A novel rat CVB1-VP1 monoclonal antibody 3A6 detects a broad range of enteroviruses

1 SCIENTIFIC REpORTS | (2018) 8:33 | DOI:10.1038/s41598-017-18495-4 www.na u e.com/scien i ic epo s A no el a CVB1-VP1 monoclonal an ibody 3A6 de ec s a b oad ange o en e o i uses Niila V. V. Saa inen1, Ju a E. Laiho1, Sa ah J. Richa dson 2, Ma ie Zeissle 2, Vi ginia M. S one3, Va pu Ma jomäki5, Tino Kan oluo o5, Ma c S. Ho wi z6, Ami babak Sioo y-Khojine1, Anni Honkimaa1, Minna M. Hankaniemi1, Malin Flods öm-Tullbe g 3, Heikki Hyö y1,4, Vesa P. Hy önen 1,4 & Olli H. Lai inen1 En e o i uses (EVs) a e common RNA i uses ha cause diseases anging om ash o pa aly ic poliomyeli is. Fo example, EV-A and EV-C i uses cause hand- oo and mou h disease and EV-B i uses cause encephali is and myoca di is, which can esul in se e e mo bidi y and mo ali y. While new accines and ea men s o EVs a e unde de elopmen , me hods o s udying and diagnosing EV in ec ions a e s ill limi ed and he e o e new diagnos ic ools a e equi ed. Ou aim was o p oduce and cha ac e ize new an ibodies ha wo k in mul iple applica ions and de ec EVs in issues and in i o. Ra s we e immunized wi h Coxsackie i us B1 capsid p o ein VP1 and hyb idomas we e p oduced. Hyb idoma clones we e selec ed based on hei eac i i y in di e en immunoassays. The mos p omising clone, 3A6, was cha ac e ized and i pe o med well in mul iple echniques including ELISA, immunoelec on mic oscopy, immunocy o- and his ochemis y and in Wes e n blo ing, de ec ing EVs in in ec ed cells and issues. I ecognized se e al EV-Bs and also he EV-C ep esen a i e Polio i us 3, making i a b oad-spec um EV speci ic an ibody. The 3A6 a monoclonal an ibody can help o o e come some o he challenges aced wi h commonly used EV an ibodies: i enables simul aneous use o mouse-de i ed an ibodies in double s aining and i is use ul in mu ine models. En e o i uses (EVs) belong o he amily o Pico na i idae and a e small non-en eloped i uses wi h a single-s anded posi i e-sense RNA genome. The e a e hi een en e o i us species, se en o which a ec humans. Cu en ly, nea ly 300 human EV se o ypes/geno ypes, including Rhino i uses ha e been iden i ied (h p://www.pico na i idae.com/en e o i us/en e o i us.h m, May 30 h 2017). The majo i y o EV in ec ions cause only mild common cold- ype symp oms, o a e asymp oma ic, howe e , in some cases hey can igge mo e se e e acu e and ch onic diseases such as myoca di is, ch onic ca diomyopa hy, hand, oo and mou h disease, pa alysis, encephali is and meningi is1,2. In some clinical o ms he main con ibu ing EVs ha cause a pa icula disease a e well known. Th ee polio i us se o ypes cause poliomyeli is and Coxsackie i us B (CVB) se o ypes a e he main cause o acu e i al myoca di is1. Co espondingly, EV71 and EV68 ha e ecen ly been ound o cause se e e neu al symp oms3,4. Howe e , in he case o o he diseases like ype 1 diabe es (T1D), whe e a link be ween EVs and he onse o he disease has been p oposed and se e al candida e se o ypes ha e been iden i ied5–8 he associa ion has no been ully es ablished. The e o e, i is essen ial o de elop ools and ech- niques ha a e capable o ecognizing mul iple EV se o ypes o de ec ing he ange o EVs ha could po en ially con ibu e o hese di e en diseases. The EV capsid is an icosahed on ha consis s o epea s o ou s uc u al p o eins, VP1-4. VP1, VP2, and VP3 a e loca ed on he ou e su ace o he capsid, whe eas VP4 esides on he inne capsid su ace9. The EV VP1 p o ein displays he g ea es sequence a ia ion be ween se o ypes, bu i also con ains a conse ed immunodom- inan epi ope10,11. The e o e, i is an a ac i e candida e o he de elopmen o diagnos ic ools o bo h de ec 1Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland. 2Uni e si y o Exe e Medical School, Exe e , De on, UK. 3Depa men o Medicine HS, Ka olinska Ins i u e , S ockholm, Sweden. 4Fimlab Labo a o ies, Pi kanmaa Hospi al Dis ic , Tampe e, Finland. 5Depa men o Biological and En i onmen al Science/ Nanoscience cen e , Uni e si y o Jy äskylä, Jy äskylä, Finland. 6Depa men o Mic obiology and Immunology, Li e Sciences Ins i u e, Uni e si y o B i ish Columbia, Vancou e , Canada. Co espondence and eques s o ma e ials should be add essed o O.H.L. (email: [email p o ec ed]) Recei ed: 21 July 2017 Accep ed: 12 Decembe 2017 Published: xx xx xxxx OPEN www.na u e.com/scien i ic epo s/ 2 SCIENTIFIC REpORTS | (2018) 8:33 | DOI:10.1038/s41598-017-18495-4 EVs and di e en ia e be ween EV g oups. The e a e a numbe o di e en EV an ibodies a ailable, which a y in hei species and se o ype speci ici y, such as Cox mAB 31A2 ha binds o ce ain CVB ypes12 (Laiho e al. submi ed Vi chows A chi 2017) and o he s ha eac mo e widely wi h di e en EV ypes, such as he mono- clonal an ibody 5D8/113 by Dako Cy oma ions, Denma k ( e e ed he ea e as 5D8/1) and he 9D5 (Millipo e); he las wo a e widely used in EV esea ch14. These an ibodies each ha e hei own s eng hs and sho comings wi h ega ds o sensi i i y and speci ici y12,14,15. Fo example, he c oss- eac i i y o 5D8/1 o smoo h muscle issue is well documen ed12,15, which may make he in e p e a ion o esul s mo e challenging in issues like hea and in es ine. The Cox mAB 31A2, which was aised agains he VP1 o CVB3, ecognizes CVB1 and CVB312 (Laiho e al., submi ed), bu no o he EV se o ypes. This na ow ecogni ion pa e n o 31A2 needs o be conside ed when i is used in clinical diagnos ics. Mice a e he mos commonly used animal model o s udying EV in ec ions and pa hology16,17 and due o he ac ha mos monoclonal an ibodies agains EVs a e based on mouse hyb idomas, hei use in his ology can be p oblema ic due o high backg ound caused by endogenous an ibodies (mouse-on-mouse unspeci ic binding)18. The e o e, he e is a need o no el non-mouse EV an ibodies. To add ess his, we gene a ed a new a monoclo- nal EV an ibody, clone 3A6, ha sol es he hos -species issue and ha can help o o e come some o he cu en challenges associa ed wi h o he EV-an ibodies in esea ch and diagnos ics. Objec i es Ou aim was o de elop a no el monoclonal an ibody ha ecognizes an EV g oup-speci ic epi ope loca ed in he N- e minus o VP1 p o ein and ha could be used in mul iple applica ions o s udy EVs. Ra s we e chosen o he gene a ion o he hyb idoma clones o add ess he challenges aced wi h mouse-de i ed monoclonal an ibodies which a e cu en ly widely u ilized in EV esea ch. Resul s 3A6 ecognizes CVB i uses and shows eac i i y owa ds VP1 N- e minus in ELISA. All 14 gene a ed monoclonal an ibodies we e s ongly posi i e o CVB1 ull-leng h VP1, bu binding o hea inac i- a ed i uses was de ec able only wi h clones 3A6 and 12A4 (Fig.1a). Based on he p elimina y sc eening o he clones using ELISA and immunocy ochemis y (da a no shown), i e clones we e selec ed o u he s udies: wo s aining/binding posi i ely o he N- e minus o CVB3 VP1 (clones 3A6 and 12A4) and h ee binding o he pa s o VP1 (4D12, 7C1, 9B9) (Fig.1). Fo he gene al s uc u e o VP1 p o ein and i s pa s, see Fig.2a and b. In e es ingly, none o he clones we e s ongly binding he VP1 C- e minus (Fig.1b).Ou o hese clones, 3A6 showed he highes po en ial as an an ibody wi h b oad eac i i y agains EVs as i ga e he s onges signal when pu i ied i uses we e used as an igens (Fig.1a). The e o e, we ocused ou u he e o s in cha ac e izing 3A6. Figu e 1. Reac i i y o 14 di e en monoclonal CVB1-VP1 an ibody clones agains CVB1-3 i uses and CVB1 VP1 as well as mapping o hei binding egion in ELISA. (a) Sc eening o hyb idoma supe na an s agains pu i ied CVB1-3 i uses and CVB1 VP1 p o ein. Vi us samples we e hea -inac i a ed be o e coa ing. (b) Sc eening o hyb idoma supe na an s agains CVB1 VP1, and he N- and C- e minal agmen s o CVB3 VP1. Samples we e un as duplica es, wi h 250 ng/well o an igen. The abso bance o blocked wells was sub ac ed om he sample abso bance alues be o e plo ing he da a. E o ba s show s anda d de ia ion o duplica es. In (b) N and C e e s o e minal GST usion cons uc s. Signal in ensi y di e ences be ween (a) and (b) a e due o di e ing incuba ion imes wi h subs a e. www.na u e.com/scien i ic epo s/ 3 SCIENTIFIC REpORTS | (2018) 8:33 | DOI:10.1038/s41598-017-18495-4 The 3A6 binding si e on he N- e minus o VP1 o e laps wi h he en e o i us g oup speci ic epi ope. As we iden i ied ha he epi ope o 3A6 esides in he N- e minal egion o VP1 (Fig.1) and as i is known ha he 5D8/1 epi ope also loca es in he N- e minal pa o VP110, we examined whe he 3A6 binds o he same o o a unique epi ope. To s udy his, wo pep ides (Fig.2c) p e iously used o cha ac e ize he epi ope bound by 5D8/119 we e u ilized in an Immuno luo escen assay (IFA). We used CVB3 VLP as a posi i e con ol o binding20. Pep ide IFAs con i med ha bo h he 3A6 and he 5D8/1 an ibodies ecognize he VP1 pep ide RPTNSESIPALTAAE (Fig.3a–d), howe e nei he an ibody ecognized he o e lapping downs eam pep ide PALTAVETGATNPLV (Fig.3c and d), sugges ing ha he 3A6 epi ope o e laps wi h ha o 5D8/1, and i hey a e no he same, he epi ope bound by 3A6 may be loca ed close o he N- e minus o VP1. We also s udied whe he ei he an ibody could bind o pep ide epi opes o CKB o ATP5B (Fig.3c and d), wo mi ochond ial p o eins p o- posed o c oss- eac wi h 5D8/115,19. We obse ed a weak binding o 5D8/1 o he CKB pep ide (Fig.3c), whe eas 3A6 showed no eac i i y owa ds hese wo pep ides (Fig.3d). 3A6 ecognizes CVB1-6 in Wes e n blo ing wi h simila in ensi y. In o de o compa e how 3A6 and 5D8/1 an ibodies de ec di e en EVs in Wes e n blo ing, EV-in ec ed cell lysa es and concen a ed CVB i uses we e used. Bo h an ibodies de ec ed CVB se o ypes 1-6, Echo i us 6, Echo i us 30 and Coxsackie i us A9 in in ec ed cell lysa es, bu nei he o hem s ained Polio i us 3 (PV3). PV3 VP1 has a lysine a posi ion 40, whe e in EV-B he e is an uncha ged amino acid, usually glu amine (Supplemen a y Figu eS1). This migh explain helack o binding in WB, whe e he epi ope is linea ized when compa ed o IHCP and IF. Nei he an ibody ga e unspeci ic binding o non-in ec ed cells o p o eins in ex ac s o in ec ed cells (Fig.4: lanes 1–2, 3–6 and 8–13). Unlike 5D8/1, 3A6 showed less a ia ion in signal in ensi y be ween he di e en CVB se o ypes (Fig.4: lanes 8–13) pa icula ly when compa ing hei ela i e binding o CVB5 and CVB6 (Fig.4; lanes 12 and 13 in panels b and c). 3A6 ecognizes EV-in ec ed cells in immunocy ochemis y (ICC). The abili y o 3A6 and 5D8/1 o ecognize di e en EV se o ypes was u he es ed in ch omogenic ICC and in IFA by wo independen labo a o- ies (Tampe e, Finland and Exe e , UK) using an a ay o EV-in ec ed cells (Table1). Bo h an ibodies ecognized he EV-B i uses well, bu only 5D8/1 showed some eac i i y owa ds i uses om EV-A species. In addi ion, bo h an ibodies ecognized PV3, which belongs o EV-C. Nei he o he an ibodies bound he non-in ec ed con- ol cell lines (A549, GMK, Ve o, RD and HeLa), o he Adeno i us o Human pa echo i us 1 (HPeV1) in ec ed cells (Table1). A sligh a ia ion was obse ed in he s aining in ensi y be ween IF and IHC-P (Supplemen a y Figu eS2) howe e , he o e all ecogni ion spec um was simila in bo h assays. The 3A6 also ecognized all es ed CVB1 (N = 30) s ains in he CVB1 cell mic oa ay21. To es he sui abili y o 3A6 o ecognize bo h acu ely-in ec ed and ch onically-in ec ed cells in non-pa a in embedded PFA- ixed co e slips, samples wi h acu ely CVB1-in ec ed PANC-1 cells and ch onically CVB1-in ec ed 1.1B4 cells (unpublishedmodel ha is based on p e iously published da a22) we e u ilized. The Figu e 2. Design o he VP1 cons uc s used in an ibody gene a ion and ELISA sc eening. (a) CVB3 VP1 s uc u e (PDB id 4GB3) p esen ed as ca oon model showing he N- e minal segmen (ligh blue), he co e egion (g een) and he C- e minal pa o he p o ein (da k blue). (b) Schema ic illus a ions o ull leng h CVB1 VP1 and unca ed CVB3 VP1 exp ession cons uc s used o immuniza ion and o immunoassays. (c) Recombinan p o ein cons uc s and pep ides used in he an ibody gene a ion ( ull-leng h CVB1 VP1) and in ELISA assays (all cons uc s excep Co e egion). www.na u e.com/scien i ic epo s/ 4 SCIENTIFIC REpORTS | (2018) 8:33 | DOI:10.1038/s41598-017-18495-4 co e slips we e co-s ained wi h 3A6 and dsRNA an ibody23 J2 o isualize he localiza ion o i al p o ein and dsRNA in he same cell. Bo h an ibodies wo ked well unde he condi ions es ed. 3A6 s aining was dis ib- u ed h oughou he cell cy osol whe eas J2 had punc a e pa e n (Fig.5, Supplemen a y Figu eS3). In addi- ion, dual s aining wi h 3A6 and 5D8/1 in CVB1-in ec ed Ve o cells showed ha he wo an ibodies co-localized (Supplemen a y Figu eS4). The ela i e sensi i i y o 3A6 in o he EV de ec ion me hods was es ed wi h IFA and IHC-P s aining using he p e iously published limi ed dilu ion se ies cell mic oa ay (CMA)24, which in ol es dilu ions o A549 cells in ec ed wi h CVB1. In bo h assays he an ibody de ec ed i us in ec ions in cells dilu ed o 10−3, e lec ing a sen- si i i y simila o ha as p e iously ob ained wi h se e al polyclonal EV an ibodies24. Howe e he 3A6 showed lowe sensi i i y when compa ed o 5D8/1 in IHC-P (10−6)24. 3A6 de ec s acu e CVB in ec ions in issue samples by immunohis ochemis y. To s udy he applicabili y o 3A6 in IHC, we es ed i in di e en mouse and human issue samples. In EV-in ec ed mouse panc eas, 3A6 showed a clea eac i i y owa ds CVB1 and CVB3 (Fig.6), and CVB4 (no shown), wi h no back- g ound s aining in non-in ec ed mouse panc ea a (Fig.6c and d). As shown p e iously25 he CVBs p e e en ially a ge he exoc ine issue in he mouse panc eas. Human panc eas and spleen samples om he PanFin ne wo k26 wi h unknown EV s a us we e s ained wi h 3A6, 5D8/1 and a iso ype an ibody con ol in consecu i e sec ions. These samples showed no EV posi i i y wi h hese an ibodies and he a iso ype con ol was also nega i e. Fu he mo e, hea issue om a con i med CVB in ec ion was s ained wi h 3A6 and 5D8/1: clea s aining was obse ed wi h bo h an ibodies in he in ec ed issue, bu no in non-in ec ed con ol hea (Fig.7). Figu e 3. Binding o 3A6 and 5D8/1 an ibodies o di e en an igens in immuno luo escence assay. In (a) he binding o 5D8/1 (g ey ba s) and 3A6 (black ba s) o pVP1 pep ide RPTNSESIPALTAAE (500 ng/ well) and CVB3 VLP (250 ng/well) was compa ed. (b) A dose esponse cu e o 5D8/1(black line) and 3A6 (do ed line) binding o VP1. (c) The binding o 5D8/1 and 3A6 (d) o pep ides de i ed om VP1 (VP1 – RPTNSESIPALTAAE; VP1 Chia – PALTAVETGATNPLV), CKB, ATP5B and whole i us capsid ep esen ing CVB3 VLP. www.na u e.com/scien i ic epo s/ 5 SCIENTIFIC REpORTS | (2018) 8:33 | DOI:10.1038/s41598-017-18495-4 3A6 labels VP1-p o eins in c yo-immuno EM. C yo-immuno-EM was used o s udy he VP1 con aining cellula compa men s in CVB1 in ec ed exoc ine panc ea ic cells using bo h 3A6 and 5-D8/1 an ibodies. Mouse panc eas issue showed clea signs o in ec ion 3 days a e in ape i oneal inocula ion wi h CVB1 (105 PFU/ mouse) (Fig.8). The p esence o i us in he panc eas was also con i med by PCR, which showed high le els o CVB1 (da a no shown). A low magni ica ion, mo phological changes due o in ec ion we e ob ious: apop- o ic nuclei, swollen endoplasmic e iculum, eme gence o no el memb anous s uc u es in he cy oplasm. These Figu e 4. Compa ison o 3A6 and 5D8/1 en e o i us de ec ion p o iles in Wes e n blo ing. (a) Sample o al p o eins isualized wi h he S ain-F ee echnology. (b) Immunoblo s ained wi h 5D8/1 (1:3000) and (c) wi h 3A6 (1:1000). VP1 p o eins a e ~30 kDa, depending on he EV se o ype. Sample o de om le o igh : lysa es om non-in ec ed GMK and Ve o cells (8 µg/lane; lanes 1 and 2), CVA9, Echo i us 6, Echo i us 30 and Polio i us 3 (Sabin) in ec ed cell lysa es (8 µg/lane; lanes 3–6), ladde (lane 7) and concen a ed and quan i ied CVB1–6 (0,66 µg) (lanes 8–13). Vi us Cell line An ibody Species Se o ype S ain Ra an i-VP1 mAb Mouse an i-VP1 mAb Clone 3A6 Clone 5D8/1 EV B CVB1 ATCC GMK ++ +++ EV B CVB2 ATCC GMK ++ +++ EV B CVB3 ATCC GMK ++ +++ EV B CVB4 ATCC GMK ++ +++ EV B CVB5 ATCC GMK +++ +++ EV B CVB6 ATCC GMK ++ ++ EV B Echo3 PB-E3DiT23 GMK + + EV B Echo4 ATCC GMK ++ + EV B Echo6 ATCC GMK +++ +++ EV B Echo9 ATCC GMK ++ ++ EV B Echo11 ATCC GMK +++ + EV B Echo30 ATCC A549 +++ +++ EV B CVA9 ATCC GMK ++ +++ EV B CVA9 PB-CVA9V59 RD ++ ++ EV A CVA2 PB-CVA2V38 RD − + EV A CVA4 PB-CVA4V36 RD − + EV A CVA5 PB-CVA5V43 RD − ++ EV A CVA6 PB-CVA6V303V RD — + EV A CVA10 PB-CVA10V2530 RD — + EV A CVA16 PB-CVA16V55 RD — + EV A CVA16 ATCC Ve o — — EV A EV71 PB-EV71Hus GMK — — EV C PV3 Sabin GMK ++ ++ Adeno C VR846 HeLa − − HPeV HPeV1 ATCC A549 − − Table 1. 3A6 mainly ecognizes EVs o he B species. A cell mic oa ay including 20 di e en EVs and wo non-EV i uses (Adeno i us C and Human Pa echo i us 1) was immunos ained wi h 3A6 in wo independen labo a o ies o e i y he ecogni ion spec um o he an ibody. The esul s o ch omogenic immunos aining by 3A6 a e shown in compa ison o hose ob ained wi h 5D8/121,24 a e also shown. Recogni ion scale: +++ = s ong, ++ = mode a e, + = weak, − = nega i e. www.na u e.com/scien i ic epo s/ 6 SCIENTIFIC REpORTS | (2018) 8:33 | DOI:10.1038/s41598-017-18495-4 ubulo esicula s uc u es, which a e cha ac e is ic o in ec ed cells27, we e an ibody posi i e (3A6 and 5D8/1) and no de ec ed in non-in ec ed cells. 3A6 does no neu alize CVB1. No neu alizing ac i i y o CVB1 was obse ed wi h any o he 14 cul u e hyb idoma supe na an s. Also, no neu aliza ion ac i i y was seen in he o al an ise a o he a s immunized wi h VP1. Figu e 5. 3A6 ecognizes en e o i us in pe sis en ly in ec ed 1.1B4 cells. Con ocal images o PFA- ixed ch onically CVB1-in ec ed 1.1B4 cells (a–d) and non-in ec ed 1.1B4 con ol cells (e) double s ained wi h 3A6 ( ed) and dsRNA an ibody J2 (g een). Me ged images o he double s ained in ec ed- and non-in ec ed cells a e isualized in (d and e), espec i ely. www.na u e.com/scien i ic epo s/ 7 SCIENTIFIC REpORTS | (2018) 8:33 | DOI:10.1038/s41598-017-18495-4 Discussion EV in ec ions a e common, especially in child en, and hei clinical mani es a ions ange om subclinical o se e e o e en a al ou comes. Mos EV in ec ions a e mild, bu ools a e equi ed o de ec ing EVs om clinical samples, including blood, ce eb al spinal luid, s ool and in his ologic sec ions in mo e se ious in ec ions. As mul iple EV ypes a e linked o diseases like i al myoca di is, encephali is and ype 1 diabe es1,28, eagen s ha a e capable o de ec ing a b oad ange o di e en EV ypes wi h bo h high speci ici y and sensi i i y a e needed. Recen ly, he b oad-spec um EV an ibody 5D8/1 om Dako has been egula ly used in bo h clinical and esea ch se ings. This an ibody is a mouse monoclonal ha ecognizes an epi ope wi hin he capsid p o ein VP111,19. 5D8/1 mainly iden i ies EV-B i uses, including CVBs and echo i uses, bu also many species A EVs and polio i us 3 o he C species. Howe e , in non-op imal condi ions 5D8/1 has been epo ed o c oss- eac wi h some cellula p o eins such as CKB and ATP5B12,15. As such, he e is an ongoing e o o p oduce be e an ibodies wi h supe io selec i i y and speci ici y due o he limi in he numbe o speci ic an ibodies ha ec- ognize a b oad- ange o EVs. Wi h his aim, he new b oad- eac i e a monoclonal an ibody 3A6 was gene a ed. 3A6 de ec s all CVBs, along wi h o he EV-Bs (Table1 and Fig.4). In addi ion o b oad- eac i i y, we aimed o p oduce an an ibody ha will u he suppo he indings made wi h o he EV an ibodies, and imp o e de ec ion in di e en expe imen al models, including he mouse. The 3A6 an ibody was success ully alida ed o use in di e en me hods including Wes e n blo ing, pep ide IFA, immuno-TEM, IHC-p and IFA. IHC-P and IFA me hods we e es ed o pa a in and PFA- ixed samples o di e en o igins. 3A6 wo ked well unde he acu e in ec ion se ings, bo h in i o and in i o de i ed models as well as wi h a pe sis en in ec ion in i o model. As a a an ibody, he 3A6 will be especially ad an ageous in mouse models, since no c oss- eac i i y o mouse cellula p o eins was obse ed. We also es ed 3A6 alongside 5D8/1 in human issue samples, some o which had been p e iously con i med posi i e o EVs1. In human sam- ples 3A6 did no show non-speci ic binding in non-in ec ed issues and showed a compa able signal o 5D8/1 in in ec ed issues (Fig.7). 5D8/1 showed eac i i y o smoo h muscle ha was no seen wi h 3A6 (no shown). We also obse ed ha an igen e ie al a pH 9 using T is-bu e esul ed in non-speci ic binding o 3A6 o ed blood cells in some cases, bu his could be a oided by using ci a e bu e a pH 6. We showed ha 3A6 and 5D8/1 co-s ain he same cellula a eas (Supplemen a y Figu eS4), which ende s 3A6 as a alid diagnos ic ool eplica ing he esul s achie ed wi h 5D8/1. Fo ins ance: double s aining wi h 3A6 and 5D8/1 issues in IFA could help alida e incohe en indings as ue posi i es o nega i es o example in sec ions wi h smoo h muscle issue. Figu e 6. 3A6 ecognizes CVBs in in ec ed mouse panc ea a. CVB1- and CVB3-in ec ed mouse panc ea a we e s ained wi h 3A6 by IHC-P and IF (a and b), espec i ely. Posi i e signal om he i us ecogni ion is indica ed by b own (a) and ed colo s (b). The non-in ec ed mouse panc ea a (c) and (d) we e used as nega i e con ols. (a,c and b,d) ha e he same magni ica ion. Blue colo in (b) and (d) is DAPI, which s ains he cell nuclei. www.na u e.com/scien i ic epo s/ 8 SCIENTIFIC REpORTS | (2018) 8:33 | DOI:10.1038/s41598-017-18495-4 The e a e ela i ely ew s udies showing he high-de ail mo phological changes occu ing in EV in ec ions27. He e, we show se e al changes ela ed o in ec ion and eplica ion o EVs in he cy oplasm o acina panc e- a ic cells, including nuclea apop osis, endoplasmic e iculum swelling and no el memb anous s uc u es in he cy oplasm con aining CVB1 VP1 e idenced wi h ei he 3A6 o 5D8/1. Impo an ly, we also show ha 3A6 can be used in combina ion wi h he dsRNA mouse an ibody J2 o simul aneously isualize he localiza ion o i al p o ein and eplica ing RNA wi hin in ec ed cells (Fig.5 and Supplemen a y Figu eS3). These esul s oge he demons a e ha 3A6 can be used o s udying he in acellula localiza ion o he i uses. In Wes e n blo ing, he 3A6 ecognizes he six CVB se o ypes wi h simila sensi i i y, whe eas CVB6 de ec- ion wi h 5D8/1 could ha e been missed, wi h a sho e exposu e ime (Fig.4b and c). Thus, he condi ions in some assays, such as Wes e n blo ing, seem o a o 3A6. Addi ionally, 3A6 has al eady p o en o be e y use- ul in ou own quali y con ols when cul u ing and pu i ying en e o i uses29 and hei VP1 p o eins (Saa inen, unpublished). Al hough 5D8/1 is a b oadly eac i e an ibody, i canno neu alize CVB1 in ec ions in i o13. Also 9D5 lacks neu alizing capaci y, which is unde s andable since i s epi ope, jus like ha in 5D8/1, is no exposed in an in ac i us pa icle14. Simila ly, none o ou 14 indi idual monoclonal an ibodies a ge ing VP1, o polyclonal se a om VP1-immunized a s, could neu alize CVBs. This indica es ha ecombinan p o eins which do no ep esen he Figu e 7. Compa ison o 3A6 and 5D8/1 de ec ion o i us in neona e hea issue wi h IHC-P. Rep esen a i e images o Clone 5D8/1 (a,c and e) and 3A6 (1:700) (b,d and ) s aining in con ol hea (a and b) and hea om neona es wi h ei he a le hal cul u e-con i med CVB2 in ec ion (c and d) o CVB4 in ec ion (e and ). Scale ba 20 μm. www.na u e.com/scien i ic epo s/ 9 SCIENTIFIC REpORTS | (2018) 8:33 | DOI:10.1038/s41598-017-18495-4 na i e EV capsid may no be e icien in he gene a ion o an ibodies capable o blocking i al in ec ion. The abo e obse a ions ques ion he a ionale o VP1-based subuni accines o p e en ing EV in ec ions28. As he numbe o b oadly- eac i e and se o ype-speci ic EV an ibodies expands, he cons uc ion o a panel o an ibodies ha o e lap in hei abili y o bind di e en EVs he de ec ion o g oup and se o ype speci ic EV in ec ions will imp o e. Macca i e al.14 sugges ed ha simul aneous use o 5D8/1 and 9D5 allows o he iden- i ica ion o en e o i us g oups based on hei di e ences in hei abili y o bind o di e en EVs. Adding 3A6 o his panel, which does no bind o EV-A i uses would make such a es mo e accu a e. Impo an ly, binding di e ences could be u ilized o cons uc a mul iplexing applica ion based on biosenso s o iden i y a wide ange o EV se o ypes om liquid samples. Figu e 8. Immunolabeling o CVB1 in ec ed mouse issue in c yo-immuno EM wi h 3A6 and 5D8/1. Sec ions om CVB1 in ec ed (a–d) and non-in ec ed (e, ) mouse panc eas exoc ine issue acina cells h ee days pos in ec ion: labelled wi h an ibodies 5D8/1 (panels a, b and e) and 3A6 (panels c, d and ). In ec ed issues (a,c) show g oss changes du ing in ec ion, and panels on he igh (b,d) show de ails o cy oplasmic memb anous s uc u es posi i e o CVB1 VP1. Do s indica ed by a ows a e p o ein A gold labeled an ibodies. A eas o no el memb anous s uc u es o med du ing in ec ion a e indica ed by as e isks (a,c).