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Journal of Virological Methods 171 (2011) 141–148

Contents lists available at ScienceDirect

Journal of Virological Methods

journa l homepage: www.e lsev ier .com/ locate / jv i romet

etection of infectious myonecrosis virus using monoclonal antibody specific toand C fragments of the capsid protein expressed heterologously

reerat Kunanopparata, Parin Chaivisuthangkuraa, Saengchan Senapinb,c, Siwaporn Longyanta,ombat Rukpratanpornd, Timothy W. Flegelb, Paisarn Sithigorngula,∗

Department of Biology, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, ThailandCENTEX Shrimp, Faculty of Science, Mahidol University, Bangkok 10400, ThailandNational Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, ThailandCenter of Excellence for Marine Biotechnology at Chulalongkorn University, National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok 10330, Thailand

rticle history:eceived 19 June 2010eceived in revised form 14 October 2010ccepted 19 October 2010vailable online 26 October 2010

eywords:apsid protein

mmunohistochemistrynfectious myonecrosis virus (IMNV)

onoclonal antibodyenaeid shrimpenaeus vannamei

a b s t r a c t

The gene encoding the capsid protein in ORF1 of the genome of infectious myonecrosis virus (IMNV) (Gen-Bank AY570982) was amplified into three parts named CP-N (nucleotides 2248–3045), CP-I (nucleotides3046–3954) and CP-C (nucleotides 3955–4953). The CP-N fragment was inserted into expression vectorpTYB1 while CP-I and CP-C were each inserted into expression vector pGEX-6P-1 for transformation ofBL21 E. coli strain. After induction, intein-CP-N (84 kDa), glutathione-S-transferase (GST)-CP-I (60 kDa)and GST-CP-C (62 kDa) fusion proteins were produced. They were separated by SDS-PAGE and elec-troeluted before immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbsspecific to CP-N and one MAb specific to CP-C were selected for use for detection of natural IMNVinfections in Penaeus vannamei by dot blotting, Western blotting and immunohistochemistry. There wasno cross-reaction with shrimp tissues or common shrimp viruses including white spot syndrome virus(WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Penaeus monodon nucleopolyhedrovirus

estern blot(PemoNPV), Penaeus stylirostris densovirus (PstDNV) and Penaeus monodon densovirus (PmDNV). Thedetection sensitivities of the MAbs were approximately 6 fmol/spot of purified recombinant intein-CP-N protein and 8 fmol/spot of GST-CP-C as determined by dot blotting. A combination of all three MAbsresulted in a twofold increase in sensitivity over use of any single MAb. However, this sensitivity wasapproximately 10 times lower than that of one-step RT-PCR using the same sample. Immunohistochem-ical analysis using MAbs specific to CP-N and CP-C in IMNV-infected shrimp revealed intense staining

ymph

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. Introduction

Outbreaks of disease due to infectious myonecrosis virus (IMNV)ere first reported from Brazil in Pacific white shrimp Penaeus

Litopenaeus) vannamei in 2002 (Poulos et al., 2006; Pinheiro et al.,007). The outbreaks of infection spread to Indonesia in early006 (Senapin et al., 2007). IMNV is an unenveloped, icosahedralirus with a diameter of 40 nm. The genome consists of a single,ouble-stranded RNA molecule of 7560 bp comprising two open

eading frames (ORF1 and ORF2). The second half of ORF1 encodescapsid protein with a molecular mass of 106 kDa (Poulos et al.,

006). Mortalities from IMNV infection in cultivated P. vannameiange up to 70% (Andrade et al., 2008). Apart from P. vannamei

∗ Corresponding author. Tel.: +66 2 664 1000x8511; fax: +66 2 260 0127.E-mail addresses: paisarn@swu.ac.th, paisarn sithi@hotmail.com

P. Sithigorngul).

166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2010.10.015

oid organ, gills, the heart, hemocytes and connective tissue.© 2010 Elsevier B.V. All rights reserved.

infected naturally, two farmed shrimp species include P. monodonand P. (Litopenaeus) stylirostris were infected experimentally (Tanget al., 2005). However, no mortalities were reported for these lattertwo species. More recently, the wild shrimp Penaeus (Farfantepe-naeus) subtilis has also reported to be susceptible to IMNV infection(Coelho et al., 2009).

Gross signs of IMNV infection in P. vannamei include necro-sis in muscular tissues primarily in the distal abdominal segment.These are visible as opaque, whitish, discoloration. Histologicalexamination of tissues infection with IMNV reveals severe necrosisof muscle, fibrocytic inflammation and the appearance of cyto-plasmic inclusions and lymphoid organ spheroids. However, thediagnosis of infection with IMNV based on clinical signs and his-

tological examination is not adequate, since other pathogens suchas Penaeus vannamei nodavirus (PvNV) (Tang et al., 2007) and Vib-rio (Longyant et al., 2008a) and a number of other abiotic factorssuch as hypoxia, sudden changes in temperature or salinity canalso cause muscle whitening. A highly specific and sensitive in situ

1 Virolog

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ybridization method has been described for detection of IMNVnfection in shrimp tissues with no gross signs of IMNV infectionTang et al., 2005). Recently, more sensitive molecular methods foretection of IMNV infection were developed including RT-PCR andested RT-PCR assays (Poulos and Lightner, 2006), real-time RT-PCRAndrade et al., 2007) and RT-loop-mediated isothermal amplifi-ation combined with a lateral flow dipstick (Puthawibool et al.,009).

Although PCR-based methods have been used widely foretection of several shrimp viruses, they are not suitable forond-side detection by farmers. In contrast, simpler serologi-al based methods using monoclonal antibodies (MAbs) haveeen developed for detection of various shrimp pathogens suchs white spot syndrome virus (WSSV) (Poulos et al., 2001;nil et al., 2002; Chaivisuthangkura et al., 2004), Yellow headirus (YHV) (Sithigorngul et al., 2002), Penaeus monodon denso-irus (PmDNV) formerly called hepatopancreatic parvovirus (HPV)Rukpratanporn et al., 2005), Penaeus monodon nucleopolyhe-rovirus (PemoNPV) formerly called monodon baculovirus (MBV)Boonsanongchokying et al., 2006), Taura syndrome virus (TSV)Longyant et al., 2008b; Chaivisuthangkura et al., 2009), Penaeustylirostris densovirus (PstDNV) formerly called infectious hypo-ermal and hematopoietic necrosis virus (IHHNV) (Sithigorngult al., 2009), Vibrio harvyei (Phainphak et al., 2005), and V. algi-olyticus (Sithigorngul et al., 2006a). Although, serological basedethods have lower detection sensitivity than PCR methods,

hey provide a field-friendly, inexpensive way to detect and con-rm causative pathogens during disease outbreaks with highccuracy and in a manner (e.g., test strips) suitable for uset the pond side by unskilled personnel (Powell et al., 2006;ithigorngul et al., 2006b, 2007a,b). Since the complete genomicequence of IMNV is available (Tang et al., 2005), the aim ofhis study was to use the capsid protein of IMNV expressedeterologously to produce monoclonal antibodies for simple

mmuno-detection of IMNV. The ultimate aim was to use the MAbor further development of rapid immunochromatographic testtrips.

. Materials and methods

.1. Viral preparation

P. vannamei infected with IMNV naturally were obtained fromshrimp farm in the Situbondo District of East Java Province in

ndonesia. They were preserved in 95% ethanol at the farm andransported to Bangkok and verified as IMNV-infected by RT-PCRSenapin et al., 2007). The cephalothorax part of the shrimp wasxed further in Davidson’s fixative and processed for immunohisto-hemical analysis. Part of the abdominal muscle was homogenizedn 1% Triton X-100 in PBS (0.15 M phosphate buffered saline, pH.2) for dot blot and Western blot assays, and part of the abdominaluscle was used for nucleic acid preparation.Uninfected P. vannamei (∼15 g) verified by RT-PCR (Senapin

t al., 2007), were obtained from a farm nearby Bangkok and useds negative control in various assays.

.2. Nucleic acid extraction

Abdominal muscle from P. vannamei infected with IMNV wasomogenized in lysis buffer (50 mM Tris–HCl [pH 9], 100 mM EDTA,

0 mM NaCl, 2% SDS; Flegel pers. comm.), and nucleic acid wasxtracted from 200 �l of homogenate using a high pure viral nucleiccid kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) asescribed in the product manual. The extracted nucleic acid wastored at −70 ◦C.

ical Methods 171 (2011) 141–148

2.3. Cloning and expression of the IMNV capsid protein gene

The IMNV coat protein gene located in the ORF1 of IMNV genome(GenBank accession number AY570982) was amplified into threeparts called CP-N (nucleotides 2248–3045), CP-I (nucleotides3046–3954) and CP-C (nucleotides 3955–4953). For expression ofCP-N, the primers IMNVF22 (C CAT ATG ATT GTT TCA ATG GAA AATC) and IMNVR819 (G GAA TTC TTG TAG TGC AGT TGC TGG) withadded restriction sites (underlined) were used to amplify the CP-Nregion by PCR with Pfx polymerase (Invitrogen, Carlbad, CA, USA)using double stranded DNA prepared by GenScript Corporation (NJ,USA) as the template. The PCR protocol consisted of initial denatu-ration at 94 ◦C for 2 min followed by 35 cycles of 94 ◦C for 15 s, 57 ◦Cfor 1 min, 68 ◦C for 1 min and a final extension at 68 ◦C for 15 min.The PCR product was cloned into an expression vector pTYB1 (NewEngland, Biolabs, Hert, UK) at NdeI and EcoRI sites and transformedinto E. coli strain BL21 (DE3).

For expression of CP-I, primers IMNVF820 (CG GGA TCC GCT GCAAAA GAG GGT GCT CG) and IMNVR1728 (G GAA TTC TTG CAT TGAACT CCA CGA AAA C) with added restriction sites (underlined) wereused to amplify the CP-I region by RT-PCR with the Superscript One-step RT-PCR system (Invitrogen) using IMNV cDNA as the template.For cDNA synthesis, RNA extracted from IMNV-infected shrimpwas reverse transcribed using SuperScript III reverse transcriptase(Invitrogen), an IMNVR primer (G GAA TTC TTA TAC TGT TGC TGTCGC TTG), and Escherichia coli RNaseH. The PCR condition consistedof initial denaturation at 94 ◦C for 2 min followed by 35 cycles of94 ◦C for 15 s, 57 ◦C for 30 s, 68 ◦C for 1 min and final extension at68 ◦C for 15 min. The PCR product was cloned into the expressionvector pGEX-6P-1 (GE Healthcare, Björkgatan, Uppsala, Sweden) atBamHI and EcoRI sites and transformed into E. coli strain BL21.

For expression of CP-C, primers IMNVF1729 (CG GGA TCC GGTAGT ATT GCA CCA GCA ATG) and IMNVR (G GAA TTC TTA TACTGT TGC TGT CGC TTG) with added restriction sites (underlined)were used to amplify the CP-C region by PCR with Pfx polymerase(Invitrogen) using double stranded DNA, prepared by GenScriptCorporation (NJ, USA) as the template. The PCR condition consistedof initial denaturation at 94 ◦C for 2 min followed by 35 cycles of94 ◦C for 15 s, 57 ◦C for 1 min, 68 ◦C for 1 min and final extensionat 68 ◦C for 15 min. The PCR product was cloned into an expres-sion vector pGEX-6P-1 (GE Healthcare) at BamHI and EcoRI sitesand transformed into E. coli strain BL21. The integrity of the openreading frames of the three recombinant plasmids was verified byDNA sequencing.

2.4. Preparation of recombinant CP-N, CP-I and CP-C

Escherichia coli BL21 strain transformed with plasmids CP-N-pTYB1, CP-I-pGEX-6P-1 or CP-C-pGEX-6P-1 was cultured inLuria–Bertani (LB) broth to the exponential phase. Expressionof recombinant protein was induced with 1 mM isopropyl-�-d-thiogalacto-pyranoside (IPTG) for 4 h. After centrifugation at4000 × g for 20 min at room temperature, the bacterial pelletwas resuspended in a buffer containing 100 mM NaH2PO4, 10 mMTris–HCl, 8 M urea pH 8, and 1 mM phenylmethylsulfonyl fluo-ride (PMSF) and sonicated until a clear lysate was obtained. Thelysate was separated by 12% sodium dodecyl sulfate polyacry-lamide gel electrophoresis (SDS-PAGE). After treatment with 0.3 MKCl, recombinant fusion proteins called intein-CP-N, GST-CP-I andGST-CP-C were excised and collected in dialysis bags. Recombinantproteins were eluted with a Transblot apparatus (BioRad, Hurculis,

CA, USA) at 70 V for 6 h, dialyzed and concentrated using a vacuumconcentrator (Savant, Farmingdale, NY, USA). Protein concentra-tion was determined by the Bradford assay (Bradford, 1976). Therecombinant protein solutions were adjusted to 0.5 mg/ml, dividedinto small aliquots and stored at −70 ◦C.

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.5. Immunization

A solution combining intein-CP-N, GST-CP-I and GST-CP-C pro-eins (1:1:1 mixture) with complete Freund’s adjuvant in a 1:1 ratioas injected intra-peritoneally into four Swiss mice at 0.05 mg pro-

ein per mouse. Mice were injected subsequently with the sameroteins mixed with incomplete Freund’s adjuvant three moreimes at 2-week intervals. One week after the fourth injection,

ouse antisera were collected and tested against lysates of E. coliontaining intein, intein-CP-N, GST, GST-CP-I or GST-CP-C proteinsy Western blotting. The most-responsive mouse was boosted sub-equently 3 days before hybridoma production. The use and animalare were performed as indicated in “the guidelines on animal userotocol” under the regulation issued by the National Committeen Laboratory Animals, The National Research Council of Thailand.

.6. Production of monoclonal antibodies

A cell fusion protocol was adapted from the method devel-ped by Köhler & Milstein (1976) with modifications described byosmann et al. (1979). Spleen cells (∼108 cells) were collected

rom the immunized mouse and fused with P3X myeloma cells∼107 cells) with 40% polyethylene glycol. Fusion products fromne mouse were plated onto 30 microculture plates (96 wells/plate)n RPMI-1640 medium supplemented with 20% fetal calf serum and% HAT (Gibco, Grand Island, NY, USA). After 12 days, identificationf positive cultures by screening methods including dot blotting,estern blotting and immunohistochemistry were performed as

escribed below, hybridomas were cloned by the limiting dilutionethod and stored in liquid nitrogen.

.7. Specificity testing

.7.1. Dot blottingLysates of E. coli BL21 containing intein, intein-CP-N, GST, GST-

P-I or GST-CP-C and muscle homogenate samples (1 �l/spot) fromninfected or IMNV-infected shrimp were applied to nitrocelluloseembranes. These were baked at 60 ◦C for 10 min and incubated

n hybridoma conditioned medium from cultures diluted 1:20 in% Blotto blocking solution (1% nonfat dry milk, 0.1% Triton X-100

n PBS) for 4 h. After extensive washing in 0.1% blocking solution,he membranes were incubated in horseradish peroxidase-labeledoat anti-mouse gamma immunoglobulin heavy and light chain-pecific antibody (GAM-HRP, BioRad) at 1:1000 dilution for 4 h. Theembrane was then washed for 5 min in 0.1% blocking solution and

ncubated in a substrate mixture containing 0.03% diaminobenzi-ine (DAB), 0.006% hydrogen peroxide and 0.05% cobalt chloride inBS (Sithigorngul et al., 2002).

.7.2. Western blottingLysates of E. coli BL21 containing intein, intein-CP-N, GST, GST-

P-I or GST-CP-C and muscle homogenate samples from uninfectedr IMNV-infected shrimp were separated by 12% gel SDS-PAGEccording to the method described by Laemmli (1970). Samplesere electrophoresed for 3 h at 60 V and one part of the gel was

tained using Coomassie brilliant blue R-250. For Western blotnalysis, the samples resolved by SDS-PAGE were transferred ontoitrocellulose membranes using a Transblot apparatus (BioRad).itrocellulose membranes were incubated in 1% blocking solution

or 10 min and treated with MAbs or mouse anti-recombinant CProtein antiserum (preabsorbed with E. coli lysate containing intein

nd GST at 1:5000 dilution) for 4 h. After extensive washing in 0.1%locking solution, the membrane was incubated with GAM-HRP at:1500 dilution for 4 hr. The membrane was then washed exten-ively as before and incubated in a substrate mixture containing.006% hydrogen peroxide, 0.03% DAB, 0.05% cobalt chloride in PBS.

ical Methods 171 (2011) 141–148 143

2.7.3. ImmunohistochemistryCephalothoraces from alcohol fixed P. vannamei specimens

infected naturally with IMNV were fixed in Davidson’s fixativesolution for 24 h before processing for paraffin sectioning. Serialsections (8 �m thickness) of tissues were prepared and processedfor indirect immuno-peroxidase staining using MAb. Peroxidaseactivity was visualized by incubation with 0.03% DAB and 0.006%hydrogen peroxide in PBS. Preparations were counterstained withhematoxylin and eosin y (H&E), dehydrated in graded ethanolseries, cleared in xylene and mounted in Permount (Sithigorngulet al., 2002). Positive reactions were visualized as brown colorationagainst pink cytoplasm and purple nuclei.

2.8. MAb class and subclass determination

Classes and subclasses of the mouse immunoglobulins producedby hybridomas were determined by sandwich ELISA using MouseMonoAb ID Kit-HRP (Zymed Laboratories Research, San Francisco,CA, USA).

2.9. Cross-reactivity testing

Shrimp samples infected with PemoNPV, PmDNV, PstDNV,TSV, WSSV and YHV were processed for paraffin sectioningand immunohistochemistry using MAb specific to IMNV. Resultswere compared to those from MAbs specific to PemoNPV(Boonsanongchokying et al., 2006), PmDNV (Rukpratanporn et al.,2005), PstDNV (Sithigorngul et al., 2009), TSV (Longyant etal., 2008b), WSSV (Chaivisuthangkura et al., 2004) and YHV(Sithigorngul et al., 2002).

2.10. Sensitivity testing with recombinant IMNV capsid proteins

Purified CP-N-intein, GST-CP-I, GST-CP-C proteins were dilutedserially with PBS, spotted onto nitrocellulose membranes and pro-cessed for dot blotting using the IMNV-specific MAbs generatedin this study. The last dilution yielding a clear positive result wasdetermined.

2.11. Comparison of sensitivity between MAb and one-stepRT-PCR using IMNV-infected shrimp samples

The sensitivity of IMNV detection in shrimp infected natu-rally was determined using MAbs specific to CP-N or CPC or acombination of them. The IMNV-infected shrimp samples werehomogenized in 0.1% Triton-X-100 in PBS, twofold serially dilutedwith PBS and 1 �l of each dilution was spotted onto nitrocellulosemembrane and processed for dot blotting as described above. Thelast dilution of shrimp homogenate yielding a clear positive resultwas determined. The same shrimp homogenate was also dilutedtenfold serially with an uninfected shrimp sample extracted with0.1% Triton-X-100, and 1 �l of each dilution was tested for IMNVby one step RT-PCR (Senapin et al., 2007).

3. Results

3.1. Capsid protein gene cloning and expression

The expected PCR amplicons of 811, 924, and 1014 bp wereobtained for the CP-N, CP-I and CP-C genomic regions (Fig. 1).

Expression of intein-CP-N, GST-CP-I and GST-CP-C was visualizedby Coomassie blue staining and the expected bands of molecularmasses 84, 60 and 62 kDa, respectively, were obtained (Fig. 2A,lanes 2, 4 and 5). After the recombinant bands were cut fromthe gel, eluted and concentrated, highly purified fusion proteins

144 A. Kunanopparat et al. / Journal of Virological Methods 171 (2011) 141–148

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Table 1MAbs specific to N and C fragments of IMNV.

MAbs (isotype) Sensitivity (fmol/spot) Western blot IHC Specificity

FGo(

ig. 1. Ethidium bromide stained gels of the expected PCR amplicons correspondedo IMNV capsid protein fragments: (1) CP-N (811 bp), (2) CP-I (924 bp) and (3) CP-C1014 bp). M = DNA markers.

2–4 mg/10 SDS-PAGE) were obtained and they were used at.5 mg/ml protein for immunization.

.2. MAb production

After the fourth immunization with the combination of intein-P-N, GST-CP-I and GST-CP-C, all four mouse sera (1:5,000 dilution)eacted well to CP-N, CP-C and natural IMNV capsid protein butot with CP-I. Serum from one mouse demonstrated the strongest

ig. 2. SDS-PAGE and Western blot analysis of antiserum and monoclonal antibody speST-CP-I, (5) GST-CP-C and muscle homogenate from (6) uninfected or (7) IMNV infectedr transferred to nitrocellulose membranes and treated with (B) mouse anti-CP-IMNV anb) intein-CP-N, (c) GST, (d) GST-CP-I, (e) GST-CP-C, (f) IMNV capsid protein, and (h) hem

IMN7 (IgG1) 6 +++ +++ CP-NIMN12 (IgG2a) 6 +++ +++ CP-NIMC1 (IgG1) 8 +++ ++ CP-C

immunoreactivity and this mouse was used for hybridoma pro-duction. Approximately 1600 hybridoma-containing wells wereobtained and approximately 40 wells gave positive binding resultswhen screened initially with combination of E. coli lysates contain-ing CP-N-intein, GST-CP-I and GST-CP-C. Positive hybridoma cloneswere further screened by dot blotting and Western blotting againsteach E. coli lysate containing intein, GST, intein-CP-N, GST-CP-I orGST-CP-C and muscle homogenate samples from uninfected andIMNV-infected shrimp. Immunohistochemical analysis was carriedout using cephalothorax sections from IMNV-infected shrimp. TenMAbs specific to CP-N and five MAbs specific to CP-C were iso-lated and cloned to establish cell lines. No MAb specific to CP-I wasobtained.

All MAbs belonged to IgG classes. Only three MAbs, IMN7, IMN12(specific to CP-N) and IMC1 (specific to CP-C) gave good results withvarious immunoassays described above (Table 1) and they wereused for subsequent experiments.

3.3. Specificity of MAb

All MAbs bound to a single band either recombinant proteinintein-CP-N or GST-CP-C and to a band at 106 kDa in musclehomogenates from IMNV infected shrimp by Western blotting(Fig. 2C and D). Similar results were obtained by dot blotting. MAbsspecific to CP-N and CP-C bound intensely to E. coli lysates contain-ing intein-CP-N or GST-CP-C, respectively, and muscle homogenatefrom IMNV-infected shrimp. They did not bind to E. coli lysatecontaining intein, GST or GST-CP-I or to muscle homogenate fromuninfected shrimp (Fig. 3).

By immunohistochemical analysis, both MAbs specific to CP-N and CP-C showed strong immuno-reactivity in the muscle, gill,heart, lymphoid organ, connective tissue (Fig. 4) and intertubu-lar cells of the hepatopancreas (not shown) from IMNV-infectedshrimp, similar to that observed previously by in situ hybridization

(Tang et al., 2005). Therefore, immunohistochemical analysis canbe used to confirm IMNV infections in a manner similar to in situhybridization.

By immunohistochemical analysis, none of the three MAbsexhibited cross-reactivity to tissues from uninfected shrimp or

cificity. Lysates of BL21 E. coli containing (1) intein, (2) intein-CP-N, (3) GST, (4)P. vannamei were electrophoresed, then (A) stained with Coomassie brilliant bluetiserum or (C) MAbs IMN12 or (D) IMC1. M = standard marker proteins. (a) Intein,

ocyanin.

A. Kunanopparat et al. / Journal of Virolog

Fig. 3. Dot blot analysis of MAb specificity. Lysates of BL21 Escherichia coli con-taining (1) intein, (2) intein-CP-N, (3) GST, (4) GST-CP-I, (5) GST-CP-C and musclehomogenate from (6) uninfected or (7) IMNV infected P. vannamei were spotted(1 �l/spot) onto a nitrocellulose membrane and treated with MAbs (A) IMN12 or (B)IMC1.

Fig. 4. Immunohistochemical analysis of MAb specificity. IMNV-infected P. vannamei tissstained with eosin or (column B) stained only with hematoxylin and eosin. Strong immmuscles, (2) gills, (3) the heart, (4) connective tissue surrounding the midgut, and (5) the

ical Methods 171 (2011) 141–148 145

from shrimp infected with WSSV, YHV, TSV, PmDNV, PstDNV orPemoNPV (data not shown).

3.4. Sensitivity testing

Detection sensitivity of MAbs IMN7 and IMN12 determinedby dot blotting using purified IM-N-intein was ca. 500 ng/ml(500 pg/spot), and this was equivalent to 6 fmol/�l of antigen. Com-bination of MAbs IMN7 and IMN12 resulted in a 2 times increasein detection sensitivity for intein-CP-N (Fig. 5D column a), indicat-ing that the two MAbs bound to non-overlapping epitopes. Similar

results were also obtained by indirect immunoperoxidase ELISAusing the combined antibodies in plates coated with purified intein-CP-N (data not shown).

MAb IM-C1 showed similar detection sensitivity for purifiedGST-IM-C, ca. 500 ng/ml (500 pg/�l), which was equivalent to

ues were treated with MAbs IMN12 (column A) or IMC1 (column C) and counter-unoreactivities were exhibited similarly with both MAbs as brown staining in (1)lymphoid organ. Pictures in rows 1–4 are the same magnification.

146 A. Kunanopparat et al. / Journal of Virological Methods 171 (2011) 141–148

Fig. 5. Sensitivity of IMNV detection by dot blotting using MAbs against CP-N and CP-C, and PCR. Lysate of (a) BL21 E. coli containing intein-CP-N, (b) GST-CP-C and (c) musclehomogenate from IMNV infected P. vannamei were serially diluted with muscle homogenate from uninfected P. vannamei and spotted onto each square of nitrocellulosemembrane. The last square of each column was spotted with lysates of BL21 Escherichia coli containing (I) intein, or (G) GST or (U) muscle homogenate from uninfected P.v combs RT-PCd and tf

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annamei. Each nitrocellulose membrane was treated with a single MAb or with aame sample of IMNV-infected muscle homogenate was diluted and processed forilution factors of the bacterial lysates containing intein-CP-N (a) or GST-CP-C (b),rom IMNV infected P. vannamei (c).

fmol/spot (Table 1). It could be used to detect natural IMNV capsid

rotein with similar sensitivity to IMNV7 (Fig. 5C).

In binding of the MAbs to natural IMNV capsid protein by dotlot assay, MAb IMN7 showed slightly stronger immunoreactivity1:40) than IMN12 and IMC1 (Fig. 5A–C column c). Combina-ion of the two MAbs specific to CP-N and one specific to CP-C

ination of MAbs: (A) IMN7, (B) IMN12, (C) IMC1, (D) IMN7 + IMN12 + IMC1. (E) TheR. *The lowest limit of detection for each method. The numbers on the left side arehe numbers on the right side are the dilution factors of the muscular homogenate

resulted in a 2 times increase in detection sensitivity (Fig. 5D

column c).

To compare the sensitivity of IMNV detection by dot blottingwith that by one-step RT-PCR, the same shrimp homogenate extractused for dot blots (i.e., 0.1% Triton-X-100 extract) was used for theRT-PCR tests. A clear positive band could be observed at 1:1000

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ilution (Fig. 5E). Therefore, in comparison with one-step RT-PCR,he sensitivity of dot blotting using a combination of three MAbsas approximately 10 times less sensitive. In this case, the sensi-

ivity of dot blotting was close to that for one step RT-PCR. Nucleiccid extraction using a commercial kit as described in Section 2.2as not performed, since the idea was to compare the sensitivity

f both methods using the same sample preparation.

. Discussion

Three MAbs specific to N and C terminal fragments of IMNVapsid protein were generated, however, MAb specific to CP-I wasot obtained. Recently, MAbs specific to IMNV capsid protein wereenerated from recombinant His-tag IMNV corresponded to aminocid positions 300–527 of capsid protein (Seibert et al., 2010). Thisrotein fragment overlapped with the GST-CP-I fragment. Theon-responsive of mouse immune system to CP-I fragment maye due to the fact that immunogenicity of CP-I is less than that ofST, CP-N and CP-C. Since several attempts on immunization ofST-CP-I alone yielded only antibodies specific to GST but not toP-I. All MAbs obtained from this study could be used for success-

ul detection of IMNV infection by immunohistochemistry. Themmunoreactivity results are similar to that observed previouslyy in situ hybridization (Tang et al., 2005). Therefore, immuno-istochemical analysis can be used to confirm IMNV infections

n a manner similar to in situ hybridization. Long storage of theamples in ethyl alcohol (over two years) followed by re-fixationith Davidson’s fixative before processing seemed to have no

ffect on the immunoreactivity in immunohistochemical analysis.rolonged storage of shrimp tissues in Davidson fixative (10ays) did not have a deleterious effect on the in situ hybridizationeaction (Andrade et al., 2008). The level of sensitivity of MAbspecific to CP-N and CP-C determined by dot blotting using puri-ed recombinant proteins was approximately 500 pg/�l similaro that previously reported for the MAbs against VP19 of WSSV1.2–5 fmol/�l; Chaivisuthangkura et al., 2010), VP28 of WSSV500 pg/�l; Anil et al., 2002, and 625 pg/�l by Makesh et al., 2006),P3 of TSV (400–800 pg/�l; Longyant et al., 2008b) and capsid pro-

ein of PstDNV (300 pg/�l; Sithigorngul et al., 2009). The detectionimit of dot blotting using a combination of three MAbs againstP-N and CP-C was twofold higher than that of a single MAb. It

s our belief that the IMNV specific MAb generated in this studyan be used to develop a simple IMNV detection kit similar to themmunochromatographic strip used in WSSV and YHV detectionits, since all three MAbs bound to non-overlapping epitopes of thentigen.

cknowledgements

This work was supported by the National Center for Geneticngineering and Biotechnology (BIOTEC) Thailand to CENTEXhrimp, Mahidol University. The authors are also indebted to farm-rs in Indonesia who provided IMNV infected shrimp samples.

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