Rev. Inst. Med. trop. São Paulo 31(4):228-234, julho-agosto, 1989.
A N A L Y S I S O F T HE S P E C I F I C I T Y O F H U M A N A N T I B O D I E S T O A N T I G E N S O F L E I S H M A N I A B R A Z I L I E N S I S B R A Z I L I E N S I S
Aoi M A S U D A (1) , Suel i Fátim a d o N A S C I M E N T O (1), Carme m Silvi a G U E R R A (1) , Gláuci a d a Silv a P A R A N H O S (1 ) & Antoni o Walte r F F R P ^ " ? *
S U M M A R Y
The antigenicity o f promastigote s o f Leishmani a braziliensis braziliensi s ( L . b . braziliensis) treated with 1 % sodiu m desoxycholat e i n 1 0 mM Tris-Hc l pH 8. 2 was analysed b y immunoblo t usin g a s probe s sera fro m America n cutaneou s leishmaniasis ( A C L ) , America n viscera l leishmaniasi s ( A V L ) , schistosomiasis , malaria an d Chagas ' disease . The A CL sera reacted constantl y wit h a 60 kD band . No reactivit y t o thi s protei n wa s observed wit h sera from th e othe r disease s above mentioned indicating tha t th e 6 0 kD protein may b e used in serodiagnosi s for A C L. K E Y W O R D S : Antigen s o f Leishmani a braziliensi s braziliensis ; Serodiagnosis o f American cutaneou s leishmaniasis .
I N T R O D U C T I O N The leishmaniase s compris e a comple x o f
protozoal parasite s whic h caus e visceral , cutaneous an d mucocutaneou s diseases . A number o f biologica l characteristic s hav e bee n identified b y whic h th e leishmania s ca n b e classified a s differen t species , suc h a s electrophorectic mobilit y o f isoenzymes7 1 5, th e buoyant densit y o f nuclea r an d kinetoplas t D N A5, b y analysin g sequenc e homologie s i n kinetoplast D N A 1 o r b y th e us e o f D N A probes3. Also , monoclona l antibodie s hav e been show n t o differentiat e specie s an d subspecies o f leishmania s whic h cause s th e A C L1 3 1 4, greatl y advancin g th e understandin g of th e antigenicit y o f th e leishmanias , thu s facilitating th e identificatio n an d characteriz -ation o f component s potentiall y usefu l i n immunoprofilatic procedures 4 o r havin g th e possibility o f interferin g i n parasit e hos t interactions16.
For serodiagnosis , B A D A R Ó e t al.2, have described th e us e o f a solubl e antige n i n micro-enzyme linke d immunosorben t assa y ( E L I S A ) t o antibodie s i n A V L whic h give s sensitive an d specifi c tes t abl e t o eliminat e cross-reactivity wit h Trypanosoma cruzi . More recently, S A N T O S e t al.1 7 characterize d tw o polypeptides o f 11 9 k D an d 12 3 k D o f Leishmania donovan i chagas i whic h d o no t cross-react with Chagas'diseas e sera . I n regar d to A C L a 7 2 k D protei n specifi c fo r L.b . braziliensis, whic h i s recognize d b y ser a obtained fro m mucocutaneou s leishmaniasis , was described11. Most recently U L R I C H e t al.1 9 showed tha t seria l absorptions o f th e sera with well characterize d strain s ca n turn ou t E L I S A assay, usin g whol e formali n treate d parasites , specifically reactiv e t o certai n strains . However, a wel l characterize d Leishmani a species specific antigen fo r th e serodiagnosi s o f
(1) Laboratóri o d e Sorologi a e Imunologi a d o Institut o d e Medicin a Tropica l d e Sã o P a u l o , S P , Brasil.
Address fo r correspondence : D r . Antoni o Walte r Ferreira . Institut o d e Medicin a Tropica l d e Sã o Paulo . A v . D i . Enéa s d e Carvalho A g u i a r , 470 . C E P 05403 Sã o P a u l o , S P , Brasil.
cutaneous an d mucocutaneou s leishmaniase s which doe s not cross-reac t with Trypanosoma cruzi is yet to b e found.
The A C L ar e disease s endemi c i n Brazi l and ar e mainl y cause d b y thre e differen t protozoan parasite s (Leishmani a braziliensis, braziliensis, Leishmania braziliensis guyanensis and Leishmani a mexican a amazonensis ) and their prevalenc e areas often overla p with areas endemic fo r Chagas'disease , posin g a serious problem fo r th e interpretatio n o f immuno -diagnostic tes t fo r leishmaniasis.
The wor k aime d t o identif y antigen s specifically reactiv e wit h antibod y o f patient s with American cutaneous leishmaniasis .
M A T E R I A L S A N D M E T H O D S Sera
A tota l o f 12 4 ser a wer e analyse d in thi s study an d they wer e distributed a s follows: Group l a 2 3 sera o f patient s wit h cutaneou s leishmaniasis wer e obtaine d a t "Institut o d e Medicina Tropica l d e Manaus" ; 9 sera o f patients wit h cutaneou s leishmaniasi s and 1 0 sera o f patient s wit h mucocutaneou s leishmaniasis wer e obtaine d a t "Hospita l da s Clínicas d a Faculdad e d e Medicin a d a Universidade d e São Paulo".
Group l b 1 0 sera fro m patient s wit h leishma-niasis caused by L. b. braziliensis were obtained at "Universidad e Federa l d a Bahia " (kindl y provided by Dr. Robert o Badaró) .
Group I I 2 0 ser a o f patient s wit h Chagas'disease, 1 2 patients wit h A V L ; 1 0 ser a of patient s wit h malaria ; 1 0 sera o f patient s with schistosomiasis .
Group I I I 2 0 ser a obtaine d fro m clinicall y healthy individual s (controls) .
Antigen Preparatio n
Leishmania braziliensi s braziliensi s ( M H O M / B r / 7 5 / M 29 3 strain ) wer e cultivate d in Schneider' s medium supplemente d with 2 0 % fetal cal f serum8. The parasites were harvested in late Log phase, washed five times (3.00 0 x g, 4 ° C , 1 5 min) i n phosphat e buffered salin e pH 7.2 (PBS) . Th e parasite s wer e incubate d wit h 1% sodiu m desoxycholat e in 1 0 mM Tris-HC l
pH 8. 2 containin g 1 0 ^g/m l of NTos y 1L -phenylalanine chloromethy l ketone-TCP K (Sigma Chemical , Co ) a t a concentratio n o f 101 0 parasites/m l after vigorou s vortexing , th e extract wa s centrifuged a t 40,00 0 x g , 4 ° C , 6 0 min. Th e supernatan t wa s take n an d protei n content wa s determined b y the Lowry method12. Micro-enzyme immunoassay ( E L I S A)
Fifty microlite r o f th e antige n extract was placed i n microtite r plat e well s a t a concentration o f 2 0 ^g/m\ i n carbonat e buffe r 0.01 M p H 9. 6 an d incubate d overnigh t a t 37°C. Th e well s wer e washe d wit h PB S and blocked with 5 % skim milk i n PB S (SK-PBS) . Then, th e tes t ser a i n 10 0 \A amount s wer e added t o eac h wel l i n doublin g dilution s an d incubated fo r 1 h a t 37°C . After , th e well s were washe d wit h 0 . 5 % solution o f P B S -Tween 2 0 and incubate d wit h a 100M 1 o f goa t anti-human Ig G ( 1 / 4 0 0 0 ) o r goat-antihuma n IgG ( 1 / 2 0 0 0 ) conjugate d t o horseradis h peroxidase fo r 1 h a t 37°C . Th e reaction wa s developed wit h orthophenilen e diamino -b e n z i d i n e - H202 (Sigm a Chemical , C o ) , stopped wit h 2. 5 N H2S 04 an d well s content s were rea d a t 49 0 n m i n Dynatec h Mini-reade r (Dynatech Lab.) .
Immunoblot
The antige n extrac t a t a concentration o f 500 Mg/m l dilute d i n sampl e buffer containin g 1% sodiu m dodecy l sulfate, urei a 1 M, 0. 5 M DL-dithiothreitol, 1 0 % glycerol an d 0 . 0 0 2 % bromophenol i n Tris-HC l buffe r p H 6. 8 was placed ont o a 1 0 % polyacrylamid e gel1 0. Afte r electrophoresis, the proteins were transferred t o nitrocellulose membran e (Millipore ) i n a transblotting chambe r containin g Tri s (hydroxymethil amin o methane ) 2 5 mM , glycine 19 2 mM an d 2 0% methano l at pH 8.31 8. After transferin g an d blocking the fre e bindin g sites o f th e membran e wit h 5 % S K - P B S , vertical strips were cu t an d the test sera diluted 1/40 i n 5 % SK-PBS wer e incubate d fo r 2 h at room temperature . Then , the y wer e washe d with PB S an d incubated with goat anti-huma n IgG ( 1 / 2 0 0 0 ) o r Ig M ( 1 / 1 0 0 0 ) conjugate d t o horseradish peroxidas e fo r 1 h a t roo m temperature. Afte r washing , th e reactio n wa s
developed with diaminobenzidine-H202 (Sigma Chemical, Co).
R E S U L T S E L I S A
The 4 2 sera o f patient s wit h cutaneou s or mucocutaneous leishmaniasi s were titrate d b y E L I S A usin g a s th e limi t o f reactivit y th e arithmetic mea n o f th e absorbanc e obtaine d from 2 0 norma l ser a dilute d 1/4 0 plu s 2
standard deviation s (cu t of f = 0.32). B y th e results show n i n fig . 1 , w e observe d that th e titer o f Ig G anti-leishmanial antibodie s i n th e sera wa s low. Onl y 2 sera in 4 2 samples tested were positiv e a t 1/64 0 dilution . A t 1/4 0 dilution al l sera wer e positiv e an d thi s result s were confirmed by indirect immunofluorescence assay (IFA ) using antigens of promastigote s o f L. b . braziliensi s fixed i n 2°7o formalin .
The 1 0 ser a o f patient s diagnose d a s cutaneous leishmaniasi s cause d b y L . b . braziliensis wer e teste d a t a dilutio n o f 1/4 0 (Fig. 2) . Th e reactio n wit h peroxidas e conjugated t o anti-huma n Ig G showed tha t 3 sera wer e negativ e an d th e anti-leishmania l antibodies wer e detected i n thes e patients wit h the anti-huma n Ig M peroxidas e conjugate . Among th e 1 0 ser a teste d fo r Ig M anti -leishmanial antibodies 1 serum was negative ho-wever, thi s seru m showe d a stron g reactivit y when th e reactio n wa s performe d wit h anti -human Ig G peroxidase conjugate.
3 h 1/40 1/4 0 CONTROL ' 1/160 AC L -CONTROL SLb b » Ig M Z Z T CONTROL SLb• Ig G Z Z b L
Fig. 1 — Frequenc y of I g G anti-leishmania antibodie s tite r to promastigote s o f L . b . brasiliensi s extrac t determine d b y E L I S A i n 4 2 ser a o f patient s wit h America n cutaneou s leishmaniasis ( A C L ) . Control : 2 0 ser a o f health y indivi -duals.
Fig. 2 — I g M an d I g G ami I eishmani a antibodie s t o pro -mastigote o f L . b. braziliensi s extract determine d b y E L I S A in 1 0 sera of patient s wit h leishmaniasi s caused b y L . b. bra -ziliensis an d i n 6 ser a o f health y individuals .
Immunoblot
The immunoblo t analysi s indicate d tha t the 4 2 ser a o f patient s wit h cutaneou s an d mucocutaneous leishmaniasi s (Grou p la ) showed a complex pattern of reactivit y (Fig.3) . However, band s o f 6 0 k D an d 6 7 k D wer e constantly reactiv e wit h thes e sera . Also , w e observed tha t patient s wit h cutaneou s lesion s (Fig. 3 , trac k 1 t o 6 ) showe d lowe r intensit y bands, with the 6 0 kD protei n tha n th e sera o f patients wit h mucocutaneou s lesion s (Fig . 3 , track 7 to 13 ) when detecte d wit h anti-huma n IgG conjugated to peroxidase. From the 1 0 sera
of patient s wit h leishmaniasi s caused by L . b . braziliensis (Group lb), 8 sera recognized the 60 kD protei n when reacted with anti-human, Ig M immunoglobulin (Fig . 4 , trac k 1,2, 5 t o 10) , with the anti-human Ig G immunoglobulin 4 se-ra showed the 60 kD band (Fig. 4, tse-rack 6 to 9), thus onl y 3 sera ha d th e Ig M an d Ig G specific antibodies t o th e 6 0 k D protei n (Fig . 4, trac k 6,7,8). I n general , th e reactivit y o f thes e sera obtained with the anti-human Ig G immuno-globulin wa s also weak fo r th e othe r proteins . In fig . 5 i t i s show n th e immunoblo t analysis o f th e cros s reactiv e antigen s o f L . b .
braziliensis extract s wit h ser a o f patient s bearing other parasiti c diseases . I n trac k 1 and 2 are represented the reactivity wit h normal sera (negative control) an d with a pool o f ser a fro m patients wit h mucocutaneou s leishmaniasi s (positive control) respectively . The pool of ser a from patient s with A VL reacted with the 67 kD and 4 3 k D protei n (trac k 3) . The reactivity o f sera obtaine d fro m patient s wit h Chagas ' disease which wer e grouped by th e tite r o f he -magglutination tes t t o Trypanosom a cruz i i s shown in tracks 4 to 8. We observed that sera of chagasic patient s wit h tite r o f 1/256 0 recogni-zed 6 specifi c band s i n L . b . braziliensi s extract bu t n o reactivit y wa s observed with th e 60 kD protein, antibodies titer of 1/1280 , 1/64 0 and 1/32 0 (track s 5 to 7 respectively) showed 2 band around 23 kD band and the sera with titer of 1/16 0 onl y a weak band of 2 3 kD detected. Schistosomiasis sera showe d a very fain t ban d of 6 7 kD (trac k 9 ) ant th e ser a of patient s wit h malaria presente d no reactivit y (trac k 10) .
D I S C U S S I O N
In thi s stud y we observed that mos t o f th e patients wit h A C L have low titer s o f specifi c
anti-leishmanial antibodie s whe n teste d b y E L I S A usin g promastigote antige n extracte d i n detergent (Fig . 1) and by I F A. Also, i n the wel l characterized ser a obtaine d fro m patient s which th e parasite s coul d b e isolate d an d classified a s L. b . braziliensis , we were able to detect specifi c anti-leishmanial antibodies onl y at 1/4 0 dilutio n o f serum . Moreover, w e have to searc h fo r Ig M an d IgG immunoglobulin i n order t o detec t th e anti-leishmania l antibodie s in ser a o f al l th e patients . B y thes e results we conclude tha t w e ar e no t abl e t o us e hig h dilution o f seru m to overcom e the proble m o f cross reactivity amon g A C L, A V L and specially with Chagas'diseas e sera . Therefore , w e analysed the antigenicity of promastigotes of L . b. braziliensi s extracte d i n detergen t b y immunoblot procedure . The A CL sera showed a comple x patter n o f reactivit y wit h thi s extract. However , we observed that al l 4 2 sera with chroni c cutaneou s o r mucocutaneou s lesions recognized a 60 kD and a 67 kD protei n (Fig. 3) . In th e grou p o f patient s i n whic h th e parasites coul d b e isolated , 2 ou t o f 1 0 sera failed t o reac t with the 6 0 kD protei n (Fig . 4) . This i s no t a surprisin g resul t sinc e a more specific serodiagnosis method usually leads to a lower sensitivit y i n th e test I t i s likely tha t th e 60 kD antige n will no t b e a subspecies specific
antigen sinc e th e 2 3 ser a obtained i n Amazo n region where the presence of L . b. braziliensis is rare6 als o recognize d th e 6 0 k D protein . These results suggest s that th e 6 0 k D protei n may b e useful i n th e diagnosi s of A C L since sera from patients wit h the othe r diseases above mention -ed did no t recognize d th e 6 0 kDband (Fig . 5).
We wer e no t abl e t o detec t reactivit y wit h the 7 2 kD protei n which ha s been described b y L E G R A N D e t al.1 1 wit h ou r antige n extract . This ma y b e du e t o differenc e i n th e antige n preparation a s well a s in th e L . b . braziliensi s strain used.
Species an d subspecie s specifi c antigen s have bee n characterize d b y hiperimmunesera 9
or monoclona l antibodies 1 3 1 4. However ,
methods fo r serodiagnosi s showin g thes e specificities hav e no t bee n described . Th e failure to fin d a more specific antige n coul d b e due to th e source of antige n utilize d in the tests. In general , th e studie s hav e bee n don e wit h promastigotes obtaine d i n culture which ar e no t the stage that grow i n the vertebrate host. Thus , it i s possibl e tha t th e specie s specific antigen s recognized b y huma n ser a are epitope s presen t in amastigotes .
R E S U M O
Análise d a especificidad e d e anticorpo s huma nos a antígenos de Leishmania braziliensis bra -ziliensis
A antigenicidad e d e promastigota s d e Leishmania braziliensi s braziliensi s tratada s com desoxicolat o d e sódi o 1 % tampã o Tris H C l 1 0 mM p H 8. 2 foi determinad a po r immu noblot usand o soro s de pacientes co m leishma niose cutânea e mucocutânea, leishmanios e vis -ceral, esquistossomose , malári a e doenç a d e Chagas. O s soros de pacientes co m leishmanio -se cutânea e mucocutânea apre-sentara m reaçã o positiva co m um a band a d e 60 kD. Nã o s e ob-servou reatividade par a esta fração e m soros de pacientes co m outra s doença s parasitária s aci -ma mencionadas , indicand o qu e est a pod e se r utilizada n o sorodiagnóstico d e leishmaniose te-gumental
A C K N O W L E D G E M E N T S
The author s greatefull y acknowledg e Dr . Roberto Badar ó fo r kindl y provindin g sera o f
patients wit h specific diagnosis of leishmaniasi s caused b y L . b . braziliensis . Paul o d e Oliveir a and Rivaldo de Souza Cardoso for th e excellen t Technical assistanc e and Ceei Corrêa Godoi d e Angelis an d Eunic e Bonfim Pint o fo r editoria l assistance.
This wor k wa s partiall y supporte d b y Fundação de Amparo à Pesquisa do Estad o de São Paul o - 86/2389, by th e U N D P / W O R L D / B A N K / W H O Specia l Programme fo r Research and Trainin g i n Tropica l Disease s (I D - 87 0 250); b y Laboratóri o d e Investigaçã o Médic a -H C - F M U S P - (LIM-48) ; an d b y F I N E P (43.88.0689/00).
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