Production and diagnostic applications of monoclonal antibodies against porcine circovirus
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(2) 260. fã Á;æBÁ"sVÁßÆÁBÁ;'. þ'b PCV¢ 6"Î "æöBê ß ªçÃç ¾ ÷Ò' ²Ò &V>æ p~ [2, 32]. ~æò "ö F¶îöB ²ÎW Ãçj ßûb ~º F¶î*W²ÎWÃê(postweaning multisystemic wasting syndrome: PMWS) ¶¾ öB ¾rb { B ê , F#, Ò j j jj ¾¢öBê >î [7, 10, 17, 18, 20]. î÷f 6ê' Ú76², ^ ¦, n., JÒ Ò ~ ªçÃçj ßûb ~ /W B~ ãÖ öÒ N 10% ;ê¾ ãÖöº 50%ræ . î÷ö 6"B "æ~ Gn ÷æbº â*. «&, G j«W *", Ë", *îW öZ &VB . " ö PMWS Ãçj ¾æÚº "æ~ çöB PK-15 ^ "öB F¾ PCVf F*' Ò ö'b B circovirus& æ³'b ªÒ> {Nb B îÚ PCV _f PCV æ« î÷ö 7º j ~º ©b rJr [4]. PMWS Ãçj ¾æ Úº "æ¦V ªÒB PCV *öº 96% ç~ " VB çÿWj ~¾~ genotypej ;W~º > , PK-15 ^"öB F¾ PCVfº 80% ~~ " VB çÿWj B :ʪ {> î [13, 24]. F*¶ f öW~ N¢ :ûb PMWS Ãçj ¾æÚº "æ¦V ªÒB îÚ PCV ªÒ"º PCV-2, Ò PK-15 ^"ö B F¾ PCVº PCV-1b ««>î [13, 24]. PCV-2¢ {~V *B *7;7Ú»(indirect immunofluorescence test: IIF) [3, 4], çz"ï »(immunohistochemistry assay: IHC) [22, 27], çÚv Ç»(in situ hybridization: ISH) [22, 27], Ò 7Î ²»(polymerase chain reaction: PCR) [12, 14] ~ &æ ê O» BB>Ú ÒÏ> ® . ß® PMWS &N PCV strainö ~ î÷ B 5 ªö & ¢ >¯~V *Bº PCV-1" PCV-2¢ 6ê > ®º type-specific .Ó' ê» BB j>' b º> ® . öBº PCV-1" PCV-2¢ 6ê > ®º ' '~ typeö & ß Ú¢ Ö~ ßWj «~, PCV-2ö ~ î÷~ êö ®ÚB Ú~ ÏWj {~¶ þj >¯~& .. Òò 5 O» :Êf ^ þö ÒÏB PCV-1 (PCV-1 6"B PK-15 ^ "; ATCC-CCL33)" PCV-2 (ISU-31 strain)º '' ã>~"¦ööB ª·Aj &" ÒÏ~& .. ISU-31 strainf PCV-1 J">Ú ®æ pf "æ Ë ^"(PCV-1 free PK-15 cell line)¢ ÒÏ~ à Vb PK-15 ^º 10% ²j.Ó(fetal calf serum: FCS, Invitrogen, Carlsbad, USA)" B& FB alpha minimum essential medium (α-MEM, Invitrogen)¢ ÒÏ ~ 5% CO2, 37oC~ zÛê ~öB V·~& . Ú Öj *~ ^[ö ÒÏB myeloma ^ º BALB/c mouse F¾ z^ SP2/0 ^¢ Ï ~&b 10~20% FCS& F>Ú ®º RPMI 1640 V æ¢ ÒÏ~ :Ê V·^f ?f ~öB V·~& . :Ê~ V· 5 B>ªÒ [ V·B PK-15 ^¢ trypsinj Ï~ ²z Î ê 10% FCS FB α-MEMö ¦FÊ PCV2¢ 1.0 m.o.i& >ê 7«~& . 1* O ê PCV2& 6"B PK-15 ^¢ 5×104 cells/ml~ ³ê . ~ ^V·Ï flaskö I 5% CO2, 37oC~ V·V öB 18* ÿn V·~& . 18* V· ê, V· j B~ 300 mM D-glucosaminej Î&~ 37oCö B 30ª ÿn >wV . >w  ê D-glucosamine j B~ α-MEMb ^¿ r 10% FCS& F>Ú ®º α-MEMj I ?f ~öB 48 * ÿn V·~& . Mouse ö ÒÏ PCVº 6 "B çV·j ÒÏ~ Tischer ~ O» [32] ö V¢ B> ªÒ~& . *7;7Ú»(indirect immunofluorescence test: IIF)ö ÒÏ plateº 0.1 m.o.i PCV-2& 6"B PK-15 ^¢ well 5×104 cells Î& ê *f ?f O»b D-glucosamine ¾Ò~ V·~ j^Êb ; ê -20oCö &~B j º ÒÏ& . .PVTF ^[ö ÒÏ mouseº 4-6 "_~ BALB/c strain j ÒÏ~&b ö ÒÏ öf B> ªÒ PCV-1, PCV-2 Ò baculovirusöB B*B PCV-2~ ORF2 Wî [1]j ÒÏ~& . ''~ ö (50 µg/ml) j ÿï~ Freund's complete adjuvantf ¾ b Ê mouse ; Ú 1N 7«~, 2" ê ?f öj Freund's incomplete adjuvantf ¾ b~ ?f ã 2N 7«~& . 2" ê, 2N 7«" ?f O»b 3N 7«~, 2" ê ; b¦V j.~ ''~ : Êö & Ú&¢ IIF»b G;~& . Ú && ^[~Vö Ϫ ãÖöº ;BB öò j ; b 7«~ 3¢ ê jË^ j ~ ^[j ~& . Ú&& Ôj ãÖöº 3N.
(3) "æ z:Êö & Ú Ö 5 ê' wÏ. 7«" ?f O»b º& 7«~& . *f ? ; Ú Êº O»" ÷¯~ mouse V*j »~V *~ Coyle [9]" Orlikf Altaner [26]~ O »j >;~ ?f ¢_~ BALB/c mouse~ footpadö 3¢ *Ïb 4² 7«~&b 1Nº Freund's complete adjuvantf 2N¦Vº Freund's incomplete adjuvantf b öj ÒÏ~& . Ú Ö 5 ·W IZCSJEPNB FB ^[f ''~ öb B mouse~ jË^ 6º BBâ*.¦V &j ^f SP2/0 ^¢ polyethylene glycol (PEG) 1500j ÒÏ~ ¢>' ^ [ O»ö V¢ ¯~& [16, 19]. ^[  b ^f 20% FCSf HAT (50 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine)& FB ^ V·ö ¦FÎ r 96-well çV· plateö well 100 µl O ª"~ 37oC, 5% Özê² ~ö B V·~, V· ê 3, 5, 7 5 9¢ö îÚ Væ v~~& . Hybridoma& well~ 30% ç Ã~&j r (^[ ê 10-15¢) PCV-1" PCV-2ö &~ ß 'b >w~º ·W hybridoma¢ Fê~& . PCV1" PCV-2ö ß'b >w~º Ú¢ Ö~º · W hybridomaº IIF»b r" ? FB~& . ¯, PCV-1" PCV-2 '' 6"Î 96-well plate¢ : Ê 6" ê 48*ö 80% acetoneb 10ª* ; Ê ê ¦ï hybridoma V· ç[ 100 µl ¢ ''~ :Ê 6"Î wellö IÚ 37oCöB 1* ÿn >wV . 1* >w ê, PBS 3² ^ ¿~ FITC-conjugated goat anti-mouse IgG +IgM (Jackson ImmunoResearch Laboratories, West Grove, USA)j Î&~ 30ª ÿn ?f NêöB >w V . 30ª ê plate¢ PBS 3² ^¿~ ;7* ãb &V~ ;7j ¾æÚº well~ hybridoma¢ PCV ß Ú Ö^" 6;~& . '' ~ PCVö &~ Ú¢ Ö~º ©b {B hybridomaº limiting dilution»b 2² ç ~ ¢ b {B hybridoma~ ¢¦º Úî² ö &Ë~ ¢¦º ÃB pristaneb Ò 6·B BALB/c mouseö ; Ú 7«~ Ϫ ·~ > & ;W>îj r >¢ j~ -20oCö &~. þö ÒÏ~& . Ú ßW « (1) Isotype { ÖB Ú~ isotypef Ig isotyping kit (SigmaAldrich, St. Louis, USA)¢ Ï~ BÒ~ ÒÏ F. 261. ö V¢ hybridoma V· ç[j ÒÏ~ β »b {~& . (2) WßW { Úf PCV-2 W"~ >wWj {~V * ~ Towbin [34]~ O»b western blotj r" ? ~& . ¯, PCV-2/ORF2 F*¶ Ò baculovirus 6"B Sf9 cell lysate¢ sample buffer (60 mM Tris-HCl pH 6.8, 2% SDS, 25% glycerol, 1.44 mM 2-mercaptoethanol, 0.1% bromophenol blue)f 1:5~ jN DÚ 100oCöB 5ª* y denaturationV . ¢ 10% SDS-polyacrylamide gelöB 2* ÿn 80V * V'ÿj r gelj ªÒ~ transfer buffer (15.6 mM Tris base, 120 mM glycine, pH 8.1-8.4)ö 30ª* æV . Ò transfer bufferö æB ¹f filter paper f nitrocellulose membrane (NENTM Life Science Products, Boston, USA) Òö gelj IÚ semi transblot (Bio-Rad laboratories, Hercules, USA)¢ Ï~ 13VöB 20ª* *V . Nitrocellulose membranej 5% non-fat dry milk/tris buffered saline (10 mM Tris-HCl, 150 mM NaCl, pH 7.5: TBS)öB ~!J ÿn blocking Î ê TBS ;~² z 3² ^¿~& . Blocking  membranef Z strip ; . ê ¦Ò~ ¶ ~º Ú~ V· ç[f C~æ p, Ò >º 1:500-1:2,000b C~ 1* ÿn. NöB z >wÊ TBS 3² ^¿~& . 2 N Ú alkaline phosphatase-conjugated goat antimouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, USA)j 1:2,000b C~ 1* >wÎ Ê TBS 3² ^¿~& . BïB NBT/BCIP stock solution (Kirkegaard & Perry Laboratories) 200 µlj alkaline phosphatase buffer (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 5 mM MgCl2) 10 mlö DÚ blottingB Wî~ Bïö ÒÏ~& . (3) "æ :Ê "~ >wNZ Ò ÖB Úf "æ :Ê ¯, "æ V^VÃê:Ê, "æ2:Ê, "æ æ:Ê Ò "æ *"W*Ë":Êf~ v N>w NZf *7;7Ú»b {~& . (4) Ú~ ç ÚöB~ >wW Ò ÖB Ú~ ç ÚöB~ >wWj Ò~ V *~ ªçÃç 5 PCR»b PCV-2 6" { B "æ~ çj Ï~ *7;7Ú»" ç z"ï»(immunohistochemistry assay: IHC)j ~.
(4) fã Á;æBÁ"sVÁßÆÁBÁ;'. 262. & . *7;7Ú»f PCV-2 6" "æ~ çj j ~ ÿÖç B(TBSTM; Triangle Biomedical Sciences, Durham, USA) ê -20oCöB ÿÖ B 6 µm vþ ÿÖ.Þj ò PBST(0.05% Tween 20, 0.01M phosphate buffered saline, pH 7.2) ^¿~ ÿÖ j B ê Ú~ j ß' Öj V B~V *~ 3% BSA-PBS 30ª* blocking ¾Ò~ & . 1N Ú PCV-2 ß Ú¢ 30ª* > wVb, 2N Ú FITC-conjugated anti-mouse IgG+IgMj >wV . IHC¢ *~ PCR»b PCV2 6" {B "æ~ çf 10% neutral buffered formalin Ïb 48* ÿn ;Î ê ¢>' O»ö ~~ ç j &j~& . "ï *ö ç .Þf xylenej ÒÏ~ wax¢ B~&b 0.5% " Öz>²& F>Ú ®º methanol 30ª* ¾Ò~ ç Ú~ peroxidase~ Wj B~ 5% normal goat serumb 30ª* ¾Ò~ Ú~ j ß' Öj VB~& . Vö 1N Ú PCV-2 ß Ú ¢ NöB 30ª* >wÎ ê 2N Ú biotinylated goat anti-mouse antibody (Vector laboratories, Burlingame, USA) 30ª* >wÎ ê *öBf ?f O»b ^ ¿~ ABC reagent(avidin DH-biotinylated horseradish peroxidase complex, Vector laboratories)¢ NöB 30 ª* >wÎ ê Bï (0.6 mg/ml DAB, 0.03% H2O2, 0.01M PBS, pH 7.2)b 5-10ª* >wÎ ê Ã~> ^¿~ haematoxylinb & "ï~& .. Ö. ". Ú~ Ö B> ªÒ PCV-1b Î mouse¢ ÒÏ~ ^[j >¯~ 6B~ PCV-1 ß Ú¢ Ö~& . 6 9B~ PCV-2 ß Ú 7 4B ~ Úº B> ªÒ PCV-2¢ öb, Ò 5B~ Úº baculovirusöB B*B PCV2~ ORF2 F*¶ Öbj öb ÒÏ ©î . PCV-1 ß Úº IIF»b PCV-1 6" PK-15 ^öBò ;7 ¾æ > PCV-2 ß Úº PCV-2 6" PK-15 ^f PCV-2/ORF2 F*¶ Ò baculovirus& 6"B Sf9 ^öBò ;7 ¾æ Ò (Fig. 1). Ú~ ßW ÖB Ú~ ßWf Table 1" ? . PCV-1 ö & Ú 6B 7 4B~ isotypef IgMîb IgG1" IgG2a& '' 1BO î . PCV-2 ß . Fig. 1. Reactivity patterns of PCV-2 specific monoclonal antibody, 4H10 by indirect immunofluorescence (IIF) test. PCV-2 specific monoclonal antibody, 4H10 was reacted with: A; PCV-1 infected PK-15 cells, B; PCV-2 infected PK-15 cells, C; mock infected Sf9 cells, D; PCV-2/ORF2 recombinant baculovirus infected Sf9 cells.. Ú 7 IgA, IgG2a, IgG2b& '' 1B, 2B 5 1B ¾æÒ 5Bº IgMb ¾æÒ . PCV-1ö ß' 6B~ Úº Îv PCV-1b 6"Î PK-15 ^öBº IIF»b ·W>w ¾æÒb¾ PCV-2 6"Î PK-15 ^f PCV-2~ ORF2 F*¶ Ò baculovirus 6"Î Sf9 ^ öBº rW>wb ¾æÒ . 6 Îv PCV-2 6" {B "æ çöBê *7;7Ú» 5 çz"ï»bº Îv rW>wb ¾æÒ . >ö PCV-2 ß Úº PCV-1b 6"B PK-15 ^öBº rWb ¾æÒb¾ PCV-2 6" Î PK-15 ^f PCV-2~ ORF2 F*¶ Ò baculovirus 6"Î Sf9 ^öBº ·W>wb ¾ æÒ . ÖB Úf PCV-2 W"~ >wWj { ~V *~ PCV-2/ORF2 F*¶ Ò baculovirus¢ ÒÏ~ western blotting¢ Ö", PCV-1 ß Úº Îv rWb ¾æÒb PCV-2 ß Ú~ ¢¦º ·Wb ¾æÒ (Table 1, Fig. 2). ÖB Úf "æ :Êf~ >wW j IIF»b Ò Ö"º Table 2f ? . PCV-1 ß Úº PCV-1 ~ :Êfº > wj ~æ p~b PCV-2 ß Ú 6 PCV2 ~ "æ :Êfº >wj ~æ p~ ..
(5) "æ z:Êö & Ú Ö 5 ê' wÏ. 263. Table 1. Characterization of monoclonal antibodies (MAbs) against porcine circovirus (PCV) Reactivity by the following tests MAb 4E2 7A10 7E9 10E8 14F11 15C9 1A11 1B5 3C2 4C7 4F2 4H10 6C6 7B4 7G2. 1. Immunogen. 2. Route. IIF3. Isotype PCV-1. PCV-2. Sf9 cell. Cryo. WB4. IHC5. PCV-1. IP IP IP FP FP FP. IgG2a IgM IgG1 IgM IgM IgM. +6 + + + + +. − − − − − −. − − − − − −. − − − − − −. − − − − − −. − − − − − −. PCV-2. IP IP IP FP. IgA IgG2b IgG2a IgM. − − − −. + + + +. + + + ++. NT NT NT ++. − − + ++. NT7 NT NT ±. PCV-2/ORF2. FP FP FP FP FP. IgM IgG2a IgM IgM IgM. − − − − −. + + + + +. + ++ + ++ +. + ++ + NT ++. ++ − ++ − +. ± ± ++ NT ++. 1. PCV-1: porcine circovirus type 1, PCV-2: porcine circovirus type 2, PCV-2/ORF2: recombinant baculovirus-expressed ORF2 protein of PCV-2. 2 IP: intraperitoneal inoculation, FP: foot-pad inoculation. 3 IIF: indirect immunofluorescence test using PK-15 cells infected with PCV-1 or PCV-2, Sf9 cells infected with PCV-2/ORF2 recombinant baculovirus (Sf9 cell), cryosection from PCV-2 positive porcine lymphoid tissue (Cryo). 4 WB: western blot analysis using PCV-2/ORF2 recombinant baculovirus infected Sf9 cell lysates. 5 IHC: immunohistochemistry assay using PCV-2 positive porcine lymph node and lung tissues. 6 −: negative, ±: weak positive, +: positive, ++: strong positive. 7 NT: not tested.. Ú~ ê ÏW ÖB Ú~ PCV-2 êö ®ÚB Ï & ËWj Ò~V *~ PMWS ªçÃç 5 PCR»b PCV-2 6" {B "æ~ çj Ï~ IIFf çz"ï»j Ö", 2B~ Ú 6C6" 7G2º ·W &.Ób ÒÏ polyclonal antiPCV-2 .Ó" ÿ¢~² çz"ï»b PCV2 6"^¢ ß'b ¦Â~& (Fig. 3). 6 3B ~ Ú 4C7, 4H10, Ò 7G2º IIF»b PCV2 6" öç ^¢ { ~² ¦Â~& (Fig. 4).. . Fig. 2. Western blot analysis of PCV-2 specific monoclonal antibodies. M: molecular weight marker, 1: PCV-2 positive serum, 2: PCV-2 negative serum, 3: MAb 4C7, 4: MAb 6C6, 5: MAb 3C2, 6: MAb 1A11, 7: MAb 1B5.. V. Porcine circovirus 2 (PCV-2)º F¶î*W²ÎW Ãê(postweaning multisystemic wasting syndrome: PMWS)j ¾æÚº "æ¦V Û'b {>Ú PMWS¢ FB~º 1N' öÚ rJ^ ®b, "öº PMWS öê "æb¦"ÃÃê(porcine dermatitis and nephropathy syndrome: PDNS), ÃWZ.
(6) fã Á;æBÁ"sVÁßÆÁBÁ;'. 264. Table 2. Reactivity patterns of monoclonal antibodies (MAbs) with other porcine viruses by indirect immunofluorescence (IIF) test MAb 4E2 7A10 7E9 10E8 14F11 15C9 1A11 1B5 3C2 4C7 4F2 4H10 6C6 7B4 7G2 1. Reactivity with the following viruses by IIF1 PCV-1 PCV-2 PRRSV PPV + + + + + + − − − − − − − − −. − − − − − − + + + + + + + + +. − − − − − − − − − − − − − − −. − − − − − − − − − − − − − − −. PRV TGEV − − − − − − − − − − − − − − −. − − − − − − − − − − − − − − −. Reactivity patterns of MAbs were confirmed by IIF test using cultured cells infected with porcine circovirus type 1 (PCV-1), porcine circovirus type 2 (PCV-2), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine rotavirus (PRV) and transmissible gastroenteritis virus (TGEV).. Fig. 4. Confirmation of PCV-2 infection by indirect immunofluorescence (IIF) test using PCV-2 specific monoclonal antibodies. Lung tissues infected with PCV-2 were reacted with PCV-2-specific monoclonal antibody 4C7(A) and 7G2(B). Arrow indicates PCV-2 infected cells by IIF test.. Fig. 3. Immunohistochemical staining of porcine lymph node infected with PCV-2. Lymph node infected with PCV-2 was reacted with PCV-2-specific monoclonal antibody 6C6(A) and 7G2(B).. ÒWöZ(proliferative and necrotizing pneumonia: PNP), FÂWê*Ã(congenital tremors) 5 ®Ë · ;~ î÷" &N ®º ©b º;> ® [11, 15]. 6 PCV-2ö 6">Ú ®Úê PMWS~ Ãçj ¾æÚæ pº &ªç; ;~ 6" Ò~V r^ ö ªç¦Ò 6º PCV-2~ {òbº PMWS~ ê Ϫ~æ pf ©b rJ^ ® . V¢B î ÷j ê~º öº *»" ?f PMWS~ *;' ªçÃçj {~ ªçÃç {B 6" "æ~ ËVöB ß÷æj {~ ÷æöB PCV-2¢ { ~º ©j j>'b º~ ® [28]. PCV-2¢ {~º O»b PCV-2 Ö~ Ò¢ ë~º 7 βê>w»" ç Ú hybridization» Ò ç Ú PCV-2 ö~ Ò¢ ë~º çz" ï»" *7;7Ú» BB>Ú ÒÏ> ® [3, 4, 12, 14, 22, 27]. ß® ç Ú PCV-2 öj {~ V *~ çz"ï»" *7;7Ú»ö Ò.
(7) "æ z:Êö & Ú Ö 5 ê' wÏ. Ï>º Úº PCV-1" B vN>wj ~æ p PCV2f ß'b Ò ¸f affinity >w > ®º ßWj æî ®Ú¢ . V¢B, þöBº PCV1" PCV-2~ 6ê 5 ¦ï»j BB~V *~ PCV1" PCV-2ö ß'b >w~º Ú¢ Ö ~ ~ ßWj «~ êö Ï &ËWj Ò~& . Ú¢ Ö~V *~ mouse¢ ÊV *~ B> ªÒ PCV-1" PCV-2¢ Milstein" Kohler ~ O» [19]ö &~ intraperitoneal route '' 7« ʺ O»" Coyle [9]" Orlikf Altaner [26]~ O »j >;~ ?f öj foot-pad 7«Êº O» j ÷¯~ ^f myeloma~ [j Ö ", 2&æ O»ö ®ÚB PCV-1" PCV-2ö ß' Ú Ö^~ WjNöº. ¦ N& ìîb¾ foot-pad 7«Î mouse¢ ÒÏ~ ^ [j ãÖ WB Ú~ isotypef &¦ª IgM typeb ¾æÒ . º Ú W";öB . Vöº IgM type " ªj>¾ êö ;~ isotypeb æ~~º foot-pad 7«Î mouse~ ã Ö, Î ê 12-14¢ö ^[j ~ Vöº jç ôf ^öB class switching ¢Ú¾æ pf F " IgM type WB ©b ÒòB . 6 intraperitoneal route 7« ãÖ, mouse ö 'Úê 6"~ V* ²º>º > foot-pad 7« ãÖöº ^[ ræ 2" Ϫ~ 4"~ V* j » > ®î . ÖB Ú¢ B>ªÒ~ ;BB Ú ÒÏ r IgG typef " protein A columnj Ï~ B> ;B~&º "öº IgM typeê IgG type" îR&æ B>~² ;B > ®º * O» [25] BB>Ú Ú¢ Ö~º mouse¢ foot-pad 7«B ʺ O» ; Ú Êº O» Î"' ©b 'B . öB ÖB 6B~ PCV-1ö & Ú (4E2, 7A10, 7E9, 10E8, 14F11, 15C9)º Îv PCV-1b 6"B ^öBº ·Wb ¾æÒ . ~æò PCV2 6"B ^, PCV-2/ORF2 F*¶ Ò baculovirus 6"B Sf9 ^ Ò PCV-2/ORF2 F*¶ Ò baculovirus 6"B Sf9 cell lysate¢ Ï western blot öB Îv rWb ¾æÒ 6, PCV-2 6" { B "æ~ çöB *7;7Ú»" çz" ï»b Îv rWb ¾æ¾ . Ú& Î v PCV-1ö ß' ©j { > ®î . >ö PCV-2ö & 9B~ Úº PCV-2ö 6"B ^ f PCV-2/ORF2 Ò baculovirusö 6"B Sf9 ^ öB ·Wb ¾æ > "æ :Ê 5. 265. PCV-1ö 6"B ^öBº >w ¾æ¾æ pj . Îv PCV-2ö ß' ©b {>î . V¢B . Úº PCV-1" PCV-2¢ 6ê~ j ¦ Â~º FÏ~² ÒÏF © . 9B~ PCV-2 ß Ú 7 5B(3C2, 4C7, 4F2, 6C6, 7G2)º PCV-2/ ORF2 F*¶ Ò baculovirus 6"B Sf9 cell lysate ¢ Ï western blotöB «{ band¢ { > ® îb¾ 4B(1A11, 1B5, 4H10, 7B4)~ ÚöBº ÚÆ bandê { > ìî . ß® 2B~ Ú(4H10, 7B4)~ ãÖ PCV-2/ORF2 F*¶ Ò baculovirus 6"B Sf9 ^öB Ö ; ·W>w j ¾æîb¾ western blotöBº rWb ¾æ © f .ç~æ á Ö"î . º Úö ~~ æ>º epitope& western blotj *~ sample j ¾Ò r ç~ æz¢ .¾~ z ç Úö ~~ æ>æ á~V r^b º; > ® . f FÒ Ö"º Ö:Ê nucleocapsid Wîö & Ú¢ Ï öªC þö B B ' ® [6]. conformational epitope ö V æzº 6"B ^¢ [35S]cysteine¾ [35S]methionineb æ ê immunoprecipitation O» j ÒÏ~ Ú¶ ;ê ÖF > ®j ©b ÒòB . ÷W6; &¦böB &æ ^ 6º :Ê öj ß' "6~² ¦Â~V * ê» B Bö Ú& wÏ>B ¢ Ï Î² », *7;7Ú» Ò formalin ; çj Ï çz"ï» ~ ' êO» &æ ^W 5 :ÊW î÷ êj *~ BB> î [8]. McNeilly [22, 23]f PCV-2ö ß' Ú¢ Ö~ ~ ßWj «~ Ú¢ Ï~ PMWS¢ ê > ®º in situ hybridization»" çz"ï»j jv ~& b PCV-2¢ ¦Â > ®º ELISA»j BB~& . öBê ÖB Ú~ PCV-2 êö ® ÚB Ï &ËWj Ò~V *~ ªçÃç 5 PCR »b PCV-2 6" {B "æ~ â*." öç j Ï~ çz"ï»j Ö", 2B~ Ú 6C6" 7G2º £*~ j ß>wj ¾æî b¾ PCV-2 6"^¢ Î"'b ¦Â~& . ©f ·W &Ú ÒÏ polyclonal anti-PCV-2 .Ó" ~ ÿ¢ >&b PCV-2 6"^¢ ¦Â~ öB ÖB Ú& PCV-2 6" êBB FÏ~² ÒÏF > ®rj B~& . ß® PCV-2 ê ö ·W &Ú ÒÏ> ®º polyclonal antiPCV-2 .Ój òº PCV-2~ Ãf j>' . ~ æò PCV-2¢ Ãʺ ÒÏ>º PK15 ^~ &.
(8) fã Á;æBÁ"sVÁßÆÁBÁ;'. 266. ¦ª PCV-1b J" >Ú ®Ú PCV-2¢ B>~² Ãʺ ôf ^B6 &v> ® . F öB Ö PCV-2 ß Úº polyclonal anti-PCV-2 .Ój & > ®º. ¦ ~~¢ &æ ® . >ö 3B~ Ú (4C7, 4F2, 4H10)º PCV-2 6"^f >wf ~&b¾ "à ^öB j ß' >w ~² ¾æ¾ ê « {~æ p~ . V¢B Ú¢ PCV-2 6" êBB ÒÏ~V *ö j ß' >wj ²z > ®º O» ÖF J>Ú¢ © . 6 ? f PCV-2 6" öçj Ï~ *7;7Ú»b PCV-2 6"^¢ { Ö", 3B~ Ú (4C7, 4H10, 7G2)º £*~ j ß>wöê ®~ PCV-2 6"^¢ { ~² ¦Â~ PCV-2 6" êBB FÏ~² ÒÏF > ®rj { > ®î . Ú¢ Ï PCV-2 ê»j Ïz ~V * Bº ôf ¢ PCV-2 ~ &¦bö &~. ê»"~ "6ê 5 ßW~ jv & jº~ , ¢ *~ öB ÖB Ú 7G2¢ ÒÏ~ çz"ï»" *7;7Ú»b PCV-2¢ {~º & *Ò ê¯7 .. Ö. . F¶î*W²ÎWÃê~ öÚ rJê "æ z:Ê .Ó; 2 (PCV-2)º Úº b * ^ê 'b "æö B~ ·î ÖëöB. ¦ b¢ .¾~ ®º ©b rJ^ ® . öBº PCV ö ß'b >w~º Ú¢ Ö~ ~ ßWj «~, PCV-2 ê» BBö wÏ~& . "æ z:Ê .Ó; 1 (PCV-1), PCV-2 6º baculovirusöB B*B PCV-2~ ORF2 Wî Î mouse¢ ÒÏ~ ¢>' Ú ÖO»j ¢¦ >;~ ^[j ~ PCV-1" PCV-2ö '' ß' Ú¢ Ö~º hybridomaº *7;7 Ú»j Ï~ FB~& . ÖB 16B~ Ú 7, 6Bº PCV-1ö ß' ©îb 9Bº PCV-2ö ß' ©î . 6 Úº Îv þö ÒÏ "æ :Êfê >w~æ pj '' PCV-1" PCV-2ö ß' Úªj { > ®î . ¢¦ PCV-2 ß Úº PCV-2 6 "B "æ çöB *7;7Ú» 5 çz" ï»b PCV-2f ß'b >w~& . ç~ Ö" Ú" r öB ÖB Ú f PCV-1" PCV-2~ 6ê 5 ÷W6;ö FÏ ~² ÒÏF ©b ÒòB .. ^^ò 1. fã , ;ÎF, fÒî, ;'. ÚöB ªÒB " æ z:Ê 2;~ F*¶ "VB ªC 5 B *. &:ʲæ. 2003, 33, 151-160. 2. Allan, G. M., McNeilly, F., Cassidy, J. P., Reilly, G. A., Adair, B., Ellis, W. A. and McNulty, M. S. Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pigs fetal material. Vet. Microbiol. 1995, 44, 49-64. 3. Allan, G. M., McNeilly, F., Kennedy, S., Daft, B., Clarke, E. G., Ellis, J. A., Haines, D. M., Meehan, B. M. and Adair, B. M. Isolation of porcine circoviruslike viruses from pigs with a wasting disease in the USA and Europe. J. Vet. Diagn. Investig. 1998, 10, 3-10. 4. Allan, G. M., McNeilly, F., Meehan, B. M., Kennedy, S., Mackie, D. P., Ellis, J. A., Clark, E. G., Espuna, E., Saubi, N., Riera, P., Botner, A. and Charreyre, C. E. Isolation and characterization of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland. Vet. Microbiol. 1999, 66, 115123. 5. Bassami, M. R., Berryman, D., Wilcox, G. E. and Raidal, S. R. Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, and chicken anemia virus. Virology. 1998, 249, 453-459. 6. Choi, K. S., Nah, J. J., Ko, Y. J., Choi, C. U., Kim, J. H., Kang, S. Y. and Joo, Y. S. Characterization of antigenic sites on the rinderpest virus N protein using monoclonal antibodies. J. Vet. Sci. 2003, 4, 57-65. 7. Clark, E. G. Post-weaning multisystemic wasting syndrome. In Proceed of the American Association of Swine Practitioners. pp. 499-501. 1997. 8. Constantine, N. T. and Lana, D. P. Immunoassays for the diagnosis of infectious Diseases. In Manual of clinical microbiology, pp. 218-233, 8th ed. ASM press, Washington, DC, 2002. 9. Coyle, P. V., Wyatt, D., McCaughey, C. and O'Neill, H. J. A simple standardised protocol for the production of monoclonal antibodies against viral and bacterial antigens. J. Immunol. Methods. 1992, 153, 81-84. 10. Daft, B., Nordhausen, R. W., Latimer, K. S. and Niagro, F. D. Interstitial pneumonia and lymphadenopathy associated with circoviral infection in a 6 week-old pig. In 39th Meeting of the American Association Veterinary Laboratory Diagnosticians. p.32. Little Rock, 1996..
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