Production of monoclonal antibody to 45 kDa somatic protein of Trichuris suis

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(1)³¤¥Èó. * .* a. (2004) 44 4 Korean J Vet Res(2004) 44(4) : 625~635. æÞÏ~L%B]ö9îö&RV]ÚÖ. «ãÁB« Á*z> Á;« ÁJ;

(2). . . *Ê ¾ÿ>÷ö ]z¶ÿ>£®() :þzÒj() *Þ&L >~& (²Òß: 2004j 9ú 15¢) 1. 2. 3. 4. Production of monoclonal antibody to 45 kDa somatic protein of Trichuris suis Jong-Kyung Lee, Jong Tae Kim1, Hunsu Seo2, Jong-Yeol Park3 and Hee-Jeong Youn4* J.E.S., Seoul 153-811, Korea Evergreen Animal Hospital, Seoul 151-887, Korea 2 Pfizer Animal Health Korea Ltd., Seoul 143-210, Korea 3 Bayer Korea Ltd, Korea 4 College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea (Accepted= September 15, 2004) 1. Abstract : Trichuris suis does not excrete eggs during larval stage as well as in particular adult stage. It is impossible to diagnose by use of fecal examination method in those periods. Therefore, serological diagnostic method can be very useful for those stages. In order to produce monoclonal antibody, specific somatic and secretory-excretory (SE) antigens of T. suis were identified and analyzed by SDS-PAGE and Western blot. Monoclonal antibody-producing hybridoma cells were cloned, which were made of popliteal lymph node of BALB/c mice immunized with a 45 kDa somatic antigen of T. suis. Five clones (1B9, 2C4, 2C5, 2D7 and 2D8) showing strong responses to T. suis antigens were selected and the isotype identified. All monoclonal antibodies were IgG1 isotype and the light chains were κ chain. Established monoclonal antibodies reacted specifically to somatic and SE antigens of T. suis and did not cross-reacted to antigens of Ascaris suum, Trichuris vulpis, or Trichinella spiralis. The sensitivity of somatic and SE antigens against these monoclonal antibodies were significant (p<0.01) associated with those of positive and negative sera. Key words : Trichuris suis, somatic, secretory-excretory antigens, monoclonal antibody, western blot, antigen-capture sandwich ELISA. * . VÏj;(Subclass: Adenophorea [Aphasmidia]), ö* ÒÏ(Order: Enoplida), ÞÏ"(Family: Trichuridae), Þ Ï³(Genus: Trichuris)ö ³ [3]. "æÞÏ~ 6"Nf F#, æöB "æ FÏ ~ 7öB 50-70% ;ê ¸ ÒòÎNöBê £ 33% ~ ãB' ¶ j .¾~º ö B [19, 21]. "æ. "æÞÏ(Trichuris suis)f ^ê'b 6Ò ª~ " "æ~ Ë" ÖËö V~º ÏbB [9], ª ~'b ÿbê(Kingdom: Animalia), F;ÿb^ (Phylum: Nemathelminths), FÏ;(Class: Nematoda), 3. *Corresponding author: Hee-Jeong Youn College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea [Tel: +82-2-880-1267, Fax: +82-2-888-0659, E-mail: [email protected]]. 625.

(3) 626. «ãÁB«Á*z>Á;«ÁJ;. ÞÏf GW . jGî~ 50-70% 7öB BÒ> ®b , *;'b 21¢ JÒ¢ ¢bÚb j6"îö j Â~¢_ £ 5¢* æ> ~ v Ö ö&& 3 Ã&~ ß® "öº ·îË Vëz>º º^öB ¾æ¾º ãB' b& * ^ ê'b * 20Û ö ® [21]. ÖÒ¾¢~ ãÖ, *~ Vë ·îËöB~ ÞÏ 6 "Nf 4.1% >î [1]. ¾ 6"Nf Ï ¦ ¦Ò»b ¯ Ö" V r^ö Ï¦j VÂ ~æ pº W? ê~ 6"j 6n B 6" Nf R ¸j ©b ÒòB . º;j & Ë& ~º ¢fB >ö "v~ ×K BÎ îÒöB~ "æÞÏ 6"Nj 8 ¢ ÿn Ò Bö ~~ 6.7% &b¾ ¶îÒöB ¦Â>æ p~~ "æÞÏ Ï ¦ ¶îÒ : rb ÿ~º GWîÒöBº 20% ¾ ¦Â>î /W "æÞÏÃb >~ öÒ& B~& ~&, "æÞÏ ÷öW ; V ÏbB î² rJæ ® ~& [4]. 6 ~ã ^B ~ ×K BÎ îÒ& Ë~ B /W "æÞÏ 6" ² ^B>Ú zV r^ö BÎ ×K îÒ¢ ÖÒ¾¢ ¢3 · ¢~ ã Ö "æÞÏÃb öÒî ô B~& ~& [31]. ÞÏÃ~ ê»bº, ÞÒ~ ãB' F r ^ö *Òræ 6Ò ÒÏ> ®º Ï¦ ¦Ò O» ® b¾, O»f Ï¦ VÂ>æ pº V~ ê ®&Ë~ ã 6"ö ~ ¾æ¾º ÞÏ 6"Ã j ê~¾ 6ê êj ~º º ôf ÚJæ ®. . ãÖöº .Ó', ' O»j Ï ê » Ö FÏ~ [21]. *Ò "æÞÏ öò jî¢ çÏ(Taenia hydatigena), Neospora caninum, FÎÏ (Thichinella spiralis) ~ VÏ 6"Ãö &~ ' ê O»j Ï & ·~² > ® b, ö Î²G;»(enzyme-linked immunosorbent assay; ELISA)~ O» Ï> ®. [5, 11, 12, 23]. öBº W? ê¢ Î ê~ Þ Ï 6"Ãö &~ V~ ªæ ¦Ò»¾ .Ó' ê» z ;{~ FÏ îÚ ê»j BB ~¶ ~ V» j Ï~& . ¯, " æÞÏ~ Úöb B îÖÊ~ Òfâ*.~ ^ j îÖÊ >«^f [BB "æÞÏ Ú¢ Ö~& ELISAf Western blottingb ßWj Ò~&b, FÒ FÏ~f~ vN> w(cross-reaction) ¦¢ Western blottingb {~ & .. Òò 5 O». þÿ> BÞ&v þÿbÒGËöB ª·Af 4"_~ BALB/c mouse 6îÒf ³â¦ ã>~"¦öö B ª· Af 4~6"_~ ICR mouse 12 îÒ¢ ÒÏ~ & . Úö # ªj 8Jö j (1) "æÞÏ" "æ²Ï~ ö ãVê Â ê»ËöB ê»B "æ~ &Ëj .B ~ "æÞÏ(T. suis)" "æ²Ï(Ascaris suum)~ WÏ j ²>~ Greenspon [18]~ O»ö V¢ öj B~& . VöB áf "æÞÏ WÏ £ 400îÒ f "æ²Ï WÏ 5îÒº Úö(somatic antigens)j ò º Ï~& "æÞÏ WÏ £ 600îÒf "æ² Ï WÏ 25îÒº ªj . VJö(SE antigens)j áº ÒÏ~& . Hill (1994)~ O»ö ~~ B~&. [20]. -² ò öf ö ªCö ÒÏ~& . Ò îÖÊ j * "æÞÏ~ ö Bº Pupo (1999)~ O»ö ~~ Ö~ îÖÊö 7«Ê º ÒÏ~& [26]. ' VÏ~ ªj . VJöf 37 C, 5% CO V·V(Forma Scientific)öB penicillin (10,000 units/ml), streptomycin(25 µg/ml)" amphotericin B 5 Fungizone 0.85%(Gibco)& Î&B RPMI1640b V·~B 24* *Ïb Væ¢ ²>~& . 14 kDa ~ ª¶ïj <º bîj R"Êº >Rïj Ï ~ ²> Væ¢ 4 C, 3N Ã~>öB 12*î Ã ~>¢ :ÞÚ " 48* ÿn RCV . O»b 14 kDa V ~~ ª¶ïj <º bîj B~ ÿÖV(lyophilizer, âöï úæ îÚç)¢ Ï~ ³» ê öb ÒÏ~& . (2) BÞÏ" FÎÏ~ ö BÞÏ~ öf ãVê 7«ö ®º ³ËöB ê »Ò~ &Ëj .B~ BÞÏ~ WÏj ²> ê ç V~ O»ö V¢ öj B~&, FÎÏ~ öf BÞ&v >~"& VÏ v öB ê&~ ® º FÎÏb¦V B öj ÒÏ~& . (3) "æªæÚ~ öWî ªÒ "æÞÏj 'b 6"Î 14îÒ~ 6"î" 14îÒ~ Z6"î~ ªæj >~& . "æÞÏ~ 6 "V ¶Ïj F Ï¦j Ñ ®öº îÒ 500 B, Ò 1"¢ *Ïb 100BO ã 6"Î ê ªæ¦Ò¢ Û~ EPG 208-3569(1163+963)B~ Ï¦ . o. 2. o.

(4) æÞÏ~ 45 kDa ]öWîö &R V]Ú Ö. &VB 14B~ ªæþ2òj ·W &b ÒÏ ~& . Ï¦j 6"Êæ pf & 9îÒ 7 Ï¦ ¦ÒÖ" Ï¦ {>æ pf ªæþ2j rW & b ÒÏ~& . ªæ j 100 ml~ ELISAÏ ê jÏ(coating buffer; Na CO 1.59 g, NaHCO 2.93 g, Ã~> 1 liter, pH 9.6)" 10:1 DÚ ÚÒ¢ ¦Ú *çj ò ¢ 5,000ÜgöB 30ª* öªÒ ê ç[j >~ coproantigenb ÒÏ~& . Lowry protein assay [17]¢ Ï~ Wî ³ê¢ G ;~& ö ³ê& 1 µg/ml >ê ELISAÏ ê jÏb C~& . ELISA microplate(Nunc)~ ' wellö 100 µl O ª" ê ELISA¢ ~& . ö ;ï Lowry protein assay [17]¢ Ï~ "æÞÏ Úö ~ Wî ³ê¢ G;~&b SDS-PAGE~ ¾¢Þ gelç~ Z Úö Ú ®º Wî~ ³êº r" ? G;~& . ¯, bovine serum albumin(BSA)j 1 mg/ml~ ³ê C ©" *öB ò "æÞÏ~ Úöj SDS-PAGE~ BSA¢ standard ~ image analysis¢ Ï~ bandç~ Wî ³ê¢ Ö;~&. . Wî ³ê& 1.00 mg/ml& >ê B 3N Ã ~> C~ 7 0.2 ml(Wî ³ê 200 µg)¢ ~ îÖÊ " *7;7Ú»j ¯~V * öb ÒÏ~& . ö ªC 2. 3. 3. (1) Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) 12% acrylamide gel( 3.33 ml, 4 stock 2.5 ml, acrylamide 4.17 ml, 10% APS 50 µl, TEMED 5 µl) dual gel caster loading 4oC overnight . Separating gel , Pupo [26] 10 µl . marker prestained marker(Sigma) .. Ã~> Ü jò ê ö Ê ~ ïËöB V. f ê ~ O»ö ~~ *öB áf ' ö ¢ *V' ÿ~ ß' Z¢ {~& r ª¶ï ¢ ÒÏ~&. (2) Western blotting 10 µl 2 sample buffer 8 Pupo [26] TBS 3 BCIP/NPT phospatase substrate (KPL) .. ' ö ¢ Ü f DÚ ª* 7û ¾Ò Ê ~ O»ö ~~ Ú¢ ² ^¿ ê ¢ BïB Z¢ {~&. VÚ Ö (1) îÖÊ ;BB "æÞÏ~ Úö" complete Freund's. 627. ¢ ÿïb DÚ 0.1 ml O 2îÒ~ 5" _ îÖÊ~ ·ã êæ~ B: ö 1 ml "Ò V¢ Ï~ 1N 7«~&, 2" êö incomplete Freund's adjuvant(Gibco)¢ Df Úöj 2N 7«~ V . 2" êö Úö 0.1 ml¢ RÒ; ö "Ò~& . 6 ;BB "æÞÏ Úöj SDSPAGE~ 45 kDa Vö ~º 10B~ band(protein ~ · 150 µg)¢ >~ "æÞÏ" "æ²Ï~ ööBf ?f O»b ¾² ¦>Ú 2îÒ~ 5"_ BALB/c îÖÊ~ ·ã êæ~ B: ö 1 ml "ÒV¢ Ï~ 0.1 ml O 1N 7«~&, 2" êö ÿ¢ O»b ò öj 2N 7«~ V . 2" êö ÿ¢ O»b ò öj 3N 7«~&. . îæï 7« 3¢ êö ;V îÖÊ¢ ; ê 'FÎ* &~ RÒ; êÂ>ê Ê 26-gauge "Ò:¾" 1 ml "ÒV¢ Ï~ îÖÊ R Ò; öB .j j~& . . ö & *7 ;7Ú >w»j ~&b Ö"& ·W îÖÊ~ Òfâ*.(popliteal lymph node)j j~ [ö ÒÏ~& . *7;7Ú >w»j *Bº 100% methanol ^¿ MTS(multi-spot microscope slide)ö *öB & j Úöj 10 µl/well~ ·b smear ê V 7 öB ~ acetoneb 10ª* ; ê -30 CöB &~ ÒÏ~& . 6 öj êö~ ;Î ê & 7~ MTS slide¢ PBS(pH 7.4) 10ª* 3² ^ ¿~& . [ 10-15¢ ê [^& Ïª® ¶¦ well ~ V·j j~ 37 C, humidified atmosphere incubatoröB 45ª* >wÊ PBS 10ª* 3² ^ ¿~& . 2N Ú FITC-conjugated goat anti-mouse IgG(Jakson immunoresearch)¢ ÒÏ~&b PBS¢ Ï 1:160b C~ 37 C, humidified atmosphere incubatoröB 45ª* >wÎ ê PBS 10ª* 3² ^ ¿~& . -² &jB slideº mounting medium(0.5 M carbonate buffer:glycerol(1:9))b mounting~& ; 7*ãb ;7 FZ¢ &V~& . (2) >«^(myeloma cell) 5 feeder cell V· : Myeloma cell Ú Ö [^ ·Wö ÒÏ >« ^º P3-X63Ag14 ^ [ 2"*ö Ú î² öB âÚÚ Salah [29]f j [7]~ O»ö V¢ V·~&b, [ æ *¦V 8-azaguaninb ¾Ò~ òæ ^¢ B~& . ^>& 4Ü10 B& >ê C~ ¢ ^ [ö ÒÏ ~& . adjuvant(Gibco) BALB/c. o. o. o. 6.

(5) «ãÁB«Á*z>Á;«ÁJ;. 628. (3) Feeder cell. [^ ·W" (cloning)r ÒÏ feeder cell f 4-8"_~ ICR îÖÊ~ ; ö^, îÖÊ ¢ ~ 70% ethanol îÖÊ~ Ú¢ ²ë ê Z'b ¦~ b¦¢ B ï ¾ê Ê 18 gauge "Ò:¾j Ï~ HAT supplement(0.1 mM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine; Sigma)f 10% .Ó Î&B N&Ú DMEM(HAT DMEM) 10 ml¢ ; Ú "«~& . ¦¢ ¦# ² ^¿~ "« ;j ²>~ B Ã ~> 30.* .¢ ÏÎ ê 1,200 rpmb 5ª * öªÒ~ [ 5 ~ *ö 96-well microplateö 100 µl/wellO ª"~ V·~& . (4) ^ [ îæï 7« 4¢ ê îÖÊ¢ B êæ~ b¦ ¢ B~ êæ nã "GÚ~ Òfâ*.j Z' b j~& .Ó Î&>æ pf N&Ú DMEM ö TÎ ¹ 26-gauge "Ò:¾j Ï~ â*.j ^. ê â*¢ ¦FV . ¦Ff 50 ml centrifuge tube TÎ 1-2ª* ;~Î ê ç[j > ~& ¢ .Ó Î&>æ pf N&Ú DMEMb 1,200 rpmöB 5ª* 3® ö Ê ^¿~& . *B â*¢ ¦FB cell~ B>¢ { ê *f ÿ¢ O»b &j >«^f 5:1(2Ü10 B(â *):4Ü10 B(>«^)) ;ê~ jN b~ ^ [j ~& [7, 26]. (5) [^~ 5 > Ö [^~ ßÚ Ö ¦¢ .j'b ¦Ò~ V *~ *f ? *7;7Ú >w»j ~&b, Western blottingj ~& . *7;7 Ú >w»" Western blotting ¦Ò Ö", ·W ^ "º Salah [26]" j [7]~ O»ö V¢ Western blottingb Ú Öj {~& -20 CöB &~ ÒÏ~& . (6) Ú~ ¦ª ;B 5 isotyping îÖÊ~ ;j Û~ áÚ >¢ Salah [26]" j [7]~ O»ö V¢ Bï~ &V~& . (7) vN >w ¦Ò. FÒ ö"~ vN >w þf *f ? Western blotj Ï~ ~& . ÒÏ öf B ÞÏ, "æ²Ï 5 FÎÏ~ Úö" ªj .VJö î . 7. 6. o. VÚ ßW ¦; (1) Î²G;»(enzyme-linked immunosorbent assay, ELISA) *~ O»b &j "æÞÏ~ Úö" ªj.V Jöj Björkman [10]" j [7]~ O»ö V¢ ELISA reader¢ Ï~ 405 nmöB 7ê¢ G;~ & . 1N Úº 45 kDaö ~º Ú, " æÞÏj 6"B áf 14îÒ~ "æ.Ó" "æ ÞÏ 6">æ pf 14îÒ~ "æ.Ój ÒÏ~&b , ''j PBS-Tween 20b 200V C~ ÒÏ~ & . G;B ' ö~ OD 8f x-test¢ Ï~ Û ê' F~Wj ªC~& . (2) Indirect competitive ELISA. "æÞÏö & Ú¢ 10 µg/ml & >ê C ê, "æÞÏ~ Úöj OÎ 96-well polystyrene ELISA microplateö *öB &j "æÞ Ï~ Úöj 1, 10, 100, 1000 5 10000 ng/ml C C" þ IÚ "î . Alkaline phosphateconjugated goat anti-mouse IgG¢ PBS-Tween 20b 5,000V C~ ' wellö 100 µl O Î& ê, 37 C öB 1* ÿn >wV . Vî( ρ-nitrophenyl phosphate(PNPP), diethanolamine 1 ml, MgCl 0.006 g, Ã ~> 9 ml) j IÚ " BïB 405 nmöB 7 ê¢ G;~& . o. 2. Ö . VÚ~ ßW (1) SDS-PAGEö ~ "æÞÏ~ Wî ªC ' öj *V'ÿ ê silver stain" Coomassie blue "ï~ Wî ª³j {~& . "æÞÏ~ Ú Wî 5 ªj . VJWîf 20~280 kDa ÒöB ª ³j {>îb, "º Wî~ Vº ÚWî~ ãÖ 215, 103, 69, 45, 38, 32, 25, 22 5 20 kDa î, ªj .VJWî~ ãÖ 38, 33 5 20 kDa î. (Fig. 1). (2) Ú~ Ö 5 isotyping SDS-PAGEöB 45 kDa Zö >Ú ®º Wî ïf 15 µg î . "æÞÏ~ Ú¢ Ö~V *~ Úöj Î îÖÊ~ Òfâ*.^ ¢ [Î Ö" 192 wells 7 C 58 wells~ n; hybridoma¢ áj > ®î . *7;7Ú >w» b Ú Ö FZ¢ ¦Ò Ö" 7 23 wellsö ®º Ç«^& "æÞÏ Úöö ß'b.

(6) æÞÏ~ 45 kDa ]öWîö &R V]Ú Ö. >w~º Ú¢ Ö~& . SDS-PAGE~ 45 kDa Vö ~º band¢ Ï~ Î îÖÊ. Fig. 1. Electrophoretic analysis of somatic and secretoryexcretory proteins of T. suis. Lane M: molecular weight marker, Lane 1:somatic proteins, Lane 2:secretory-excretory proteins. Lane 1 and 2 were stained by silver staining. Lane. 3:somatic proteins stained with Coomassie blue. Arrow indicated the band on the molecular weight markers.. 629. ~ Òfâ*.^¢ [Î Ö" 192 wells 7 C 148 wells~ n; Ç«^¢ áî . *7;7 Ú >w»" Western blottingb Ú Ö FZ ¢ ¦Ò Ö" 7 114 wellsöB "æÞÏö & ß'b >w~º ©b ¾æÒ . ·W wells 7 * 7;7Ú >w»" Western blotting Ö" >wW ±f clone 5 B(1B9, 2C4, 2C5, 2D7, 2D8)¢ F ~ 2N ~& . 5B 7 Western blotting Ö " Z& ;~² ¾æ¾º 2C4 clone F¾ Ú ¢ Ú~ ßW ¦Ò 5 antigen-capture ELISA¢ * biotinylated antibody ÒÏ~& rb Z & ;~² ¾æÂ 1B9 clone" 2D8 clone F¾ Ú¢ antigen-capture ELISA¢ * coating antibody ÒÏ~& (Fig. 2). "æÞÏ 45 kDaö ~º ö j Î îÖÊ~ Òfâ*.^f îÖÊ >« ^¢ [B áf Ú 7 >wW ±f 5 B(1B9, 2C4, 2C5, 2D7, 2D8)¢ isotyping Ö" Îv IgG1 subgroup îb light chainf Îv κ-chain î . (3) Úf FÒ ö"~ vN >w ¦Ò 2C4 clone F¾ Ú¢ Ï "æÞÏ(T. suis), "æ²Ï(A. suum), BÞÏ(T. vulpis) 5 FÎÏ(T. spiralis)~ Úö, ªj .VJöö & Western blotting Ö" "æÞÏ~ Úö" ªj.VJöö &~ 45 kDa Vö ~º ß' Z¢ { > ®î. Fig. 2. Immunoblotting of the somatic proteins and SE proteins of T. suis reacting to monoclonal antibodies against the 45 kDa antigen. Lane M, molecular weight marker. Lane 1, 3, 5, 7, 9 are somatic proteins of T. suis reacted with monoclonal antibodies 2C4, 1B9, 2D8, 2C5, 2D7, respectively. Lane 2, 4, 6, 8, 10 are SE proteins of T. suis reacted with 2C4, 1B9, 2D8, 2C5, 2D7, respectively. Arrow indicates the molecular weight marker..

(7) «ãÁB«Á*z>Á;«ÁJ;. 630. Fig. 3. Immunoblotting of the somatic and SE antigens of T. suis and other parasite antigens for reacted with 2C4 monoclonal antibody. Lane M, molecular weight marker. Lane 1, T. suis somatic antigens reacted with antiserum raised in mouse. Lane 2, T. suis somatic antigen reacted with 2C4. Lane 3, T. suis SE antigen reacted with 2C4. Lane 4, Ascaris suum somatic antigen reacted with 2C4. Lane 5, A. suum SE antigen reacted with 2C4. Lane 6, Trichuris vulpis somatic antigen reacted with 2C4. Lane 7, T. vulpis SE antigen reacted with 2C4. Lane 8, Trichinella spiralis somatic antigen reacted with 2C4. Lane 9, T. spiralis SE antigen reacted with 2C4. Arrows on the left side indicate the molecular weight markers and the arrow on the right side indicates 45 kDa molecular weight marker.. b¾ VÏ~ Úö" ªj.VJö~ ãÖ Z ¢ { > ìî (Fig. 3). (4) Ú~ ßW þöB Ö "æÞÏ ö 45 kDaö >w~ º monoclonal antibodyf "æÞÏ~ Úö 5 ªj.. VJöö & >w;ê¢ {~V *~ 2C4 f "æÞÏ~ ·W 5 rW.Ób Î²G;» j >¯~& . ' ö f >w~º OD meansÛSD 8 f "æÞÏ WÏ~ Úö OD8 0.378Û0.017î , "æÞÏ WÏ~ ªj. VJö OD8 0.273Û. monoclonal antibody (ELISA) 2C4 monoclonal antibody. Table 1. Comparison of observance values for Trichuris suis somatic and secretory-excretory antigens to 2C4 monoclonal antibody and to mouse negative sera by ELISA Trichuris suis adult antigens No of Examination. Somatic Ag. SE Ag. 2C4 Mab positive serum Mouse negative serum 2C4 Mab positive serum 1 2 3 4 5 6 7 8 9 10 ODmean±SD *OD values at 405 nm **P<0.01. 0.358* 0.351 0.367 0.372 0.391 0.379 0.380 0.399 0.383 0.401 0.378±0.017**. 0.081 0.098 0.096 0.108 0.080 0.071 0.065 0.127 0.111 0.112 0.095±0.020. 0.256 0.277 0.278 0.265 0.280 0.273 0.277 0.269 0.275 0.280 0.273±0.008. Mouse negative serum 0.102 0.088 0.085 0.083 0.128 0.104 0.083 0.076 0.112 0.079 0.094±0.017.

(8) æÞÏ~ 45 kDa ]öWîö &R V]Ú Ö. î (Table 1). Ò ' ö ·W.Ó" > w~º OD meansÛSD 8f "æÞÏ WÏ~ Úö OD8 0.293Û0.086î, "æÞÏ WÏ~ ªj.V Jö OD8 0.231Û0.027î . ' ö rW. Ó" >w~º OD meansÛSD 8f "æÞÏ WÏ~ Úö OD8 0.095Û0.020î, "æÞÏ WÏ~ 0.008. 631. ªj . VJö OD8f 0.094Û0.017î (Table 2). · W OD8f cut-off value(negative OD means+3SD)~ OD 8 ç~ 8b 6;~& . "æÞÏ~ Úö" ª j.VJö 2C4 monoclonal antibodyf >w~º OD 8f rW.Ó~ OD8" jv~ " r ê F~ W(P<0.01) ®rj {~&b, 6 ·W.Ó~ OD. Table 2. Comparison of OD values for somatic and secretory-excretory antigens to positive and negative sera by ELISA Trichuris suis adult antigens No of Examination. Somatic Ag. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 ODmean±SD. SE Ag. Positive serum. Negative serum. Positive serum. Negative serum. 0.323* 0.342 0.289 0.209 0.221 0.223 0.412 0.422 0.224 0.341 0.175 0.189 0.334 0.398 0.293±0.086**. 0.081 0.098 0.096 0.108 0.080 0.081 0.067 0.127 0.111 0.112 0.097 0.054 0.119 0.093 0.095±0.020. 0.233 0.256 0.212 0.222 0.259 0.215 0.198 0.199 0.186 0.265 0.236 0.239 0.276 0.238 0.231±0.027. 0.102 0.098 0.065 0.073 0.128 0.104 0.083 0.076 0.100 0.079 0.099 0.097 0.118 0.088 0.094±0.017. *OD values at 405 nm **P<0.01. Table 3. Comparison of observance values of coproantigens Trichuris suis infected and noninfected pigs to 2C4 clone, positive serum and negative serum by ELISA Infected pigs Abs Fecal no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 OD mean±SD. Positive serum. Monoclonal antibody. 0.389* 0.403 0.512 0.356 0.203 0.281 0.229 0.561 0.406 0.390 0.382 0.408 0.401 0.198 0.366±0.107**. 0.426 0.412 0.345 0.219 0.392 0.391 0.551 0.499 0.561 0.402 0.509 0.598 0.398 0.222 0.423±0.115. *OD values at 405 nm **P<0.01. Noninfected pigs Negative serum. Abs Fecal no.. Positive serum. Monoclonal antibody. 0.091 0.141 0.101 0.108 0.097 0.081 0.081 0.099 0.093 0.078 0.076 0.049 0.065 0.121 0.092±0.023. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 OD mean±SD. 0.102 0.098 0.109 0.078 0.097 0.102 0.111 0.096 0.083 0.127 0.123 0.093 0.133 0.088 0.103±0.016. 0.099 0.143 0.092 0.101 0.179 0.098 0.121 0.219 0.089 0.079 0.128 0.203 0.168 0.093 0.129±0.046.

(9) «ãÁB«Á*z>Á;«ÁJ;. 632. Fig. 4. Standard curves determined by indirect competitive ELISA for somatic antigens of T. suis using 45 kDa monoclonal antibody. The titer microplate was coated with somatic antigens of T. suis (100 µg/ml) and somatic antigens of T. suis was competed with monoclonal antibody (10 µg/ml).. 8 ê ¸² ¾æÂ ©j {~& . (5) ªæöö & Î²G;»(ELISA) þöB Ö 45 kDaö ~º Ú f "æÞÏ~ ªæÚöB ªÒ ªæöö & > w;ê¢ {~V *~ 2C4 clone Ú, "æ ÞÏ~ ·W 5 rW.Ób Î²G;»(ELISA) j >¯~& . "æÞÏ 6"î~ ªæ ò& 2C4 clone Úf >w~º OD meansÛSD8f 0.423 Û0.115î, ·W.Ó" >w~º OD meansÛSD 8 f 0.366Û0.107îb, rW.Ó" >w~º OD meansÛSD8f 0.092Û0.023î . "æÞÏ Z6"î ~ ªæò& ·W.Ó 5 2C4 clone Úf > w~º OD meansÛSD 8f '' 0.103Û0.016 5 0.129 Û0.046 ªæö" rW.Ój >wÎ 8" F Ò~& . "æÞÏ~ ªæö 2C4 clone Úf >w~º OD8j rW.Ó~ OD8" jv~ " r ê F~W(P<0.01) ®rj {~&b, 6 ·W.Ó~ OD 8 ê ¸² ¾æÂ ©j {~ & (Table 3). (6) Ú~ ö¦ÂË Ú~ ³ê¢ 10 µg/ml >² ~ Ú~ &¢ indirect competitive ELISA»b G;~&~ : ö~ · 100 ng/ml ræ ¦Â > ®î (Fig. 4).. V. Ï¦ ¦Òö ~ VÏ 6"~ êf ÞÒ~ ã B'æò Ï¦ VÂ>æ pº V~ ê 5 ã. 6"ö ~ ¾æ¾º ÞÏ 6"Ãj ê~º º ÚJæ ôV r^ö *Ò VÏ~ .Ó' êö & & B® ê¯> ®b, º V Ï6" ê ªæÚ Ï¦ ¦Âö ~ ê~ 6 j j > ® [21]. ¯, 6" ê¢ê W? êö Bº Ï¦ ¦Â ®&Ë~&b¾, .Ó' O »~ Ïf Ï¦j ¦Â > ìº V~ 6"j ê ~º Ö FÏ~² ÒÏF > ® [10]. æ ÞÏÃ~ êf .Ó'¾ ' O»b ¦Â Nj ¸¢ > ® . b'b Úº ê ~ ÿîW" ßW ®bæ î÷~ ê¾ ¢ Ï ö ªC 6º ªÒ ö 6Ò 'Ï> ®. [2, 7, 25, 29]. þöBº "æÞÏ~ Úö" ªj .VJöj Ï~ ÞÏÃ êö Ï > ®º ' Ú¢ Ö~& ELISAf Western blotb "6 êf ßWj ªC~& . 7 >w ±f Ú¢ F ê Ú¢ ÒÏ antigen- capture sandwich ELISA V»j Ï~ "æ.Ó 5 "æª æÚ~ ö ¦Âj Û "æÞÏÃ~ ' ê ö & 'ÏWj ¦Ò~& . Hill [21]f "æÞÏ~ WÏj Ï~ 20 kDa ª³öB ¾æÂ ªj . VJö~ ßWj {~&b , 6 îÖÊ ÞÏ~ WÏj Ï ªj .VJö ~ ª³j 85~105 kDaöB {~& ~&, æ [6]f "æÞÏ~ WÏ 5 B 3V ¶Ï~ Úö " ªj . VJö~ FÒ ª³j {~V *~ * V'ÿÎ ê silver stainj Ö", 20~80 kDaö B ª³j {~&b ' ª³f 80, 45, 32, 28, 20 kDaöB {~& ~& . þöB {B 20 kDaf 45 kDaö ~º ª³f Hill [21]" æ [6] ª³" Îv ¢~~&b "æ ÞÏ~ WÏj V·~ ²> V·öB column chromatography¢ Ï~ 20 kDa secretory-excretory (SE) glycoproteinj ªÒ~&b "æÞÏ WÏj in vitroöB V·~ ªÒ zinc metalloprotease& 45 kDa V~ öb "ïO»ö ~ "æÞÏ~ stichosomeö *~~ ® º ©j { Hill [21] ~ f ¢~~& . Hill [21]f 6 "æÞÏ~ WÏj Ï ªj .VJö~ ª³j 85~105 kDaö B {~& ~&º ©f þöB { B 103 kDa ª³" FÒ~& . þöB {B ö~ ª³f Hill [21]" æ [6]~ ª³ ô ¾æÒº ©f Úö" ªj . VJö ªÒ cation exchange high-pressure liquid chromatography column~ ê¢ ~æ p~V r^ ©b Òò.

(10) æÞÏ~ 45 kDa ]öWîö &R V]Ú Ö. B . "æÞÏ 5 FÒ FÏ~ ~ ö~ ª³ö & & ô >Ú ®º Lillywhite [22]f Ò² ÞÏ WÏ~ ªj .VJö~ "º ª³ 52~54 kDa, 35~45 kDa f 17 kDa îb Úö~ ª³f ?"~ VÏö & ' >w" ÷Ò' >w 5 ?" OÚ V* æz¢ rÚÚº j ~ & . 6 vÞÏ~ ªj .VJö~ "º ª³ 43 kDa î ~&º þöB {B "æÞ Ï~ 45 kDa~ ª³"º ¢~~æ p~ . Fetterer [15, 16]f "æÞÏ ªj~º phenol oxidase¢ Ï ¢N~ öB "æÞÏ~ Úç ªC Ö" 44 kDa" 53 kDa~ ª³j {~& ~&, Drake [13]f ÞÏf ç VÏbB 6"W ®º B 1V FÏ ?"~ Ë 6ïö R~ BG ê, WÏ~ *Ú¦ö ê Ö>º stichocyte ¢ <² >º © ªj~º ö f öW ¸ 6>W ®º ?"öB OÚ j FB ~& . V¢B ª¶ 6º ªjb ~ V Ë VÏ~ ö j>' &Ò ";ö & © b º;> vÞÏ~ VJ-ªj bîf 105 kDaf 85 kDa~ "º peptidase¢ &æ ® ~& . Else [14]f complete Freund's adjuvant¢ Df v ÞÏ~ ªj .VJöb 7« NIH miceöB 7« ê >wj Ò Ö" vÞÏ~ ªj .VJöj crude antigenf öW Ö ¸² ¾æÒb 105~110 kDa, 90~95 kDa, 80~85 kDa~ "º ª³ j {~& ~& . Chan" Ko [11]º 7öB sb >«B 2,000v~ "æöB SDS-PAGEf indirect ELISA O»b FÎÏ öö & ßWj Ò Ö" 28 kDa~55 kDaö ~º ª³j {~ & ~&º Ö" f Îv þö B ·WB 45 kDa~ "æÞÏ~ ª³"º ¢~~æ p ~ . Needham [24]f VÏ~ ªj .VJöö & .V Ú >wf '· Wî¾ 6" ;êö &êì * ~' &Ë Â]~² IgG1 group © b ¾æÒ ~&, 6 ÞÏ~ antibody isotypej Ò : IgG1 group" IgG2 group >& *>' 6" ³êf &Ë 7º &ê& ®b, © f ÞÏ 6" ;~ · Ú >wj ¢b Î º Lillywhite [22]~ ¢ { ©¢ ~&º, Ö" f þöB áf Ú~ isotyping Ö"& IgG1 groupb ¾æÂ ©" F Ò Ö" ÒòB . Roach [27, 28]f B ÞÏ*~ vN>w. 633. ¾æÂ ©f stichocyte öj F~V r^ *O ö ®º stichocyte~ «¶ z ;~² >w ~& . Urban [30]f "æ²Ï 6"ö ~ f ?"~ ¢_ 6"" &N ®b, ËÚöB ² Ïj B~ê f 'Ëj Aæ pº ~ & . Homan [23]f FÎÏ~ êj * ö { þöB ªj .VJö" stichocyte F¾ ö. crude extract ê'b R Ö>~. º © ¢>'b Aj æ ® ~& FÎÏ "G FÏ~ ªj .VJöj ªC Ö" 92, 53, 48 kDaö ~º ª³j {~& ~& . Hill [21]~ ö ~~ "æÞÏ WÏ~ ªj. VJöf "æ²Ï, FÎÏ, BÞÏ, Ç²¶Ï (Toxoplama gondii), B²Ï(Toxocara canis), BÏ (Ancylostoma caninum), ªFÏ(Strongyloides sterocoralis), "æËÖ.Ï(Oesophagostomum dentatum) ~ FÏ~ 6"ö ~ ;WB Úf >w~æ pbæ .Ó ' O»~ ê öb Ï > ® ~&. . s [8]f Ú Ï "æ²Ï ªj .VJö 5 Úö ßW ªCöB 29 kDa~ ª¶ïö & Ú& "æÞÏ~ Úö 5 ªj .VJö"~ vN> wj {~¶ Western blotj ~&b¾ vN>wf ¢Ú¾æ p~ ~& . þöB áf Ú¢ Ï~ "æÞÏ, "æ²Ï, BÞÏ 5 FÎÏ~ Úö" ªj . VJö ö & Western blotj Ö" "æÞÏöBº 45 kDa Vö ~º ß' Z¢ { > ®î b¾ VÏ~ Úö" ªj .VJööBº Z ¢ { > ìî . Ö" " r "æÞÏ 6"ö ~ W? ê¾ Ï¦ VÂV~ ÞÏÃf "æÞÏ~ 45 kDa ö ~º monoclonal antibody¢ Ï~ Î² G;»(ELISA)j b êö Ï > ®j ©b ÒòB . þöB ·W 45 kDaö ~º Ú f "æÞÏö 6"B "æ~ ªæÚöB ªÒ ö W Wîö & >w ;ê¢ {~V *~ Î² G;»(ELISA)j >¯ Ö" "æÞÏ 6"î~ ª æ ò& ·W .Ó¾ rW .Ó OD8 ¸² ¾ æÂ ©j {~& . Ö"º öj ªÒ~ "æ ÞÏ~ ·W 5 rW .Ób Î²G;»j >¯ ~ ·W .Ó" >w~º "æÞÏ WÏ Úö~ OD 8(rW .Ó" >w~º OD8)j 0.30Û0.120(0.09Û 0.006)b, Ò WÏ~ ªj . VJö~ OD8j 0.24Û0.031(0.11Û0.00) æ [6]~ Ö". ê z ¸² ¾æÂ © ..

(11) «ãÁB«Á*z>Á;«ÁJ; æN^, ÆB, ;ß. .Ó' êj * æ 6. Björkman [10]f *7;7Ú»(IFAT)" ÞÏ~ Úö, VJ/ªjö~ ªÒ 5 jv. & indirect ELISA¢ Ï~ .Ó" ÖFÚ Neospora >~²æ . 1999, 39, 159-168. j c>. Sulfamethazine caninum~ Ú¢ ³® « > ®î ~& ö & VÚ~ Ö" 7. b, Chan" Ko [11]º IgG-ELISA¢ Ï~ FÎÏ ¢ Ï ELISA~ BB. BÞ&v &ö CÒ ö~ ßWj ~&b, Deplazes [12]f . BÞ. pp. 6-10, 1996. I* .¢^ 8. . VÚ Ï æ ²Ï ªj ö 5 Ú antigen-capture andwich ELISA¢ Ï~ B~ ç ö ßW ªC. BÞ&v &ö ;Ò * ¢ Ï(Taenia hydatigena)~ ªæöj ¸f ß>&öB ^. BÞ, pp. 43-44, 1999. {~&rj ~& . 9. Beer, R. J. S. Studies on the biology of the life-cycle Ö" Ú " r þöB áf of Trichuris suis Schrank, 1788. Parasitology. 1973, 67, Ú¢ Ï~ antigen-capture sandwich ELISA»b 253-262. ' êj *^ê'b ·îËö ô 10. Björkman, C(amilla)., Joakim, O., Holmdahl, M. f ãB' b¢ «® ®º "æÞÏ~ Î 6" and Uggla, A. An indirect enzyme-linked immunoassay êö & ê &Ëb ·îËöB "' (ELISA) for demonstration of antibodies to Neospira { Ïê³j ^Þ > ®² >Ú ÖW Ë caninum in serum and milk of cattle. Vet. Parasitol. çö ² V ©b ÒòB . 1997, 68, 251-260. 634. Ö . ÞÏf W? êf WÏ~ ÚÊ VöBº Ï¦ j VÂ~æ pV r^ö ¢>' ªæ¦Ò»bº 6"Ã ê ®&Ë~æ ' ê» jº~. . "æÞÏ~ Úöj Î BALB/cîÖÊ~ Ò fâ*.^f îÖÊ >«^¢ [~ [ Ç «^¢ ò î êC»" ELISA¢ Ï~ b 45 kDa~ Ú¢ Ö~& . Western blotb "6ê¢ Ò Ö" "æÞÏ~ Úö 5 ªj . VJö" ß'b >w~&b¾ "æ²Ï, BÞÏ 5 FÎÏ~ ö"~ vN>wf ¾ æ¾æ p~ . Ï¦ ·W "æöB ªæ ö ¦ÂN " .Ó Ú ö ¦ÂNj jv Ö" ªæ ö ¦ ÂN .Ó Ú ¦ÂN z ¸² ¾æÒ .. ^^ò. ;8, *9~, Bç\, Ë~, Rç^. 8ëz ·îË~ æ Ú¦8Ï 6" æz·ç Ò. >~7²æ 13, 15-19. ² ?\. Psedorabies Virus. 1989, VÚ¢ Ï Bï 2. Î²{Ö» BB 5 gP50" gP63 F*¶ R ö & . BÞ&v &ö ;Ò * ¢^. 43, 1991. BÞÒ\, pp.. >~ 8Ï. pp. 266-268, &v" 3. B"²Ò ,× BÞ;, L1987. Ë L ~ Ò ö. BÎ×K îÒö & æ 4. , , . &>~²æ. 1991, 31, 509-513. æÚ¦8Ï N^, ;ßÒ . þ&ÚöB æ²Ï(Ascaris suum) 5. ¶Ï¦(L )~ V·. &>~²æ. 1998, 38, 1.. 2. 107-117.. 11. Chan, S. W. and Ko, R. C. Specificity of affinitypurified Trichinella spiralis antigens. Vet. Parasitol. 1992, 41, 109-120. 12. Deplazes, P., Gottstein, B., Stingelin, Y. and Eckert, J. Detection of Taenia hydatigena Copro-Antigens by ELISA in Dogs. Vet. Parasitol. 1990, 36, 91-103. 13. Drake, L. J., Bianco, A. E., Bundy, D. A. P. and Ashall, F. Characterization of peptidases of adult Trichuris muris. Parasitol. 1994, 109, 623-630. 14. Else, K. J., Wakelin, D., Wassom, D. L. and Hauda, K. M. MHC-restricted antibody responses to Trichuris muris secretory-excretory (SE) antigen. Parasite Immunol. 1990, 12, 509-527. 15. Fetterer, R. H. and Hill, D. E. Localization of phenol oxidase in female Trichuris suis. J. Parasitol. 1994, 80, 952-959. 16. Fetterer, R. H. and Hill, D. E. The occurrence of phenol oxidase activity in female Trichuris suis. J. Parasitol. 1993, 79, 155-159. 17. Fields, H. A., Bruguera, P., de la Torre, N., Puig, J. and Anderson, L. J. Purity, antigenicity and immunogenicity of the hepatitis B surface antigen purified by five different methods. J. Virol. Methods. 1988, 22, 283-294. 18. Greenspon, L. W., White, J., Shields, R. S., Funer, A. and Gold, W. M. Purification of Ascaris suum antigen: Its allergenic activity in vitro and in vivo. J Allergy Clin. Immunol. 1986, 77, 443-51. 19. Hill, D. E. Protective Antigens from culture Fluids of the Adult Swine Whipworm, Trichuris suis. Research Investment Report (National Pork Producers Council). 1996..

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