R ev ista d a S o c ie d a d e B ras ileira d e M e d ic in a Tro pical 2 9 ( 5 ) : 4 8 3 - 4 8 9, set- o ut, 1 9 9 6 .
PRO D UCTIO N A N D CHA RA CTERIZA TIO N O F
M O N O CLO N A L A N TIBO D IES TO TH E ED TA EXTRA CT O F
LEPTO SPIR A IN TER R O G A N S,
SEROVAR
IC TER O H A EM O R R H A G IA E
Lilian Terezinha de Queiroz Leite, M auricio Resende, W anderley de Souza,
Elizabeth R.S. Camargos and M atilde Cota Koury
M o no clo nal ant ibo dies (M A Bs) iv ere p ro d u c e d agains t a n et by lenediam inet et raacet at e (E D T A ) e x t ra c t o f Leptospira interrogans s e ro v a r icterohaem orrhagiae b e in g c h a ra c t e riz e d by g e l p recip it at io n as IgM a n d IgG (I g G l a n d IgG 2 b ). The ED TA ex t ract w as det ect ed as s ev eral b a nd s by s ilv er s t a in in g in SD S- PA G E. I n t he W es t ern blo t t he b a nd s a ro u n d 2 0 K D a re a ct e d w ith a m o n o clo na l ant ibo dy , 4 7 B 4 D 6 , a n d w as o x id iz e d by p e rio d a t e a n d w as no t diges t ed by p ro n a s e , s ugges t ing that t he d e t e rm in a n t is o f ca rb o hy d ra t e n at ure , lm m uno cy t o chem is t ry , us ing co llo idal g o ld labeling, s ho w ed t hat a n ED TA ex t ra ct d e t e rm in a n t re c o g n i z e d b y m o n o clo na l ant ib o dy 4 7 B 4 D 6 , is lo c aliz e d u n d e r t he o ut er env elo p e o f s e ro v a r icterohaem orrhagiae. Hoe A IA B ra is e d a gains t t he ED TA ex t ract w as no t ab le to p ro t e ct ham s t ers f ro m let hal ch a ll e n ge w ith v iru len t ho m o lo go us lepto spires.
K ey - w o rds : Leptospira. M o n o clo nal ant ibo dies . P as s iv e p ro t ect io n.
Lep to sp iro sis is a z o o no sis o f w o rld -w id e d istribu tio n, m an b ein g an ac c id en tal ho st.
Sev eral attem p ts hav e b e e n m ad e to iso late le p to sp iral e n d o to x in . Lip o p o ly sac c h arid es (LPS) e x trac te d fro m le p to sp ire s sh o w e d e n d o to x ic p ro p erties'3. So m e au tho rs co u ld no t d em o nstrate any en d o to x in activ ity in LPS e x trac ted fro m le p to sp ires8 7 28. T h e EDTA extrac t fro m lep to sp ires sho w ed b io lo g ical activ ities characteristic o f en d o to xin , altho u g h th ese effec ts w ere less in ten se than th o se o f E. co li O H 1-.B4 LPS'.
Th e d ev elo p m en t o f hy brid o m a tec h no lo g y fac ilitate d th e p ro d u c tio n o f m o n o c lo n al an tib o d ies that co u ld b e u sed in id entificatio n, lo c aliz atio n o f an tig en ic d eterm inants and stu d ies o f p ro tectiv e cap acity .
D epartam entos de M icrobiologia e M orfologia do Instituto de Ciências Biológicas da U niversidade Federal de M inas G erais, Bclo H orizonte, M G e Laboratório de Ultra-Estrutura Celular do Instituto de Biofísica Carlos Chagas Filho, U niversidade Federal do Rio de Janeiro, Rio de Janeiro, RJ. This study w as supp orted in part b y Fundação de A m paro a Pesquisa do Estado de M inas G erais (FA PEM IG).
A d d r e s s t o : P ro f * . M atil d e C o ta K o u ry ., D e p t° d e M icrobiologia/ICB/U FM G , C aixa Postal 4 8 6 , 3 1 270- 901 Belo H orizonte, M G, Brasil.
Receb id o p ara p ub licação em 2 3 / 1 1 / 9 5 .
Lep to sp ire g enu s sp e c ific d eterm inant o f th e p ro te in an tig e n (G P-A g ) o f se ro v ar krem asto s rec o g niz ed b y m o n o c lo n al antib o d y w as lo c a te d o n th e s u b - s u rf a c e o f th e le p to s p ira l e n v e lo p e b y im m u n o e le c tro n m icro sco p y and sero v ar sp e c ific TM antig en (LPS) w as lo c aliz ed o n the c ell su rfac e23. Th e sam e p ro tein antig en w as lo c aliz ed o n the o u ter m em b rane o f n o n p ath o g en ic sero v ar
a n d a m a n a r. Th e LPS o f sero v ar h a rd j o w as lo c aliz ed o n the o u ter m em b rane b y m eans o f a m o no clo nal antibo d y rev ealed b y im m u no g o ld s ta in in g in e le c tr o n m ic r o s c o p y 29. T h e d eterm inant to w h ic h the M A Bs ag ainst the o u ter en v elo p e (O E) o f sero v ar c o p e n h a g e n i
w ere d irected w ere lo c aliz ed in the lep to sp iral O E b y the im m u no g o ld lab elin g te c h n iq u e “ .
M A Bs ag ainst v ario u s antig enic su b stan c es o f L ept o s pira w ere p rep ared and sh o w ed p ro tectiv e cap acity . Th e M A B o b tain ed ag ainst a g ly co lip id (O E) p assiv ely p ro tec ted ham sters f ro m l e p to s p i r a l i n f e c t i o n 20. H a m s te r s im m u niz ed w ith the M A B ag ainst sero v ar
Leite LTQ , R e s en d e M , So uz a W , C am argo s ERS, K o w y M C . P ro duct io n a n d cha ra ct eriz a t io n o f m o n o clo na l a nt ib o dies to t he ED TA ex t ra ct o / T ep to sp ira interrogans, s e ro v a r ictero h aem o rrh ag iae. R ev ista d a S o c ie da de B ras ileira d e M e d ic in a Tro pical 2 9 : 4 8 3 - 4 8 9 , set- o ut, 1 9 9 6 .
sero v ar C o p e nh a gen ? 1. H o w ev er, the M A Bs a g a i n s t th e o u te r e n v e l o p e o f s e r o v a r
C o p enhagenid id n o t p ro tec t new -b o rn g u inea p ig s fro m c h allen g e w ith v iru lent lep to sp ires16 and M A B LW 4 ag ainst sero v ar la i Fractio n 1 o f P-A g also d id n o t p ro tec t ham sters o f lethal c h allen g e20.
Th is p ap e r rep o rts the p ro d u c tio n o f m o n o c lo n al an tib o d ie s ag ain st the ED TA e x trac t o f L e p t o s p ira i n t e rro g a n s , sero v ar
i c t e ro h a e m o rrh a g i a e , their c harac teriz atio n, lo c aliz atio n and p assiv e p ro tec tio n test.
M A TERIA LS A N D M ETH O D S
B a c t e ri a l s t ra in a n d g ro w t h co n d it io n s . Lepto spira interro gans, sero var ictero haem o rrhagiae,
strain RGA , w as g ro w n in sy nthetic m ed iu m 25 at 28°C fo r 7-10 d ays and harv ested b y cen trifu g atio n at 6,800g fo r 20 m inu tes at 4°C and ke p t at -20°C until u se. Th e v iru lent strain o f lep to sp ire w as iso lated fro m a p atient w ith lep to sp iro sis in H o sp ital Sarah K u b itsc h ek ( Be lo H o riz o nte, M G , Braz il) and w as g ro w n in liq u id m ed iu m (EM JH )15. It w as id entified b y the Po ly m e rase C hain R eac tio n (PC R) as b elo n g in g to sero v ar i c t e ro h a e m o rrh a g i a e '.
P re p a ra t io n o f t he ED T A e x t ra ct . L ept o s pira
c ells w ere ex trac ted b y the EDTA m etho d o f Leiv e and Sho v lin19. Th e c ells w ere w ashed th ree tim es w ith 0.12M Tris-H C l, p H 8 , ressu sp en d ed in the sam e b u ffer and b ro u g ht to 37°C . EDTA (in the sam e b u ffer) w as ad d ed . Th e final v o lu m e c o n tain ed 0.2g , w et w eig ht, o f c ells p er m illiliter and 0.01M EDTA . A fter 4 m inu tes o f g en tle ag itatio n at 37°C , M gCl w as ad d ed (0.05M final c o n c en tratio n ) and the cells w ere cen trifu g ed at 6,800g fo r 20 m inu tes. The su p ernatant w as filtered thro u g h m illip o re filters (0.45m m p o re siz e), d ialy z ed at 4°C , first ag ainst 0.1M so d iu m p h o sp hate, pH 7.0, fo r l6 - 2 4 h and then ag ainst sev eral ch ang es o f d istilled w ater fo r 2-4 d ays. Th e no n-d ialy z ab le m aterial w as ly o p h iliz ed . H ereafter, this m aterial is term ed the ED TA extract.
P a rt ia l c h e m i c a l a n a ly s es . Th e LPS w as m easu red b y the m eth o d o f Jan d a and W o rk1", u sin g E . c o li 0 1 1 1 :B 4 (D ifc o Lab s.) as stand ard . To tal c arb o h y d rate c o n te n t w as m easu red b y the p heno l-su lfu ric acid m eth o d 5 u sin g g lu c o se as stand ard . Pro te in w as estim ated ac c o rd in g to the m o d ified m etho d o f Lo w ry u sing b o v in e alb u m in as stand ard 11’.
P ro d u c t i o n a n d s c re e n i n g o f m o n o c lo n a l a n t ib o d ie s . Fo u r w e e k s o ld fem ale BA LB/ c
m ice w ere im m u niz ed in trap erito neally (i.p .) w ith the ED TA extrac t (200p g o f LPS) o n d ays z ero and 58. Sp lee n c ells w ere harv ested fo u r d ay s later an d fu sio n w as p erfo rm ed as d e s c rib e d b y G alf re an d M ilste in 9 w ith P3X63/ A g 8653 m y elo m a cells. H y brid o m as w ere g ro w n in D u lb e c c o ’s m inim al essen tial m ed iu m p lus hy p o xanthine, am ino p terine and thy m id ine (H A T) su p p lem en ted w ith 20% fetal c alf seru m , l.Om M p y ru v ate, 1 .OmM o x alo ac etic acid , 2.OmM L-g lu tam ine, 0.1 e ac h o f a m ixture o f no n -essen tial am ino acid s, 15.OmM H EPES and 0.2 u nits o f b o v in e insu lin. H y b rid o m as w ere screened b y enzy m e-linked im m uno so rbent assay (ELISA ) u sing the ED TA ex trac t as antig en. Th ey w ere c lo n e d tw ice b y lim iting d ilutio n. Th ree M A Bs w ith hig h reactiv ity in ELISA b esid e s g o o d g ro w th characteristics, w ere se lec ted fo r further stud y.
E n z y m e - l i n k e d i m m u n o s o rb e n t as s ay . A n ind irect ELISA w as c arried o u t ac c o rd in g to A d ler et al1. M icro titer p lates w ere c o ated eith er w ith the EDTA extrac t (200n g LPS/ w ell) in a 0.06M c arb o n ate -b ic arb o n ate buffer, p H 9-6, fo r 24h at 4°C . T h e w ells w ere b lo c k e d w ith a 2% c asein -p h o sp h ate b u ffer so lu tio n, p H 7.6. Be tw e e n e ac h stag e, the p late w as su bm itted to a series o f w ashing s w ith b u ffer so lu tio n-Tw een 20. Th e w ells w e re p ro b ed w ith the sup ernatant fo r lh at ro o m tem p eratu re. A g o at anti-m o u se h o rserad ish p ero xid ase c o n ju g ate d iluted at 1/ 800 reac ted w ith the antig en-antib o d y c o m p lex fo r 45 m inu tes at ro o m tem p eratu re. T h e c o m p le x fo rm ed w as rev ealed b y 0.02M o rto p h en y len -d iam in e (M erck) d ilu ted in a 0.01M citrate b u ffer and ad d ed o f 0.03% H O fo r 15 m inu tes in the d ark. Th e reac tio n w as sto p p ed b y 4N H 2 -SO^ and read in 492p m (M ultiskan, Finlend ). T h e c u t o ff co nsid ered w as tw ic e the av erag e o p tical d ensity (O D ) o f a le p to sp ire-ab so rb ed m o u se seru m p lus o n e stand ard d ev iatio n.
M i c r o s c o p i c a g g l u t i n a t i o n t e s t (M A T ).
T h e M A T w a s p e r f o r m e d a c c o r d i n g to t h e g u i d e l i n e s o f t h e W o r l d H e a l t h O rg aniz atio n6.
Iso ty pes. The m o n o c lo n al an tib o d ies w ere id entified b y th e d o u b le im m u n o d ifu sio n m etho d 22 u sing anti-m o u se iso ty p ing reag ents (Sigm a,USA ).
Leit e LTQ , R es en de M , S o uz a W , C a m argo s ERS, K o ury M C . P ro d uc t io n a n d ch a ra ct e riz a t io n o f m o n o c lo na l ant ib o dies to t he ED TA ex t ract o / -Lep to sp ira interrogans, s e ro v a r ictero h aem o rrh ag iae. R ev ista d a S o cie d a d e B ras ileira d e M e d ic in a T ro pical 2 9 :4 8 3 - 4 8 9 , set - o ut, 1 9 9 6 .
fo u r ho u rs at 150V and eith er silv er-stained 12 o r transb lo tted o n to n itro c ellu lo se m em b ranes27. Th e m em b ranes w ere slic ed into strip s, so m e o f w h ic h w e re treated b y p erio d ate, p ro nase o r w e re le ft u n treated . O n e strip w as th e n in c u b a te d f o r 1 h o u r w ith th e an ti-ED T A ex trac t M A B. Th e o th er strip s w ere incu b ated w ith anti-ED TA p o ly c lo n al an tib o d y . Th e i m m u n o d e t e c t i o n w a s c o m p l e t e d b y in c u b atin g w ith a g o at anti-m o u se ho rserad ish p ero xid ase co nju g ate. D iam ino b enz id ine and 4-c h lo ro n ap h to l w e re u se d to rev eal the b and s11.
ED T A e x t ra c t t re a t m e n t w ith p ro n a s e a n d p e ri o d a t e . Tran sb lo tted ED TA e x trac t w as treated w ith p ro nase (100p g / m l-Pro m eg a) in PBS, p H 7.2 at 37°C fo r 24h. Perio d ate o xid atio n w as p erfo rm ed as d esc rib e d b y O m e r et al21.
E le c t ro n m icro s co p y . L. i n t e rro g a n s, sero v ar
i c t e ro h a e m o rrh a g i a e w as g ro w n in liq u id m ed iu m 15 fo r fiv e d ays and harv ested b y c entrifu g atio n at 6.800g fo r 15 m inu tes. Th e o u te r e n v e lo p e w as re m o v e d b y th ree e x p o su re s to d istilled w ate r fo llo w e d by centrifu g atio n26. T h e p e lle t w as fix e d b y 0.1% g lu tarald ehy d e fo r 15 m inu tes at 4°C and w ash ed three tim es w ith PBS p H 8 . M A B 47B4D 6 w as b o u n d to the lep to sp ires after an in c u b atio n o f o n e ho u r at 37°C . Th ree m o re w ashing step s fo llo w ed . A co llo id al g o ld g o at anti-m o u se im m u no g lo b u lin (A u ro p ro b e EM GA M 10, Ey Lab s) w as u sed as se c o n d lab e l fo r o n e h o u r at 37°C . A fter rep eated w ashing s, the p e lle t w as d ep o sited o n a c arb o n -c o ate d fo rm v ar c o p p e r g rid . Ex c e ss liq u id w as rem o v ed w ith filter p ap er. T h e m aterial w as c o ntrasted b y 2% am m o niu m m o ly b d ate and o b se rv e d , after d ry ing , in a tran sm issio n elec tro n m ic ro sc o p e at 80 KV (Z eiss).
P as s iv e p ro t e c t io n as say . Six g ro u p s o f fiv e g o ld en ham sters (three w ee k s o ld , 30 g ram s av erage w eig ht) w ere ino culated intraperito neally w ith 1m l o f cu ltu re su p ernatant and 24 ho u rs later c h alle n g ed b y the sam e ro u te w ith virulent
L. int erro gans , sero var ict ero haem o rrhagiae (107 cells/ m l). D eath s w e re rec o rd ed and the anim als sh o w ed sy m p to m s ty p ical o f acu te se v ere le p to sp iro sis (arc h ed b ac k , icteru s, hem o rrhag es).
RESULTS
S c re e n i n g o f m o n o c l o n a l a nt ib o d ie s . The p artial c h em ic al analy sis o f the EDTA extrac t rev ealed the p re se n c e o f LPS (37.8% ), p ro tein (18.3% ) and p o ly sac c h arid es (9.7% ).
Fo u rte e n h y b rid o m a c e ll lin e s w e re p ro d u c ed ag ain st the ED TA ex trac t. Th e sc reenin g w as m ad e by ELISA . Th e cu ltu re su p ernatants, tested ev ery 48 ho u rs, reacted w i t h t h e ED T A e x t r a c t o f s e r o v a r
i c t e ro h a e m o rrh a g i a e and sh o w e d an O D o f 0 . 4 1 9 t o 0 . 9 6 9 - A l l o f t h e m w e r e n o agglutinating.
Th e M A Bs w ere o f the fo llo w ing iso ty p es: 3 Ig G l (47B4D 6, 47B4D 11, 47B4F5), 7 Ig G 2b (47C 1C 4C 8, 47C 1C 4D 4, 47C 1C 5, 47C 1C 7, 47A 5G 11C 9, 47A 5G 11D 2, 47A 5B4B11) and 1 IgM (47C 1C 11). Th ree c o u ld n o t b e tested .
T h re e o f th e m w e re c h o s e n f o r th e exp erim ents, M A Bs 47B4D 6, 47C 1C 4C 8 and 47C 1C 11. This se lec tio n w as b ased o n rap id ity o f g ro w th, titers in ELISA and iso ty p e.
G el e le c t ro p h o re s is a n d im m u n o b l o t t in g a n t i g e n s a n t ig e n s . Th e SD S-PA GE o f the EDTA extrac t d em o nstrated sev eral b and s. So m e o f them m ig rated to the sam e p o sitio n s as tho se o f E. co li O H l :B 4 LPS sho w ing the sam e rep etitiv e p o ly saccharid e p attern (Fig u re 1).
Th e results o f the d eterm inatio n o f the chem ic al nature o f the an tig en ic d eterm inants o f the ED TA e x trac t b y W estern b lo t are sho w n in Fig ure 2. Th e p o ly c lo n al anti-ED TA extrac t antib o d y (Strip 1) rev ealed b and s fro m 17 to 67 kD a. W hen treated w ith p erio d ate (Strip 2), the e x trac t b an d s aro u n d 20k D a h ad th e ir intensities d im inished . Strip 3 sho w s the b and s that w ere n o t affec ted b y p ro nase treatm ent. Strip 4 sho w s the extrac t b and s rev ealed by M A B 47B4D 6. A lso M A Bs 47C 1C 11 and 47C 1C 4C 8 w ere tested and sh o w ed the sam e result. Th e b and s w ere o xid iz ed b y p erio d ate and n o t d ig ested b y p ro nase, su g g esting that the d eterm inant is a carb o hy d rate.
E le c t ro n m icro s co p y . Th e lo c aliz atio n o f the EDTA extrac t d eterm inant in lep to sp ires o f sero v ar i c t e ro h a e m o rrh a g i a e w as d o n e b y M A B 4 7 B 4 D 6 . F i g u r e 3 s h o w s a n electronmicrograph o f sero v ar ict ero haem o rrhagiae.
Leit e LTQ , R e s e n d e M , S o u z a W. C a m argo s ERS, K o ury M C . P ro d uct io n a n d characte> ~ iz atio n o f m o n o clo na l ant ib o dies to the ED TA ex t ract o f Leptospira interrogans, s e ro v a r zcterohaemorrhagiae. R ev ista d a S o cie d a d e B ras ileira d e M e d ic in a Tro pical 2 9 . 4 8 3 - 4 8 9 , set- o ut, 1 9 9 6 .
su rf ace l ay e r o f th e l ep to sp ire, u n d e r th e O E
( 56,000 X ).
P as s iv e p ro t e c t io n as s ay . T h e results o f the p assiv e p ro tec tio n assay w ith M A B 47B4D 6 is sh o w n in Tab le 1. T h e c o ntro l anim als d ied after an av erag e su rv iv al tim e o f 3-3 d ays. Th o se g ro u p s that rec eiv ed the und ilu ted m o n o c lo n al antib o d y h ad an av erag e surv iv al o f 5.2 d ay s and th o se that rec eiv ed the 1/ 10 d ilu tio ns o f 4.0 d ay s. T h e kid ney s o f the
T able 1 - Passiv e p ratec tio n o f ham sters by m o n o c lo n al an tibody 4 7 B 4 D 6 ag ain st c hallen g e tuith Leptospira in terrog an s, sero v ar ic t e r o b a e m o r r h a g ia e .______ _______
M o n o c lo n al an tib o d y N u m b er o f surv iv o rs/ Su rv iv al av erag e 47B4D 6 an im als te sted (d ay s) U n d ilu ted c u ltu re
su p e rn atan t 0/ 5 5.2
C u ltu re su p e rn atan t
(1/ 10 d ilu tio n ) 0/ 5 4.0
C o ntro l 0/ 5 3.3
Leit e LTQ , R e s e n d e M , S o u z a W. C a m argo s ERS, K o ury M C . P ro d uct io n a n d ch a ra ct eriz a t io n o f m o n o c lo na l ant ib o dies to t he ED TA ex t ract o/T eptospira interrogans, s e ro v a r z'cterohaemorrhagiae. R ev ista d a S o c ie d a d e B ras ileira d e M e d ic in a Tro pical 2 9 : 4 8 3 - 4 8 9 , set- o ut, 1 9 9 6 .
su rface lay er o f the lep to sp ire, u nd er the O E ( 56,000 X ).
P as s iv e p ro t e c t i o n as s ay . Th e resu lts o f the p assiv e p ro tec tio n assay w ith M A B 47B4D 6 is sh o w n in Tab le 1. Th e c o ntro l anim als d ied after an av erag e surv iv al tim e o f 3-3 d ays. T h o se g ro u p s that rec eiv ed the u nd ilu ted m o n o c lo n al antib o d y had an av erag e surv iv al o f 5.2 d ay s and th o se that rec eiv ed the 1/ 10 d ilu tio ns o f 4.0 d ay s. T h e kid ney s o f the
Table 1 - Passive prote ction o f hamsters by m onoclonal antib ody 47B 4D 6 agains t challenge ivith Leptospira inten-ogans. s erov ar icte rohaem orrhagiae. ________
M o n o c lo n al an tib o d y 4 7B4 D 6
N u m b e r o f su rv iv o rs/ an im als tested
Su rv iv al av erag e (d ay s) U n d ilu ted c u ltu re
su p ern atan t 0/ 5 5.2
C u ltu re su p e rn atan t
Cl/ 10 d ilu tio n ) 0/ 5 4.0
C o ntro l 0/ 5 3.3
Leite LTQ , R es en de M , S o u z a W , C a m argo s ERS, K o ury M C . P ro d uc t io n a n d ch a ra ct e riz a t io n o f m o n o c lo na l ant ib o dies to t he ED TA ex t ract o/Xeptospira interrogans, s e ro v a r icterohaemorrhagiae. R ev ista d a S o c ie da de B ras ile ira d e M e d ic in a T ro p ical 2 9 :4 8 3 - 4 8 9 , set- o ut, 1 9 9 6 .
f i g u r e 3 - Elect ro n m icro g rap h o/ Lep tosp ira interrogans, s e ro v a r icterohaem orrhagiae. 3 . 1 W ith t he o u t e r env elo p e a ft e r re ac t io n w ith M A B 4 7 B 4 D 6 a n d a co llo idal g o ld g o a t an t i- m o us e co nj ugat e. 3 0 , 0 0 0 x B a r = O .lu m . 3 . 2 w itho ut t he o u t e r env elo p e, 3 0 , 0 0 0 x .
3.3
W itho ut t he o u t e r e n v e lo p e b o u n d to t h e m o n o c lo n a l a n t ib o d y 4 7 B 4 D 6 a n d to a g o a t - a n t i- m o u s e c o llo id al g o l d co nju gat e, 5 6 , 0 0 0 x , B ai- = 0 .2 ^ m .anim als w ere cu ltu red fo r 30 d ays and no ev id en c e o f lep to sp ira g ro w th w as o b serv ed .
D ISCUSSIO N
Fo u rteen M A Bs ag ainst the EDTA extract w ere p ro d u c ed and c h arac teriz ed as Ig G l (3), Ig G 2b (7), IgM ( 1) and three w ere n o t tested . A ll o f them w ere n o n ag g lutinating . Th e M A Bs p r o d u c e d a g a i n s t t h e O E o f s e r o v a r
c o p e n h a g e n i , w ere als o n o n ag g lu tinating 16. T h e stud y o f th e c h em ic al natu re o f the antig enic d eterm inant o f the ED TA extrac t by PA GE and W estern Blo t (W B) tec h niq u es sh o w ed that this extrac t w as n o t affected b y p ro n ase , b u t w as o x id iz ed b y p erio d ate, s u g g e s t i n g a c a r b o h y d r a t e n a t u r e .
Im m u no d iffu sio n and W B exp erim ents sho w ed that M A B M UM / FI-1 o f sero v ar c o p e n h a g e n i
reac ted w ith a c arb o hy d rate d eterm inant in the
lept o s piral LPS17. Th e M A B ag ainst sero v ar
c o p e n h a g e n i also reac ted w ith an ep ito p e o f carb o hy d rate natu re w h en so n ic ated antig ens w ere d ig ested w ith p ro n ase24. T h e M A B ag ainst the O E o f sero v ar h a rd j o reac ted w ith a p ro tein d eterm inant in W B ex p e rim en ts16.
W hen lo c aliz ing the an tig en ic d eterm inant, w e o b serv ed that M A B 47B4D 6 b o u n d to the
lep t o s p ira c ell o nly after the rem o v al o f the O E, ind icating that it is u nd er this stru ctu re. The M A B ag ainst the g enu s sp e c ific p ro tein antig en (G P-A g ) o f sero v ar k rem as t o s , lab eled w ith p ero xid ase o r io d inated , also b o u n d to the su b -su rface o f the O E23. H o w ev er, the M A B ag ain st G P-A g o f n o n -p ath o g e n ic sero v ar
a n d a m a n a b o u n d to the O E2. Th e M A B ag ainst LPS o f sero v ar h a rd j o o r the O E o f sero v ar c o p e n h a g e n i b o u n d to th e o u ter m em b rane o f lep to sp ire as e v id e n c e d b y im m u no g o ld lab eling tec h n iq u es“ 29.
T h e M A B ag ainst th e ED TA e x trac t o f sero v ar i c t e ro h a e m o rrh a g ia e , w h en in o c u lated in ham sters co u ld n o t p ro tec t them fro m lethal c h alle n g e w ith v iru len t le p to sp ire . M A B ag ainst sero v ar c o p e n h a g e n i and M A B LW4 ag ainst sero v ar lai d id n o t p ro tec t n ew -b o rn g u in ea p ig s16 o r h am sters20 ag ain st le th al in fe c tio n . H o w ev er, M A B ag ain st se ro v ar
c o p e n h a g e n i co u ld p assiv ely p ro tec t g u inea- p ig s ag ainst lethal lep to sp iro sis w h en d aily ad m inistered in the d o se o f 175m g to ensu re a p ro tectiv e titer17. Th e M A Bs ag ainst sero v ar
c o p e n h a g e n i also p ro tec ted ham sters, d o g s and m o n key s24 and the M A B o b tain ed ag ainst a g ly co lip id o f th e O E (PA g) fro m sero v ar lai
p ro tected ham sters fro m lept o s pirdi in fe c tio n 20. Im m u niz ed ham sters w ith V I M A B ag ainst sero v ar i c t e ro h a e m o rrh a g i a e w ere p ro tec ted fro m lethal c h alleng e w ith the h o m o lo g o u s sero v ar31.
In c o n c lu sio n , w e hav e a n o n ag g lu tinating anti-ED TA extrac t M A B, that d id n o t p ro tec t h a m s t e r s f r o m c h a l l e n g e w i t h v i r u l e n t lep to sp ires o f the h o m o lo g o u s sero v ar. Th e d eterm inant is o f c arb o hy d rate natu re and is lo c ated u nd er the lept o s piral O E.
RESUM O
Leite LTQ , R es en de M . So uz a W , C a m argo s ERS, K o ury M C . P ro d uc t io n a n d cha ract eriz a t io n o f m o n o c lo na l ant ib o dies to t he ED TA ex t ra ct o f Lep tosp ira in terrogans, s e ro v a r z ctero h aem o rrh ag iae. R ev ista d a S o c ie da de B ras ileira d e M e d ic in a Tro pical 2 9 : 4 8 3 - 4 8 9 , set- o ut, 1 9 9 6 .
P elo teste d e p re c ip it a ç ã o f o ra m ca ra ct e riz ad o s co m o IgM e Ig G (Ig G l e IgG 2 ). A elet ro fo res e e m g e l d e p o lia crila m id a do ext rat o ED TA rev elo u div ers as b a n d a s q u a n d o co ra d a p e la p ra t a . N o “W estern blot" , as b a n d a s e m t o rno d e 2 0 kD a re ag iram co m o A cM 4 7 B 4 D 6 , f o ra m o x id ad as p e lo p erio d at o e n ã o d ig e rid a s p e l a p ro n a s e , s u g e ri n d o q u e o d e t e rm i n a n t e é d e n a t u re z a ca rb o id ra t o . O d e t e rm in a n t e re co n h e c id o p el o A cM 4 7 B 4 D 6 estã lo c aliz ado s o b o env elo p e ex t e rn o co m o rev elado p e l a i m u n o c it o q u ím ic a u s a n d o m a rc a ç ã o co m o uro co lo idal. O A cM co nt ra ext rat o ED TA do s o r o v a r icte ro h a e m o rra h a g i a e n ã o p r o t e g e u h a m s t e rs q u a n d o i n o c u l a d o s c o m l e p s t o p ira ho m ó lo ga v irulent a.
P a l a v r a s - c h a v e s : L e p to s p i ra . A n t i c o r p o s m o no c lo na is . P ro t eção p as s iv a.
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