To develop a gold immunochromatographic test strip for on-site rapid detection of lymphocystis disease virus (LCDV).
To develop a gold immunochromatographic test strip for on-site rapid detection of lymphocystis disease virus (LCDV).
Monodispersional colloidal gold and gold-labelled anti-LCDV monoclonal antibody (McAb) 2D11 were prepared and characterized by UV-visible spectroscopy and transmission electron microscopy. Gold-labelled probe was used as the detection antibody, and goat anti-mouse IgG at the control line and anti-LCDV McAb 1A8 at the test line of the test strip served as the capture antibody. The positive results could be easily judged by the presence of a red test line with naked eye within 10 min. The test strip, in good agreement with enzyme-linked immunosorbent assay and dot-blotting in sensitivity and LCDV detection, gave a detection limit of 1 μg ml−1 of LCDV and was stable for 6 months at room temperature and 12 months at 4°C.
The test strip was specific, simple and convenient for rapid detection of LCDV presenting good stability and reproducibility.
This ready-to-use test strip allows on-site rapid detection of LCDV in fish without the requirement of specialized equipments and professional personnel, which could augment the practical application for diagnosis of LCDV even in disadvantage areas.
Lymphocystis disease, caused by lymphocystis disease virus (LCDV), is a chronic viral disease characterized by the appearance of pearl-like nodules consisting of hypertrophied dermal cells in fish skin, gills, fins and even the internal organs (Sheng et al. 2007; Cano et al. 2009). It has a worldwide geographical distribution and has been detected in more than 140 species of freshwater, estuarine and marine fishes (Wolf 1988). Lymphocystis disease is rarely fatal; however, the diseased fish exhibiting deformation cannot be commercialized and are more susceptible to secondary infection by other micro-organisms resulting in a great economic loss in aquaculture (Iwamoto et al. 2002; Kvitt et al. 2008). In addition, water contamination, oxygen depletion and nutrition deficiency in fish culture may activate virus latent in asymptomatic carriers and lead to a disease occurrence (Cano et al. 2006). In this case, it is imperative for LCDV surveillance in aquaculture systems to control viral dissemination.
Various laboratory methods are available for the diagnosis of LCDV, such as indirect immunofluorescence assay, indirect dot-blot immunoenzymatic assay, flow cytometry based on cell culture inoculation, immunoblot, PCR, multiplex PCR and in situ hybridization techniques (García-Rosado et al. 2002; Cano et al. 2006, 2007, 2009; Kitamura et al. 2006). Generally, these diagnostic approaches have respective merits in terms of accuracy, sensitivity, safety and cost. However, their performances are restricted to laboratory mainly because of the requirement of highly trained personnel and specialized equipments.
Gold immunochromatographic assay (GICA), a technique based on the specific antigen–antibody immunoreactions, is a highly useful tool in diagnostics, as the assay results are directly visible to the naked eye without the requirement of specialized equipments and complicated handling procedures, providing convenience for rapid testing. These characteristics enable it feasible and accessible for on-site detection of antigens (Wang and Zhan 2006). This study demonstrated the development of a GICA-based test strip as a supplementary technique for rapid detection of LCDV in fish. The test strip utilized two monoclonal antibodies (McAbs) of distinct specificity, one LCDV-specific McAb 1A8 immobilized on nitrocellulose (NC) membrane as the capture antibody and another LCDV-specific McAb 2D11 labelled with colloid gold particles as the detection antibody. The performance of assay was very simple and the test could be completed within 10 min.
Flounders (Paralichthys olivaceus) were collected from a farm located in Qingdao, Shandong province of China, and subjected to PCR to ensure LCDV free as described previously (Zhan et al. 2010). LCDV-infected flounders were from another farm in Shandong province, and flounders suspected of LCDV infection were from Hebei province of China.
Pearl-like nodules on the body surface of LCDV-infected flounder were isolated, and LCDV purification was carried out following the methods described by Cheng et al. (2006). Skin and fins from flounder suspected of LCDV infection were sampled and vigorously homogenized in PBS at a ratio of 10% (w/v) on ice, and the supernatant fluids were collected as test solution and used for LCDV detection.
Hybridomas secreting anti-LCDV McAb 1A8 and 2D11 (Cheng et al. 2006), stored in liquid nitrogen, were revived by quickly warming in a water bath at 37°C. Each 8-week-old BALB/c female mouse was injected with 0·5 ml of liquid paraffin intraperitoneally and induced for 7 days, and then 1 × 106 of McAb 1A8 and 2D11 hybridoma clones were injected into the peritoneal cavity of BALB/c mouse, respectively. Ten days later, the mice ascites were harvested and centrifuged at 800 g for 15 min, and anti-LCDV McAbs 1A8 and 2D11 were separated from the ascitic fluids by the method of salting out with caprylic acid–ammonium sulfate as described by Ogden and Leung (1988) and stored at −80°C until use.
Colloidal gold was produced through controlled reduction of chloroauric acid following the procedures described by Frens (1973) with slight modifications. Briefly, 100 ml of 0·01% (m v−1) chloroauric acid (HAuCl4) (China) was heated to boiling for 2 min in microwave oven (Galanz Group, Guangdong, China; 700 W), and 2·0 ml of 1% (m v−1) sodium citrate solution was then added rapidly to the boiling chloroauric acid solution and reduced the heat to medium–low (280 W) for another 3 min. The colloidal gold suspension was allowed to cool down to room temperature and stored at 4°C.
The purified McAb 2D11 was dialysed against distilled water overnight at 4°C and subsequently centrifuged at 15 000 g for 30 min to get a clear supernatant for conjugation. The pH of colloidal gold was adjusted to 8·2 with 0·01 mol l−1 K2CO3 solution according to the pre-experiment. Various amounts of McAb 2D11 (0, 5, 10, 15, 20, 25 and 30 μg) were added to 1 ml of colloidal gold at room temperature, respectively, and incubated for 10 min with shaking, followed by addition of 100 μl of 10% NaCl solution and shaking for 10 min. Two hours later, the absorbance of each solution was measured spectrophotometrically at 520 nm, and the amount of protein showing maximum absorbance was confirmed as the minimum amount of McAb needed to prevent the colloidal gold from aggregating. The optimal amount of McAb used for conjugation was 20% excess of the minimum amount of McAb.
The purified McAb 2D11 (180 μg) was added to 10 ml of dispersed colloidal gold solution and agitated for 30 min. After coating, the reaction mixture was further stabilized by adding 2·5 ml of 10% bovine serum albumin (BSA) (Sigma-Aldrich Co. LLC, USA) with moderate stirring for 20 min and then stored overnight at 4°C. Following the colloidal gold–McAb probe centrifuged at 30 000 g for 30 min at 4°C, the supernatant was removed and the resulting precipitate was resuspended in 0·01 mol l−1 PBS containing 1% BSA and then centrifuged again to remove free unconjugated antibody. The pellet was resuspended in 1 ml storage buffer (0·01 mol l−1 PBS containing 1% BSA, 5% sucrose, 0·5% Tween-20 and 0·02% NaN3) and stored at 4°C prior to use.
The average diameter and dispersion of colloidal gold particles and colloidal gold–McAb probe were estimated by transmission electron microscopy (TEM). A drop of colloidal gold solution or colloidal gold–McAb conjugates was dropped on a carbon-coated TEM copper grid and allowed to air dry for 15 min, and then colloidal gold particles were observed by TEM, while colloidal gold–McAb conjugates were measured after negative staining with uranyl acetate.
The colloidal gold solution and formation of colloidal gold–McAb probe were scanned by UV-visible (UV-vis) spectroscopy (200–700 nm) using a double-beam spectrophotometer. The colloidal gold solution was monitored for both conjugation and aggregation by measuring gold solution with: no antibody, immediately after addition of anti-LCDV McAb 2D11, and after centrifugation and resuspension of the colloidal gold–McAb conjugates in storage buffer.
The test strip consisted of four components assembled together on a plastic backing: the colloidal gold–McAb conjugate pad, NC membranes, sample pad and absorption pad. Colloidal gold–McAb conjugate pad was prepared by spraying colloidal gold-labelled McAb 2D11 on a glass fibre and dried at room temperature. Purified McAb 1A8 at 1 mg ml−1 and goat anti-mouse IgG (Sigma) at 0·5 mg ml−1 in 0·01 mol l−1 PBS were dispensed on NC membrane (Whatman Inc., Clifton, USA) with 5–8-μm pore size to form test line and control line, respectively. The distance between the two lines was about 5 mm. The membrane was then air dried, blocked with 2·5% BSA in 0·01 mol l−1 PBS at 37°C for 1 h and washed three times with PBST (0·01 mol l−1 PBS containing 0·05% Tween-20) and dried again at 37°C for 1 h. For test strip assembly, the NC membrane was pasted onto the centre of the plastic plate and covered with the colloidal gold–McAb conjugate pad and the absorption pad on each side. The sample pad receiving liquid sample to be analysed was attached to the other side of colloidal gold–McAb conjugate pad (Fig. 1). Then, they were cut into 3-mm-wide pieces and stored desiccated at 4°C.
Flounder suspected of LCDV infection were detected by the developed immunochromatographic strip. About 100 μl of gill or fin supernatant fluids was pipetted onto the sample pad, or the sample pad of test strip was immersed into the test solution of which the surface must be lower than the conjugate pad, allowing the test solution to flow by chromatography through the NC membrane to the other end. The result was observed by naked eye within 10 min, and the presence of two red lines in control line and test line region indicated a positive reaction denoting the sample containing LCDV, while the presence of one red line in control line region suggested the absence of LCDV in the sample or below the limit of detection for the strip. If the red control line did not appear, the test was invalid.
The indirect enzyme-linked immunosorbent assay (ELISA) and dot-blotting as control methods were also performed on these supernatant fluids. LCDV-infected flounder and LCDV-free founder were used as positive and negative controls, respectively.
For ELISA, the 96-well EIA plates (Costar, Corning, NY, USA) were coated overnight at 4°C with serial dilutions of purified LCDV (100 μl per well) and blocked with 3% BSA at 37°C for 1 h. After washing the wells three times with PBST, the anti-LCDV McAb was added to the ELISA plates and held at 37°C for 1 h and then washed three times with PBST. Goat anti-mouse immunoglobulin conjugated with AP (1 : 20 000) (Sigma) was applied and incubated at 37°C for 1 h and washed three times with PBST. Finally, the wells were incubated with freshly prepared pNPP (p-Nitrophenyl phosphate) substrate solution for 15 min, and the absorbance of the reaction product was measured at 405 nm with a Versa Max automated microplate reader.
For dot-blotting, serial dilutions of purified LCDV were made (4 μl) and the resultant LCDV was spotted onto NC membrane and air dried. The NC membrane was blocked with 10% BSA at 37°C for 1 h and washed three times with PBST. Next, the anti-LCDV McAb was added and held at 37°C for 1 h and then washed three times with PBST, followed by addition of goat anti-mouse immunoglobulin conjugated with AP (1 : 20 000) and incubation at 37°C for 1 h. After three washes with PBST, the colour reaction was developed with NBT-BCIP (nitro-blue tetrazonium chloridez—5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) substrate solution for 15 min in the dark as described by Zhan et al.(2004).
Purified LCDV was diluted with PBS to protein concentrations of 50, 10, 5, 1 and 0·5 μg ml−1 and used as test solution for the sensitivity, and the last dilution that yielded an observable positive result was regarded as the detection limit of immunochromatographic test strip. The diluted test solution was also used for ELISA and dot-blotting simultaneously as described above, and the tissue homogenate supernatant of LCDV-free founder served as negative controls.
To determine specificity of the test strip for LCDV detection, gill homogenates of LCDV-infected P. olivaceus, infectious hematopoietic necrosis virus (IHNV)-infected P. olivaceus and turbot reddish body iridovirus (TRBIV)-infected Scophthalmus maximus (Qingdao, China) were detected using the strip. Purified LCDV served as positive controls.
Positive and negative samples of LCDV were tested three times to determine the reproducibility of test strips. The same batch or three different batches of test strips were applied.
For stability, test strips were stored at room temperature and 4°C for up to 12 months and tested by positive and negative samples at 30-day intervals.
After gold nanoparticle formation, the colloidal gold solution had a ruby-red colour. Under TEM, the colloidal gold particles were well dispersed and uniform in size and shape with an average diameter of approximately 20 nm (Fig. 2). There was no obvious difference in the size distribution of colloidal gold before and after conjugation. Upon conjugation, TEM image showed a thin white layer, called ‘halo’ effect, surrounding the surface of the nanoparticles indicating coating of the gold with McAb 2D11 (Fig. 3), which was not present prior to treatment.
Spectra of the colloidal gold solution without antibody, immediately after the addition of antibody and after centrifugation and resuspension of the colloidal gold–McAb conjugate, were recorded (Fig. 4). An absorbance peak at 520 nm in curve a, with a high symmetry and narrow width, was observed owing to surface plasmon resonance of colloidal gold particles, indicating that the prepared colloidal gold was monodisperse and had narrow size distribution. Upon addition of antibody (curve b), the surface resonance band shifted a little into the red direction as a result of colloidal gold–McAb interaction and a peak at approximately 280 nm in the conjugation system arose. After centrifugation and resuspension of colloidal gold–McAb probe, two absorption bands was observed at 524 and 280 nm as shown in curve c, suggesting that the antibody was present on the gold surface. The intensity of the surface resonance band and protein absorbance band at 280 nm decreased because of the reduction of colloidal gold concentration and removal of the unconjugated McAb molecules in solution.
Various amounts of anti-LCDV McAb 2D11 were added to colloidal gold solution. The minimum amount of McAb required for conjugation with colloidal gold particles was determined based on the measured absorbance at 520 nm, showing a maximum absorbance at approximately 15 μg of McAb (Fig. 5). To ensure complete reaction with colloidal gold particles, the optimal amount of McAb 2D11 was chosen to be 120% of the minimum amount of McAb.
In test solution containing LCDV, the virus would bind to colloidal gold-conjugated McAb 2D11, and the resulting complex would be captured by the McAb 1A8 at the test line to give a red band. The unbound McAb conjugated with colloidal gold moved across the test line to be captured by the goat anti-mouse IgG and form a red band at the control line. Immunochromatographic strip test (ICT) showed that the serial dilutions of purified LCDV at protein concentrations of 1, 5, 10 and 50 μg ml−1 yielded positive results, so the detection limit of test strip was 1 μg ml−1 (Fig. 6). The same serial virus dilutions were examined by ELISA and dot-blotting simultaneously, and the sensitivity of ELISA (Fig. 7) and dot-blotting (Fig. 8) was found to be 1 μg ml−1.
Samples from flounder P. olivaceus suspected of LCDV infection were tested positive by ICT, which were in agreement with the detection results of ELISA and dot-blotting (data not shown).
Cross-reactivity studies using gill homogenates of LCDV-infected P. olivaceus, IHNV-infected P. olivaceus and TRBIV-infected S. maximus showed that positive signals were observed in the positive control and LCDV-infected P. olivaceus, but not in IHNV, TRBIV samples.
Lymphocystis disease virus viral suspensions and the LCDV-free flounder samples tested three times with the same batch of immunochromatographic test strips showed the same positive or negative results, respectively. Similar results were obtained for the three different batches of test strips.
The test strips were stable for at least 6 months at room temperature and 12 months at 4°C without changes in sensitivity (data not shown).
Gold immunochromatographic assay is a technology combining immunology with chromatography which has been developed for several years and applied increasingly in immunoanalysis diagnosis (Sithigorngul et al. 2007; Cui et al. 2008; Zhao et al. 2010). The quality of colloidal gold particles plays a key role in the development of a GICA-based one step lateral-flow strip for antigen analysis. Appropriate size and uniformity of the colloidal gold particles can result in good performance and yield more tests per litre (Carney et al. 2006). The size and hence the property of the gold nanoparticles can be controlled through the use of different reducing agents and the speed of reduction (Frens 1973; Morrow et al. 2009). In this study, colloidal gold in ruby-red colour was synthesized with the reduction of chloroauric acid by sodium citrate at pH 8·2. As shown in TEM images, the gold nanoparticles were well dispersed and uniform in size with diameter of approximately 20 nm, revealing they were of good quality and aggregate free. Additionally, the stability of gold–McAb conjugates is important for desirable biological reactivity and optimal performance of test strip (Thobhani et al. 2010). Our research showed that the developed step strip was stable for 6 months at room temperature and 12 months at 4°C without changes in sensitivity for LCDV detection, suggesting that the synthesized colloidal gold–McAb conjugates were stable which ensured the quality, stability and reproducibility of batches of test strip.
UV-visible spectroscopy is a powerful characterization tool, which can be used to monitor the conjugation of nanoparticles and indicate adsorption of proteins onto gold with a change in absorbance peak for the nanoparticles, and the intensity and position of spectrum peak of colloidal gold solution are related to the dispersity of nanoparticles (Thobhani et al. 2010). In the colloidal gold solution without antibody, an absorbance peak at 520 nm with high symmetry and narrow width was observed, showing that gold particles were monodisperse and had similar size, which was consistent with the results of TEM. Upon addition of antibody, the surface resonance band shifted a little suggesting the McAb conjugation, and a peak at approximately 280 nm arose for the absorbance from the tyrosine and tryptophan residues in the protein of McAb (Gole et al. 2002). UV-vis provided physical confirmation that the anti-LCDV McAb had bound to the nanoparticles with well dispersity and size uniformity.
Many serological methods have been developed for rapid and sensitive detection and diagnosis of fish viruses according to immunological principles, such as ELISA, dot-blotting, immunofluorescence assay test (IFAT) and immunohistochemistry (Olesen et al. 1993; Lorenzo et al. 1996; Falk et al. 1998; Shieh and Chi 2005; Fenner et al. 2006), and McAbs against unique antigens or epitopes of antigens have been widely used (Zhou et al. 2006; Reschova et al. 2007; Chen et al. 2008). As a highly effective and economical immunological tool, McAbs assure a reliable source of antibodies and often achieve greater specificity and sensitivity than polyclonal antibodies (Shi et al. 2003; Dixon and Longshaw 2005; Reschova et al. 2007). To avoid the possible cross-reactivity and low affinity of polyclonal antibodies, two clones of McAbs that recognized different surface epitopes of LCDV (Cheng et al. 2006) were chosen to develop the colloidal gold immunochromatographic test strip for LCDV detection of fish. The test strip gave a detection limit of 1 μg ml−1 of LCDV and was in good accordance with ELISA and dot-blotting in sensitivity and in LCDV detection. Moreover, ICT had several advantageous features such as simplicity, immediate result and the fact that it could be performed anywhere at any time without prior preparation of equipment. Therefore, the rapid ICT could augment the practical application for diagnosis of LCDV even in disadvantage areas and laboratories with limited technical expertise.
The affected fish with lymphocystis disease show typical pearl-like nodules on the skin and fins, and fibroblasts are proved to be the main target cells for LCDV replication (Cano et al. 2009). Thus, fish skin and fins were homogenized and used as test samples of suspected fish. Considering sensitivity of immunological methods is confined by viral extraction, suspected positive samples tested with the test strip may show negative results. It is necessary that the tiny infection samples should be further confirmed by cell culture, histological or molecular tests. Nevertheless, the strip test is still recommended for screening of subclinical infection or greater intensity infection of LCDV to differentiate from other pathogen infections.
This study was supported by National Natural Science Foundation of China (grant 31172429, grant 31072232) and the National Science and Technology Supporting Program (grant 2012BAD17B01).