of capture antibody and reached saturation at 0.5 mg/ml. (b) The images of capture antibody at various protein concentrations used for the immobilization.
(IGSS) and Cy3-conjugated streptavidin-biotin detection system was 0.88 g/ml, 1.1 g/ml and 0.14 g/ml, respectively (Fig. 3). TCID50 of LCDV suspensions was 24.10 /40 l tested by FG cells which means 40 l LCDV suspensions of 17 times dilution is 1 TCID50 . When LCDV suspension was up to 29 -fold dilution, it was detected positive by antibody microarray with HRP-conjugated streptavidin–biotin detection system. Therefore the sensitivity of antibody microarray was 2−4.9 TCID50 /40 l for LCDV by conversion.
3.3. Specificity of antibody microarray Fig. 2. Detection limits based on different supports. Antibody microarrays based on six types of supports were incubated with LCDV diluents and the detection limits were tested. As low as 0.55 g/ml of LCDV could be detected by the antibody microarray based on agarose gel-modified slides (I) and aldehyde slides (V), whereas 1.1 g/ml of LCDV were detected by the antibody microarray based on amine slide (II), APES modified slide (III) and mercapto slide (IV), and 1.46 g/ml of LCDV by the antibody microarray based on poly-l-lysine modified slide (XI).
Cross-reactivity studies using gill homogenates of LCDVinfected P. olivaceus, IHNV-infected P. olivaceus, TRBIV-infected S. maximus and healthy P. olivaceus showed that positive signals were observed in the positive control and LCDV-infected P. olivaceus, but not in IHNV, TRBIV and healthy samples.
Table 1 Detection limit with different markers conjugated to anti-LCDV MAbs.
3.4. Shelf-life stability of agarose gel-modified slides
Markers
Detection limit of LCDV (g/ml)
FITC HRP IGSS Cy3 HRP–avidin–biotin Cy3–avidin–biotin
0.88 0.55 1.10 0.55 0.0686 0.14
a detection limit of 0.55 g/ml; while indirect sandwich ELISA was 0.28 g/ml (data not shown). Streptavidin–biotin detection system was applied to amplify the detection signal and HRP-conjugated streptavidin gave the lowest detection limit of 0.0686 g/ml. The detection limit produced by FITC, immunogold-sliver staining
We tested the stability of the agarose gel-modified slides stored under dry condition over 1 year at different temperatures (Fig. 4). The results illustrated that agarose gel-modified slides have a good capability of immobilizing protein after 1-year storage compared to the slides modified freshly, and the detection limit was still 0.55 g/ml of LCDV by using HRP-conjugated anti-LCDV MAbs as detection antibody. However, when Cy3-conjugated anti-LCDV MAbs were used as detection antibody, the background value increased with prolonged storage resulting in the decline of relative signal value, which was more obvious for the supports stored at −20 ◦ C, and as a result the microarray based on the agarose gelmodified slides exhibited a higher detection limit of 1.46 g/ml after 4-month storage at −20 ◦ C.
Fig. 3. Detection limit of antibody microarray with Cy3 (a), HRP (b) and colloidal gold (c) conjugated to anti-LCDV MAbs as detection antibody. LCDV diluents were incubated with the antibody microarrays and the antibody–antigen complexes were identified by detection antibody with different markers. The left column of (a)–(c) was positive control, and then the viral protein concentrations were 4.39, 2.2, 1.1, 0.88 and 0.55 g/ml in order for (a) and (b), and 8.78, 4.39, 2.2, 1.46 and 1.1 g/ml in order for (c).
Relative signal value
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Relative signal value, stored at RT Relative signal value, stored at 4ºC
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that 8/50 samples were detected LCDV-positive, suggesting 8 samples were infected by LCDV. Partial analysis results of antibody microarray were present in Fig. 6, showing samples III, IV, V, VI, VII and VIII were LCDV-positive and samples I and II were LCDVnegative. Additionally 8/50 samples were confirmed positive by ELISA, which had 100% agreement with the antibody microarray and the correlation coefficient (r) was 0.9853 (Fig. 6b). Only 7 samples were validated positive by IFAT and had 98% agreement (data not shown), which was also in good concordance with antibody microarray.
Relative signal value, stored at 20ºC
4. Discussion
Fig. 4. Agarose gel-modified slides stability. Agarose gel-modified slides stored under dry condition for a 1-year period still have a good capability of immobilizing protein compared to modified slides freshly with HRP conjugated to anti-LCDV MAbs as detection antibody.
3.5. Stability of antibody microarray For stability of the antibody microarray stored at 4 ◦ C and −20 ◦ C, the shelf life of capture antibody spotted on the agarose gelmodified slides was tested every 15 days. When Cy3-conjugated anti-LCDV MAbs were used as detection antibody, the relative signal value of microarrays decreased gradually after 4-month storage due to the background value increasing, especially for the microarrays stored at −20 ◦ C, though the fluorescence intensities of antibody spots were still saturated. The detection limit was 1.1 g/ml after 4-month storage. When HRP-conjugated MAbs were used as detection antibody, no obvious decrease in relative signal value was seen until 8-month storage at −20 ◦ C (Fig. 5), and the detection limit then declined to 1.1 g/ml 1 year later compared to the antibody microarray prepared freshly (0.55 g/ml). The antibody microarrays stored at 4 ◦ C were less stable and the detection limit declined to 1.1 g/ml after 6-month storage. Fig. 5 showed the relative amount of LCDV detection limit as a function of storage time at −20 ◦ C. These data indicated that capture antibody coupled onto agarose gel-modified slides and stored at −20 ◦ C was stable for at least 8 months when HRP conjugated to anti-LCDV MAbs were used as detection antibody. 3.6. Concordance comparisons of antibody microarray, ELISA and IFAT Positive samples were detected in the antibody microarray by exhibiting obvious fluorescence intensities. The results showed
Fig. 5. Variation of antibody microarrays sensitivity under −20 ◦ C storage with HRP conjugated to anti-LCDV MAbs as detection antibody for up to 1 year. The detection limit was 0.55 g/ml after 8 months of storage and 1.1 g/ml 1 year later, a comparable amount relative to antibody microarray prepared freshly.
LCDV infections pose a serious threat to the aquaculture industry and are responsible for significant economic losses worldwide (Lang, 1994; Plumb, 1993). Although it is reported that there is self-healing phenomenon in LCDV-infected fish causing tumor-like lesions to disappear when exposed to warmer water temperatures, virus particles seem to persist in asymptomatic carriers until animals become stressed resulting in a recrudescence of clinical symptoms. LCDV was still detectable in fish skin, caudal fin and eyeballs up to four weeks after all pathognomonic external signs of LCD have resolved (Cano et al., 2006). This suggests that the recovered individuals may still be infective to naive fish in the same pond. This has been suggested after detection of LCDV in the caudal fin of asymptomatic gilthead sea bream (Sparus aurata), showing reappearance of LCDV symptoms when animals are maintained under stressful conditions (Cano et al., 2007). Therefore it is important to establish rapid, convenient, and reliable methods for the tracking and monitoring of LCDV exposure to prevent further transmission and outbreaks of lymphocystis disease. Antibody microarray technology has provided new molecular diagnostic tools with a high-throughput capacity that enables a multitude of tests to be performed simultaneously (Park et al., 2005). Nowadays antibody microarray has been used in clinical practice for detection of hepatitis virus, biomarkers or surface antigen profiler of colorectal cancer, or cardiovascular risk markers (Teresa et al., 2006; Ozgur et al., 2007; Xu et al., 2007; Zhou et al., 2010). In this study, rabbit anti-LCDV antibody (0.5 mg/ml) was selected as capture antibody and the antibody microarray was developed for LCDV diagnosis, which exhibited reasonable sensitivity and specificity. Anti-LCDV MAbs were used as capture antibody in pilot research, however, purified rabbit anti-LCDV antibody, which had the same titer as anti-LCDV MAbs, could improve the microarray performance dramatically. A likely explanation is that polyclonal antibody enhances the antigen capture by binding multiple sites on the analyte (Lian et al., 2010). Agarose gel-modified slides are chosen as the supports of antibody microarray because they provide three-dimensional surface capable of immobilizing protein in high efficiency (Xu et al., 2011). In this paper, antibody microarray based on agarose gel modified slides also gave rise to a higher sensitivity. To achieve the best sensitivity of microarray on agarose gel-modified slides, different markers including colloidal gold, FITC, Cy3, HRP and two additional streptavidin conjugates (streptavidin–Cy3 and streptavidin–HRP) were examined. Fluorescent dyes are becoming more and more popular and are the universal choice for labeling and detection of molecules in microarray applications currently, and FITC is a common fluorescent probe; also Cy3, HRP and immunogold assay are also used widely in microarray technology (James et al., 2004; Yu et al., 2004; Jiang et al., 2008). In the paper, anti-LCDV MAbs conjugated to different markers were used as detection antibodies, and HRP and Cy3 conjugated anti-LCDV MAbs gave a higher sensitivity of 0.55 g/ml. Jiang et al. (2008) compared sensitivities of fluorescent and colorimetric detection methods-based protein microarray
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Fig. 6. LCDV detection in the fish gills or fins tissue homogenates. Fish gill tissue homogenates were incubated with antibody microarray and then with Cy3-conjugated antiLCDV MAbs as detection antibody. Indirect ELISA measured the same samples with dilution buffer used as a negative control. (a) Partial LCDV detection results by antibody microarray were shown, revealing LCDV-positive in III, IV, V, VI, VII and VIII samples. (b) Correlation of antibody microarray results vs. ELISA. The correlation coefficient (r) was 0.9853.
for serodiagnosis of TORCH infections, and the detection limits of IgM antibody on the microarrays were 0.48 and 0.24 g/ml, which was in accordance with this research. The use of fluorescence dye Cy3 can avoid interruption of endogenous enzyme clearly, yet the measurement yielded by HRP-conjugated MAbs can be visualized with naked eye, which is more practical in on-site detection of fish disease. Fluorescent or HRP conjugates of streptavidin are often used as amplifiers to detect biomolecules (Liu et al., 2005; Ye et al., 2005; Lucarelli et al., 2006), because biomolecules can be labeled with biotin easily, and the biotin–streptavidin interaction has been established as a representative system for amplifying signal after the hybridization (Vijaya et al., 2007). Vijaya et al. (2007) presented applications of various streptavidin–fluorophore conjugates including streptavidin–Cy3, streptavidin–Cy5, streptavidin–Alexa Fluor 555 and streptavidin–phycoerythrin, and chose streptavidin–Cy3 conjugate to enhance the detection limit of the microarray finally. In this research, the best sensitivity was produced by streptavidin–HRP. Also, it was found that the co-incubation method, covering the slides with biotin-conjugated anti-LCDV MAbs and Cy3-conjugated streptavidin simultaneously after incubation with LCDV diluent or the detected samples, gave the same fluorescent or staining signal as obtained with the conventional method. The co-incubation could simplify the operating procedures and shorten time-consuming operating procedures, which not only reduced labor costs, but also reduced sample-handing errors potentially (Park et al., 2010). The shelf life stability of antibody microarray based on agarose gel-modified slides over time is also an important practical consideration in facilitating the manufacture of sensitive and reproducible assays (Du et al., 2003; Seshi et al., 2004). In this study, the shelf life of antibody microarray was stable for at least 8 months at −20 ◦ C when detected with HRP-conjugated anti-LCDV MAbs. However, when detected with Cy3-conjugated anti-LCDV MAbs, the relative signal value decreased 4 months later as the background value increased with this phenomenon more evident when microarray was stored at −20 ◦ C. The shelf life stability of the support showed that the background value of agarose-gel modified slides stored at −20 ◦ C increased greatly after 4-month storage when detected with Cy3-conjugated anti-LCDV MAbs. It seemed that the agarose gel surface had changed after long-term frozen storage, and the dye Cy3, a small molecule, was easy to be adsorbed and hard to be washed away from the surface; however, storing at −20 ◦ C had the advantage of protecting the antibody activity on the microarray. Therefore, HRP-conjugated anti-LCDV MAbs as a detection antibody using the HRP-TMB Chromogenic system was recommended because it was both more economical and convenient. In conclusion, an antibody microarray for LCDV detection with high specificity and sensitivity was developed. The
lowest detectable protein concentration of LCDV by the antibody microarrays was 0.0686 g/ml (sample volume was 40 l/array), which was more sensitive than that of indirect sandwich ELISA (0.28 g/ml) or indirect dot–blot immunoenzymatic assay (0.5 g/ml) (Garcíarosado et al., 2002). The sensitivity was expressed as 2−4.9 TCID50 /40 l for LCDV. Furthermore, scaling down permitted low consumption of both samples and reagents, reducing the amounts of biohazardous waste as well as assay costs (Park et al., 2010). The diagnostic results of antibody microarray showed 100% agreement with conventional ELISA, and 98% agreement with IFAT which might be due to the low sensitivity of IFAT or sampling location differences. The developed antibody microarray can be utilized to detect LCDV in multiple samples simultaneously, making it suitable for aquatic animal health inspection diagnostic testing, for meeting import and export requirements of quarantine of aquatic animals, and for conducting diagnostic and epidemiological studies and surveys of LCDV infections in aquaculture and wild populations. However, considering sensitivity of immunological methods is confined by viral extraction, suspected positive samples tested with the antibody microarray may show negative results. It is necessary that the tiny infection samples should be further confirmed by cell culture or real-time PCR as complement. Acknowledgements This study was supported by National Natural Science Foundation of China (Grants. 31172429 and 31072232), and the National Science and Technology Supporting Program (Grant 2012BAD17B01). Prof. E. Scott Weber, from University of California Davis, is acknowledged for his kind assistance in perfecting the text. References Angenendt, P., 2005. Progress in protein and antibody microarray technology. Drug Discov. Today 10, 503–511. Cano, I., Alonso, M.C., Garcia-Rosado, E., Saint-Jean, S.R., Castro, D., Borrego, J.J., 2006. Detection of lymphocystis disease virus (LCDV) in asymptomatic cultured gilt-head seabream (Sparus aurata L.) using an immunoblot technique. Vet. Microbiol. 113, 137–141. Cano, I., Ferro, P., Alonso, M.C., Bergmann, S.M., Romer-Oberdorfer, A., GarciaRosado, E., Castro, D., Borrego, J.J., 2007. Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species. J. Appl. Microbiol. 102, 32–40. Cheng, S.F., Zhan, W.B., Xing, J., Sheng, X.Z., 2006. Development and characterization of monoclonal antibody to the lymphocystis disease virus of Japanese flounder Paralichthys olivaceus isolated from China. J. Virol. Methods 135, 173–180. Chinchar, V.G., 2002. Ranaviruses (family Iridovirudae): emerging coldblooded killers. Arch. Virol. 147, 447–470. Du, W.D., Xu, Z.S., Ma, X.L., Song, L.H., Schneider, E.M., 2003. Biochip as a potential platform of serological interferon ␣2b antibody assay. J. Biotechnol. 106, 87–100. Garcíarosado, E., Castro, D., Cano, I., Pérezprieto, S.I., Borrego, J.J., 2002. Serological techniques for detection of lymphocystis virus in fish. Aquat. Living Resour. 15, 179–185.
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