Received 3 March 2004; accepted 25 May 2004 Keywords: Shrimp; WSSV; Mabs; ELISA; Immunodot blot; Alkaline phosphatase

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1 Aquaculture 239 (2004) Elimination of shrimp endogenous alkaline phosphatase background and development of enzyme immunoassays for the detection of white spot syndrome virus (WSSV) Wenbin Zhan a, *, Xiaojie Wang a, Jing Chen a, Jing Xing a, Hideo Fukuda b a Laboratory of Pathology and Immunology of Aquatic Animals, LMMEC, Ocean University of China, Qingdao , China b Laboratory of Fish Pathology, Tokyo University of Fisheries, Tokyo , Japan Received 3 March 2004; accepted 25 May 2004 Abstract Enzyme-linked immunosorbent assay (ELISA) and immunodot blot assay, using monoclonal antibodies against white spot syndrome virus (WSSV) as primary antibody, and goat anti-mouse Ig serum conjugated with alkaline phosphatase (AP) as secondary antibody, were developed to detect WSSV in shrimp tissue samples. However, false-positive results were obtained and the cause was considered to be reaction of endogenous shrimp AP with ELISA and immunodot blot substrates. Four AP inhibitors were tested at different concentrations and treatment times to solve this problem. EDTA, NaHSO 3, levamisole and HEPES Na inhibited AP activity by 96.45%, 86.82%, 68.82% and 9%, respectively. In conclusion, sample pretreatment with 0.5 M EDTA eliminated shrimp endogenous AP background in ELISA and immunodot blot assays, where AP was used for detection. D 2004 Elsevier B.V. All rights reserved. Keywords: Shrimp; WSSV; Mabs; ELISA; Immunodot blot; Alkaline phosphatase 1. Introduction White spot syndrome (WSS) has caused the most severe economic loss in cultured shrimp in the past 10 years (Lightner, 1999; Hossain et al., 2001). Thus, it is very * Corresponding author. Tel.: ; fax: address: wbzhan@mail.ouc.edu.cn (W. Zhan) /$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi: /j.aquaculture

2 16 W. Zhan et al. / Aquaculture 239 (2004) important to have specific, accurate, rapid and simple diagnostic methods for its detection. Enzyme-linked immunosorbent assay (ELISA) and immunodot blot assays have been previously described (Poulos et al., 1994; Lu et al., 1996; Lightner, 1999). In developing similar try to use anti-wssv monoclonal antibodies (Mabs) (Zhan et al., 1999), we encountered false-positive results when using supernatant of shrimp tissue as samples. It was likely that the false-positive results were caused by endogenous shrimp peroxidase (POD) or alkaline phosphatase (AP) with activity similar to that of the horseradish POD or AP conjugated to the secondary antibody. In a previous publication, we succeeded in inhibiting activity of shrimp endogenous POD and eliminating its disturbance in WSSV detection (Zhan et al., 2003). Here, we tested four reagents for inhibition of shrimp endogenous AP activity that interfaced with ELISA and immunodot blot assays for WSSV based on detection by AP-conjugated secondary antibody. 2. Materials and methods 2.1. Preparation of samples Moribund shrimp (Marsupenaeus japonicus, mean weight 6.1 g) with obvious cuticular white spots were collected from shrimp farms in Shandong Province. Healthy shrimp (M. japonicus, mean weight 8.5 g) were collected from a shrimp farm in Hebei Province where no viral disease had been observed, and these shrimps showed no signs of virally induced lesions. Gills of morbid and healthy shrimp (20% w/v) were homogenized separately for 20 min on ice in glass homogenizers containing phosphate-buffered saline solution (PBS, 2.7 mm KCl, 0.13 M NaCl, 1.5 mm KH 2 PO 4, 8.1 mm Na 2 HPO 4, ph 7.4). Homogenates was centrifuged at 3000 g for 15 min at 4 jc and supernatants were collected and stored at 80 jc. A semi-purified virus solution was prepared from haemolymph of experimentally infected moribund shrimp. Briefly, haemolymph was centrifuged at 2000 g for 10 min at 4 jc, after which the supernatant fluid was added to a discontinuous sucrose gradient of 35%, 50% and 60% (w/v) and ultracentrifuged with CP 100MX Hitachi Preparative Ultracentrifuge (Hitachi. Japan) at g for 90 min at 4 jc. The virus band was collected by puncturing the tube with a syringe needle, and dialyzed overnight in PBS at 4 jc before storage at 80 jc until use (Zhan et al., 1999) Antibodies A mixture of six anti-wssv Mabs (hybridoma culture fluid) was used as the primary antibody (Zhan et al., 1999) and goat anti-mouse Ig serum conjugated with AP (Sigma) was used as the second antibody Inhibition of shrimp AP activity Supernatant solution from healthy shrimp homogenates (25 Al) was mixed with 25 Alof each inhibiting reagent solution in a 96-well microplate. The reagents included EDTA

3 (0.01 M, 0.1 M, and 0.5 M), NaHSO 3 (0.01 M, 0.1 M, 1 M), levamisole (0.01 M, 0.1 M, 1 M) and HEPES Na (0.01 M, 0.1 M, 1 M). Each treating reagent and concentration was tested separately for 0.5, 1 and 2 h. Then, 100 Al p-nitrophenyl phosphate ( p-npp) substrate (Sigma) (1 mg/ml) in the carbonate buffer solution (CBS, 0.05 M Na 2 CO 3, 0.5 mm MgCl 2, ph 9.8) was added into each well and allowed to react for 10 min before color development was stopped by addition of 50 Al 2 M NaOH. The absorbance was then measured at 405 nm using Emax precision microplate reader (Molecular Devices, USA). The supernatant solution (25 Al) in 25 Al PBS was used as the control. This experiment was repeated three times ELISA Six groups of samples were experimented by ELISA procedure. The purified virus solution diluted from 1:100 to 1: in PBS (original concentration 5.23 Ag/ml) was used as the positive control (Group 1). The supernatant solution from gills of moribund and healthy shrimp homogenates was diluted from 1:1 to 1: in PBS (original concentration 20%, w/v). Each sample was divided into two aliquots, to one was added an equal volume of 0.5 M EDTA for 1 h, while an equal volume of PBS was added to the other as control (Groups 2 5): supernatant solutions from moribund shrimps were untreated (Group 2) and were pretreated with EDTA (0.5 M for 1 h) (Group 3) separately, supernatant solutions from healthy shrimps were untreated (Group 4) and pretreated with EDTA (0.5 M for 1 h) (Group 5) separately, and PBS solution was used as the negative control (Group 6). Each sample from the six groups above was placed (100 Al) in a polystyrene microtiter well and the plates were covered and incubated overnight at 4 jc. Wells were washed three times with PBS containing 0.05% Tween 20 (PBS-T) and then blocked with 200 Al 10% albumin bovine for 1 h at room temperature, before washing with PBS-T again. After that, 100 Al primary antibody (mixture of six anti-wssv Mabs) was added into each well and incubated for 1 h at 37 jc. Next, they were washed with PBS-T three times for 5 min before Mab binding was detected with goat anti-mouse Ig serum conjugated with AP (1:20000) for 1 h at 37 jc. After washing as above, they were washed finally two times with CBS. Reaction was developed with 100 Al substrate p-npp (1 mg/ml) in CBS for 15 min before color development was stopped by adding 50 Al 2 M NaOH and the absorbance was measured at 405 nm using microplate reader. The experiment was repeated three times Immunodot blot W. Zhan et al. / Aquaculture 239 (2004) The six groups of samples were same as for the ELISA tests. Supernatant solution was diluted 1:1, purified virus solution was diluted 1:100 and the pretreatment and the controls were the same as for the ELISA. First, each sample was spotted (2 Al) onto a nitrocellulose (NC) membrane in duplicate and air-dried. The NC membrane was blocked with 10% albumin bovine for 1 h at room temperature and then washed three times for 5 min with PBS-T. The membrane was incubated in primary antibody (mixture of six anti-wssv Mabs) for 1 h at 37 jc and washed

4 18 W. Zhan et al. / Aquaculture 239 (2004) Fig. 1. Inhibition of EDTA in shrimp AP activity. (1) treated 0.5 h; (2) treated 1 h; (3) treated 2 h. again as above. Mab binding was detected with goat anti-mouse Ig serum conjugated with AP (1:20000) for 1 h at 37 jc and washed three times with AP buffer (100 mm NaCl, 100 mm Tris HCl, 5 mm MgCl 2, ph 9.5). The reaction color was developed with freshly prepared substrate solution, 66 Al nitroblue tetrazolium (NBT) and 33 Al 5-bromo-4-chloro- 3-indolyphosphate (BCIP) (Sigma) in 10 ml AP buffer for 5 min. 3. Results 3.1. Inhibition of shrimp AP activity EDTA and NaHSO 3 gave clear AP inhibition with increasing concentration but gave little change in degree of inhibition with increasing time. For EDTA at 0.5 h exposure, increasing concentration (0.01 M, 0.1 M, 0.5 M) gave a sharp increase in inhibition (4%, 25%, 92%, respectively). For exposure of 1 h, inhibitions were 8%, 27%, 94%, and for 2 h, they were 17%, 36%, 96%, respectively. At the same concentration (0.01 M or 0.1 M or 0.5 M), increasing exposure time had little effect on the degree of inhibition (4%, 8%, 17%; 25%, 27%, 36% and 92%, 94%, 96%, respectively) (Fig. 1). The inhibition pattern for NaHSO 3 was like that of EDTA except that inhibition was weaker. At the exposure time of 0.5 or 1 or 2 h at concentrations of 0.01 M, 0.1 M, 0.5 M, inhibition was 2%, 58%, 83%; 3%, 61%, 84% and 10%, 68%, 87%, respectively (Fig. 2). HEPES Na (Fig. 3) exemplifies reagents that had a poor inhibiting effect. Treatment with 0.1 M for up to 2 h caused only 4% inhibition, and the highest inhibition was only 9% at a concentration of 1 M for 2 h. Fig. 2. Inhibition of NaHSO 3 in shrimp AP activity. (1) treated 0.5 h; (2) treated 1 h; (3) treated 2 h.

5 W. Zhan et al. / Aquaculture 239 (2004) Fig. 3. Inhibition of HEPES Na in shrimp AP activity. (1) treated 0.5 h; (2) treated 1 h; (3) treated 2 h. With levamisole at 0.01 M and exposure times of 0.5, 1, and 2 h, inhibition was 52%, 58%, 69%, respectively. When samples were treated with more than 0.1 M levamisole solution, a white opacity formed, eliminating it for use with the microplate reader ELISA As shown in Fig. 4, Group 1 gave the strongest WSSV-positive reactions and group 6 was properly negative. Group 2 showed a background reaction from shrimp endogenous AP, but this was inhibited by 94% with 0.5 M EDTA in Group 3. Group 4 gave a falsepositive WSSV result also because of endogenous AP, but this too was inhibited with EDTA (group 5), the background was eliminated, and the result was negative as in Group Immunodot blot The results of immunodot blot assay are shown in Fig. 5. Purified virus solution diluted 1:100 in PBS (line 1) was used as the positive control and PBS as the negative control (line Fig. 4. Six groups of samples were experimented by ELISA procedure. Group 1: the purified virus solution diluted 1:100 in PBS as positive control. Group 2: moribund shrimp supernatant diluted 1:1 in PBS. Group 3: moribund shrimp supernatant pretreated with 0.5 M EDTA for 1 h. Group 4: healthy shrimp supernatant diluted 1:1 in PBS. Group 5: healthy shrimp supernatant pretreated with 0.5 M EDTA for 1 h. Group 6: PBS as negative control.

6 20 W. Zhan et al. / Aquaculture 239 (2004) Fig. 5. Color development in the immunodot blot assay (repeated 2 times). Line 1: the purified virus solution diluted 1:100 in PBS as positive control. Line 2: moribund shrimp supernatant diluted 1:1 in PBS. Line 3: moribund shrimp supernatant pretreated with 0.5 M EDTA for 1 h. Line 4: healthy shrimp supernatant diluted 1:1 in PBS. Line 5: healthy shrimp supernatant pretreated with 0.5 M EDTA for 1 h. Line 6: PBS as negative control. 6). The color of moribund shrimp supernatant diluted 1:1 in PBS (line 2) was deeper because it combined reactions of shrimp endogenous AP and secondary antibodyconjugated AP with the substrate (NBT&BCIP). Pretreatment of the supernatant with 0.5 M EDTA (line 3) resulted in a lighter color, as EDTA inhibited the activity of endogenous AP. Healthy shrimp supernatant diluted 1:1 in PBS gave a false-positive background reaction due to endogenous AP (line 4) but this was removed by pretreatment with 0.5 M EDTA for 1 h (line 5). 4. Discussion EDTA gave the best inhibiting effect on AP activity, and sample pretreatment with 0.5 M was enough to eliminate false-positive backgrounds in ELISA and immunodot blots. In our previous paper, EDTA had a poor inhibiting effect on shrimp endogenous POD activity. Treatment with 0.5 M for 2 h caused inhibition of only 44%, while phenylthiourea (PTU) was the most effective with 90% inhibition at 10 mm for 2 h (Zhan et al., 2003). Therefore, choice of inhibitor is dependent on the detection enzyme and the corresponding endogenous shrimp enzyme. Enzyme immunoassays are widely used for diagnosis of viral diseases because of their high sensitivity and because they permit rapid processing of large numbers of samples. The use of ELISA for detection of shrimp viral diseases has been reported by several authors (Poulos et al., 1994; Shih et al., 2001). When the antigen is prepared virus, the indirect ELISA gives good results for diagnosis and for Mab screening, but false-positive results appear whereas shrimp tissue samples are used as the antigen (Poulos et al., 1994; Zhan et al., 2003). Besides the explanation that primary antibodies (Mabs) react nonspecifically with components in normal shrimp tissue (Lightner, 1999), our experiments also show that endogenous enzymes are able likely to cause falsepositive results. Use of endogenous enzyme inhibition could be expanded to other indirect enzyme immunoassays that employ shrimp tissue samples and employ enzyme systems for detection.

7 Acknowledgements W. Zhan et al. / Aquaculture 239 (2004) This work was supported by Projects under the Major State Basic Research Development Program (G ); NSFC ( , ); National 863 Project (819-Q-08); LMMEC (200211) and Cheung Kong Scholars Program in China. References Hossain, M.S., Otta, S.K., Karunasagar, I., Karunasagar, I., Detection of white spot syndrome virus (WSSV) in wild captured shrimp and in non-cultured crustaceans from shrimp ponds in Bangladesh by polymerase chain reaction. Fish Pathology 36, Lightner, D.V., The penaeid shrimp viruses TSV, IHHNV, WSSV, and YHV: current status in the Americas, available diagnostic methods, and management strategies. Journal of Applied Aquaculture 9, Lu, Y., Tapay, L.M., Loh, P.C., Development of a nitrocellulose-enzyme immunoassay for the detection of yellow-head virus from penaeid shrimp. Journal of Fish Diseases 19, Poulos, B.T., Lightner, D.V., Trumper, B., Bonami, J.R., Monoclonal antibodies to a penaeid shrimp parvovirus, infectious hypodermal and hematopoietic necrosis virus (IHHNV). Journal of Aquatic Animal Health 6, Shih, H.H., Wang, C.S., Tan, L.F., Chen, S.N., Characterization and application of monoclonal antibodies against white spot syndrome virus. Journal of Fish Diseases 24, Zhan, W.B., Wang, Y.H., Fryer, J.L., Okubo, K., Fukuda, H., Yu, K.K., Meng, Q.X., Production of monoclonal antibodies (Mabs) against white spot syndrome virus (WSSV). Journal of Aquatic Animal Health 11, Zhan, W.B., Chen, J., Zhang, Z.D., Zhou, L., Fukuda, H., Elimination of shrimp endogenous peroxidase background in immunodot blot assays to detect white spot syndrome virus (WSSV). Diseases of Aquatic Organisms 53,