Short communication
An assay for quantification of white spot syndrome virus
using a capture ELISA
Z J Chen1, C S Wang1,2and H H Shih1
1 Department of Zoology, National Taiwan University, Taipei, Taiwan
2 Department of Life Science, National University of Kaohsiung, Kaohsiung, Taiwan
Keywords: capture ELISA, diagnosis, quantification, WSSV.
White spot syndrome virus (WSSV) is the causative agent of a shrimp viral disease which has severely impacted most of the shrimp farming regions of south-eastern Asia and presently overshadows all other disease agents as the leading cause of production losses in Asia (Flegel 1997). Although WSSV has been known since 1993, viral quantifi-cation in infected animals was hindered by the lack of a continuous cell culture system for shrimp. Recently, an in vitro system using primary shrimp lymphoid cells was used for the quantification of the Chinese baculo-like virus (CBV, now known as WSSV) from the haemolymph and gill samples of experimentally infected Penaeus stylirostris (Tapay, Lu, Gose, Nadala, Brock & Loh 1997). A competitive polymerase chain reaction (PCR) method was developed for quantification of the WSSV genome and successfully applied in analy-sing changes in virus quantity in the haemolymph during the course of infection (Tang & Lightner 2000). A monoclonal antibody (MAb), designated 6E1, recognizing an epitope on a major envelope protein, VP 28 of WSSV, has been produced and characterized (Shih, Wang, Tan & Chen 2001). In the present note, we describe the development of a double antibody sandwich, i.e. capture
enzyme-linked immunosorbent assay (ELISA) combining MAb 6E1 with polyclonal antibodies affinity-purified from WSSV-immunized rat sera. The method was applied to monitor changes in con-centration of WSSV proteins in the haemolymph during the progression of white spot syndrome.
The WSSV used in this study was obtained from infected P. japonicus collected from Ilan in north-eastern Taiwan. The head soft tissues were homo-genized at 4 °C in TNE buffer [50 mm Tris, 100 mm NaCl, 1 mm ethylene diamine tetraacetic acid (EDTA), pH 7.4] and ultracentrifuged to purify WSSV as described previously (Shih et al. 2001). An aliquot of tissue homogenate was diluted with TNE buffer and used as the inoculum for the subsequent infection trial. The purified WSSV was assayed for protein using a protein assay reagent (BIORAD Laboratories, NY, USA). Adult Wistar rats were immunized and boosted 3 weeks later with 100 lg purified WSSV. Immunoglobulin G (IgG) was purified from pooled rat sera using a HiTrap protein A HP column (Pharmacia Biotech, Uppsala, Sweden). The MAb 6E1, isotyped to IgG1, was secreted and affinity-purified from mouse ascites according to the method of Harlow & Lane (1988). Purified antibodies were also assayed for protein concentrations.
A capture ELISA was designed using MAb 6E1 as the primary, i.e. capture antibody, and rat IgG as the secondary antibody. The 96-well enzyme immunoassay (EIA) plates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 100 lL well 1 of 6E1 diluted to 2.5 lg mL 1in coating buffer (50 mm sodium carbonate, pH 9.6).
Journal of Fish Diseases 2002, 25, 249–251
Correspondence Dr Hsiu-Hui Shih, Department of Zoology, National Taiwan University, Taipei, Taiwan
(e-mail: [email protected])
249 Ó 2002
The plates were then washed three times in phosphate buffered saline (PBS) with 0.1% Tween 20 (PBST) followed by blocking with 3% skimmed milk in PBST at 100 lL well 1, for 3 h at room temperature. The plates were drained and incubated overnight at room temperature with 100 lL well 1 of viral samples. All subsequent incubations were performed at 37 °C. After washing the plates three times in PBST, 100 lL well 1of purified rat IgG diluted to 0.15 lg mL 1in 3% skimmed milk in PBST was added and the plates were incubated for 1 h. The plates were then washed and 100 lL well 1 peroxidase-conjugated goat antirat IgG (Kirkegarrd & Perry Laboratories, Inc., Gaith-ersburg, MD, USA), 1/5000 in PBST, was added and the plates were incubated for a further 1 h. Finally, following washing, 100 lL well 1 of H2O2–OPD substrate (3.5 mm o-phenylene-diamine and 0.012% hydrogen peroxide in
35 mm citrate/150 mm phosphate, pH 5.0) was added. The optical density was read at 492 nm using an ELISA reader (DigiScan, ASYHITECH, Eugendorf, Austria) after 30 min incubation.
Serial dilutions of the purified WSSV containing known amounts of proteins were applied to the capture ELISA system. Optical densities at 492 nm read from the various concentrations of the purified WSSV were plotted against known concentrations of WSSV. This plot was linear between 5.00 and 42.40 ng WSSV lL 1(Fig. 1). A regression curve was thus plotted and was used as a standard curve to determine the concentration of WSSV proteins in infected shrimp and to monitor the changes in concentration during the progress of disease. Haemolymph samples were appropriately diluted such that the protein concentrations fell within the range of the relationship shown in Fig. 1.
A time-course infection trial was performed and an inoculum prepared as described above was injected into abdominal muscle of healthy specific pathogen free (SPF) P. vannamei (average weight 10.5 g). Two replicates of 30 shrimp were injected and two other populations of 15 shrimp were similarly injected with TNE buffer as controls. After injection, shrimp were kept in aerated glass aquaria and fed with commercial feed. Water temperature and salinity were 24–28 °C and 30–33&, respectively, throughout the experiment. Three shrimp from one injection replicate and one or two shrimp from one control replicate were harvested daily until 9 days post-injection (dpi). Haemolymph collected from individual shrimp was assayed using the capture ELISA. The concentration of WSSV protein within each sample was calculated from the standard curve. Additionally, mortality 0 0.1 0.2 0.3 0.4 0 10 20 30 40 50 WSSVconc. (ng µL–1) OD at 49 2 n m
Figure 1 A standard curve of WSSV concentrations for the capture ELISA. Optical densities at 492 nm (d) were read from the various concentrations of purified WSSV through the capture ELISA and were plotted vs. the known concentrations of WSSV protein.
Days post-injection
WSSV ng µL
–
1 hemolymph
Figure 2 The concentration of WSSV pro-teins in the haemolymph of Penaeus vanna-mei and the cumulative percentage mortality during experimental infection. The concen-trations of WSSV proteins were determined by capture ELISA from P. vannamei injected with tissue extract of the WSSV-infected shrimps ( ), or with TNE buffer as a con-trol (n). The cumulative mortality was ob-served from another infection replicate injected with tissue extract (d) or TNE buffer (s). Each point represents the average of three samples from the infection group.
Journal of Fish Diseases 2002, 25, 249–251 Z J Chen et al. Capture ELISA for WSSV
250 Ó 2002
was observed daily from the other infection and control replicates.
The concentration of WSSV proteins in haemo-lymph was quantified over the course of the experimental infection (Fig. 2). The haemolymph contained 5.2 ng lL 1of WSSV proteins at 10 h post-injection (day 0). Concentrations did not vary significantly within the first 4 dpi, but were then significantly elevated to 13.8 ng lL 1at 5 dpi. The highest concentration of 14.4 ng lL 1was reached on 6 dpi. Subsequently the concentration decreased to 5.6 ng lL 1at 9 dpi, which was very close to the initial concentration. The cumulative mortality observed in injected shrimp reached 70% at 7 dpi, one day later than the highest concentration of WSSV protein in haemolymph. Cumulative mor-tality was 6.7% in the control shrimp. The increase of WSSV concentration was thus consistent with the cumulative percentage mortality.
The presence of WSSV in shrimps pre- and post-injection was tested by a two-step PCR protocol (Lo, Leu, Ho, Chen, Peng, Chen, Chou, Yeh, Huang, Chou, Wang & Kou 1996) with DNA templates extracted from gill tissues using a GenomicPrep Cell and Tissue DNA Isolation Kit (Pharmacia Biotech, Sweden). The results of the analysis showed that the experimental batch of shrimps was WSSV-free before injection but that all became infected after injection.
The most useful method to detect and quantify antigens is the two-antibody sandwich assay, i.e. capture ELISA. The assay is quick and reliable and can be used to determine the absolute amounts of protein antigen by comparing the readings with a standard curve obtained using known amounts of pure antigen (Harlow & Lane 1988).
The major advantages of this technique are that the antigen (e.g. WSSV within the shrimp haemo-lymph in the present study) does not need to be purified prior to use and that the assays are very specific. The method provided the first rapid and specific test for the detection of epizootic haemato-poietic necrosis virus (EHNV) in cultured and clinical material (Hyatt, Eaton, Hengstberger & Russel 1991). Quantification was achieved by electron-microscopy particle counting and a viral titration method. The results indicated that an optical density value of approximately 0.14 in the EHNV capture ELISA could be obtained with as few as 400 virus particles in 50 lL of sample.
The present study demonstrated that a quan-tification assay of WSSV within unpurified
samples could be developed by using capture ELISA and the concentration of WSSV proteins in haemolymph corresponded to the cumulative mortality of the artificially infected shrimp pop-ulation. Although other methods are necessary to estimate the number of WSSV particles contained in the sample, capture ELISA makes it possible to detect concentrations of WSSV proteins as low as 0.1 ng lL 1 and monitor their changes within the shrimp population. The rate of increase in the WSSV genome was much higher in the tissues than in the haemolymph as assayed by compet-itive PCR (Tang & Lightner 2000), and the improved assay developed here will be applied to quantify WSSV proteins in tissue extracts of experimentally infected shrimp.
Acknowledgements
The present study was supported by the Fisheries Administration, Council of Agriculture under Grant 89-ST-1.2-FA-04(13).
References
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Tapay L.M., Lu Y., Gose R.B., Nadala E.C.B., Brock J.A. & Loh P.C. (1997) Development of an in vitro quantal assay in primary cell cultures for a non-occluded baculo-Iike virus of penaeid shrimp. Journal of Virological Methods 64, 37–41. Received: 16 July 2001
Accepted: 28 November 2001
Journal of Fish Diseases 2002, 25, 249–251 Z J Chen et al. Capture ELISA for WSSV
251 Ó 2002
