A basic reagent for the lgM-immunofluo-

Rev. Inst. Med. trop. São Paulo

29(6) :354-360, novembro-dezembro, 1987

tEìgTlNS FOR THE DETEGTION OF lgM ANTIBODIES TO T. GONDII lN THE DIAGNOSIS OF ACUTE TOXOPLASMOSIS BY IMMUNOFLUORESCENCE

TEST.

r. T. r. NoJrMo|ro (r), s. HosHrNo-sHrMrzû (2), T. K. & M. E. (l¡iúlRGO (1)

SUMMARY

I-ectins were labeled with fluoresc€in and tried as conjugates

in

the immu. nofluorescence

(IF)

test

for

the detection

of

IgM antibodies

to

T. gondü,

in

the

diagnosis

of

acute toxoplasmosis. This approach was an attempt

to

find

atter-native reagents

for

a¡rti.human

fgM

fluorescent conjugates (AHIgMFC), which contain quite frequentþ anaibo'dies

to

toxopla,sma, as contamina¡¡ts, due

to

na!-ural

T.

gondii infections among animals used

for

imunization.

Iæntil (Lens

culinaris) lectin fluorescence conjugates (LcF€) provided most satisfactory re.

sults. Ihe

evaluation

of

LcFC carried out är a total

of

179 sera from patients

with

acute and chronic toxopla'smosis,

with

non-related infections

or

healthy

subjects, gave high values

of

relative efficienc¡r, co-positivitJ¡ and co-negativitv indices, respectively 0.989, 0.969 and 1.000, in reference to the conventional AHIgMFIC.

Moreover, three batches

of

LcfC

succressive\r prepared gave reproducible test results. Ttre advantage

of

LaFlC as an alternative reagent

for

the serodiagnosils

of

acute toxoplasmosis

is

supported by practical _aspects

_of

_its preparation.

KEY WORDS: .Anti-IgM detection; Iæctins; Acute toxoplâsmosis; I-ectin cor\iugate.

One of the main serologio markers

for

the

acute toxoplasmosis

is

the

presence

of

IgM

anti=toxoplasma antiboflies

in

serum. To

de

teet sush antibodies, immunofluorescence tests

are widely used because eaql

to perform.

Af-though presenting limitations

of

sensitivity

and qlecificit¡r, these can be oversome

by

rel. atively simple procedures

of

_{removing the} interfering factors, such as rheumatoid factor,

with

immunosorbents e a¡rd IgG antibodies to

toxoplasmar, twith protein

4c,

which afford a

high reliability

to

the immunofluorescence a.s-sary.

(1)

INTRODUCTION

(1) Instituto de Medicin¿ T¡opics¡ d€ São psuto, Btesit.

(2) rbculdade de ciênctas Farnacêuticas ds rlniyersidads de sãú paulo, Brasil.

Address lor corres¡loudelree: _Dra. Sumie Hoshi¡oshimizu. trþculdade de ciencias Fâ,rfnacêuflcas dÊ USp. Av. prof_

IJ¡eu Prestes, 580 _- Btoco A _- conjunto clas _{Qufmicas, Cidade UDiversitária. Cm 05508 São paulo, Sp, BrssU.}

354

A

basic reagent

for

the lgM-immunofluo-rescence test is a specific anti-human IgM

fluo-ressent conjugate (.{HIgMFC), usually obtain-ed by labeling with fluo,rochrome the IgG frac.

tion

of

immunized animal serum, anti-hum¿n

IgM.

Quality

of

suoh conjqgates

differ

con-sidérably, not onl¡r in respecü to the

character-istics

of

specificity and

affinity

of

the

pro.

dused antibodies

but

also, by the usua,l

pres-ence

of

contaminating anti-toxoplasma affi-þodies. These are due

to

frequent infections

NOJII\,tqIO, _det€ction I. _{of igM} T. I.; EOSIIINO.SIfIMIZU, _S.; NAGASS,FfSI,GAIÍAN,å,, T. K. & CAMARCIO, M. E. _ I.€Ct¡DS

ar¡tibodies _{to T.} gonalll in the diagnosts of acute toxopla$nosis by ¡Emunofluorescence test. Irst. Med. úrop. São P¿ulo, 29:854.860, 198?.

for

products furnished

by

teading manufactu. rers.

Some _{lectins present a much higher}

affin-ity

for

IgM

than

to

the other immunoglobu-trins

7'æ.

.å,lso,

T.

gondÍi taahyzoites

do

not present membrane sugar receptors

_for

most knor¡yn lectÍns

r¡.

fn

thsi Tray ftuorescent

con-jugates

_of

_{lectins were studied as alternative}

reagents

to

fluorochromê,labeled anti-IgM an_

tibodies,

in

the

detection

of

anti-toxoplasma

ÌgM

antibodies

in

serum.

In

spite.of

la^rge

applications in Ímmunological, biological,

chem-ical

and even clinical researches le,

it

seems that leotins have not been previousþ employed

in

the detection

_of

antibodies

for

diagnostic

purBoses.

MAIERIAI. AND METIIODS

Serum sa,rnples

_-

Ttre study was

perform-ed

irl

a total

of

179 sorum samples, 64 of which presenting a serological patern of acute

toxoplasmosis (pattern

I),

₂₆

_with

pattern

II

or

III,

corresponding

to

cilder toxoplasma

infections, 40

from

clinically healthy individ-uals and, 49

from

patients

_with

nonrelated infections, including 10 casep of Gïragas'disease,

10

of

malaria" 10

of

rnucocutaneous leishma,

niasis, 11

of

schistosorniasis mansoni and g

of

rheumatoid

arthritis.

Toxoplasmosis serol. ogic patterns were established accrording to pÌevious published griteriaa,s, on the basis of results

of

a

battery

of

tests, including IgM

and

lgG:immunofluoreseence

(ISM-IF

and

IgGIF),

passive hemagglutination

(IIA)

and complement fixation (CF) tests, perfonrred as

described ¿.

Toxoplasma antigens

_-

_T.

gondü tachy-zoites' w€re obta.ined

from

mouse peritoneal exsudates two days after inocrrlation

of

para-sites and TG180 sarcoma cells, as describedz. .A,fter agglutinating

the

peritoneal cells with pþtohemagglutining _{P and filtration}

_in

_n¡ilon wool, purified parasites were treated with

for-malin 13.

I^€cúln fluorescent codugates (LFIC)

Five

sommercially available lectins (Sigma) Ctrem. Co.,

St. IouÍs,

USA)

from

Lens culÍ-naris, Phaseolus vulgaris, Glycine 'max, Atan chis hy¡¡ogBa and Canavalia ensifstm,i¡$ were

tested,

for

selecting the most adequate

in

de, tecting

fgM

antibodies.

For the study, three batches of lentil (Lens culinaris) lectins

(f, II

and

III)

were then prepared

in

large amowrts, following the tech_ nique of HOI{¡AR,D

&

SAGT':14,15 and one batch

of Con A (Canavalia ensifonnis) as described r. Lectins were labeled.

with

fluorescein

iso-thioqyanate a,ccording to CLARtr( & SHEPAR,D 8

arr¡d lectin

protein

concentration determined

þy LO1üRY et al method r? _{with crystalline}

_bo

vine

albumin (Sigmq Chem.

Co'

St.

Louis,

USA) as standard.

Activity

of

lectins

_-

All

the lestins were characterizæd

by

their ability

in

agglutinating forrnalinized., group 0, human red blood cells.

Quantitative assays were performed

in

plastic

microtiter plates with,V-shaped v¡ells. To 0.05

ml

ûwo-fold serial dilutions of lectins

in

0.01M phosphate buffered saline solution

at

pII

?.2

(PBS), stating

from

1:2, 0.025

ml

ZVo

erytrro-c5¡tes suspension

in

.PBS were

addpd.

Sed-imentatiqn patterns

were read

as

usually, after 90 minutes,

at

room têmperature.

fmnunofluorescionce

(IF)

t€st

_-

Conven

tional assays

for

IgM-IF and IgCTIF were

cêr-ried out as described4 using commercial (IIy-land Div. Travenol Lab,, California, USA)

fluo-rescent

anti-human.fgM,

_,rchain

specific

(AHIgMFC) and anti-human IgG, Tchain

spe-cific

(AHIgGFO) conjugates. Monospecìficity

of

these conjugates was confirmed

by

immu-noelectrophoresis, as Well as absence

óf

anti-T. gondü _antibodies, as determined by i.mmu-nofluorescence assays. Optimal conditions for

tests with lectin fluorescent conjugate (IæFC)

were standardized, as later referred. To

ver-ify

the

specificity

of

I,IIC, inhibition

of

IF

tests was performed

_þ

previous addition of

saccharide

to

the

conjugate. Also,

to

verify the specificity

of

the interaction between LFC

and IgM antibodies, sera shdwing positive

r+

sults

were treated

with

2-mercaptoethanol e

a,nd

retested.

To

avoid

the

interference of rheumatoid factors

in

assays

for

IgM

antibod-ies,

all

sera were tested

by

RA latex

aggluti-,nation _test (Behrinryerke _{AG, Çermany)} a,nd

those giving positive results

were

absorbed

with

heat-aggregated huma¡¡ gamrnaglabulin

as recornmended3.

for the

NOTIMCTO, I. T. I,: HosHINo'sIfIMIzu, s.; NÀGASSE-SI,GAEAn,A, T. K. & _{c.ATIrARc¡o, M:,,8. _ I€ctiDs for t,ho}

detect'ion ot. rgM antibodies to T. Eondlt in the diagnosis iof acute toxopla,rosis bJ¡ innûofluorescence t€st. aer. Irst. Md. t¡op, sãa paulo, _{29:354_860, l9B?.}

Súatistical anat¡sis

_-

To compare results

obtained

_with

_{LFC and ÁÌIIgMFç}

_in

_the

_IF

tests, t-stirdent test and, coefficient correlation

were utilized according

to

pAIIL

_&

_WHITE

and LIITZ 22,1E.

_{For thê}

_evaluation

_of

_LFC

tests, relative efficiency, co-positiv,ity .and co-negativity indices rwere estimated r0.

REST]LTS|

.{

tota,l

of

? lectins were studied., 5 from commercial purchase and

2

prepared

_in

_our laboratory,

_for

_{the standardization}

_of

_{the me_}

thod.

Protein contents per

_milliliter

_and hemag-glutinating _{titers we¡e respectively: L.}

culina-ris

(10.8 _{mg, 1:512),} p. _vulga,ris (14.g _{mg, l:16),} G. max (10.8 _mg,

_l:g),

_{A. \ypogea (tãO mg,}

1:8),

_(I)

_(12.6 C. ensiform,is 4.0 mg,

l:2), L.

culinaris mg, l:2,048), C. ensifomnis

(I)

(5.5 mg, 1:16).

Ihe

Iast tryo

(I),

locafiy prepared,

presented _higher

_titers

_in

_relation

_to

_those commercially acquired.

Different conditions

_{of pII,}

_temperature a¡rd time were required

in

the

IF

test ,$rith

LFÌC

to

obtain comparable titers

to

those gi-ven

by

the

conventional _Á,IIIgMFC

4.

_Ttrus, serum gamples _{were irrcubated}

_with

_{T. gondit}

a,nti.gen

at

¿eC, _{25"C and B?.C}

_for

_{periods var_}

ying froarr B0 minutes

_to

2?

hours.

Best re-sults we¡e seen

at

25"C (room temperature),

from 4 to 2? hours.

I'or

_{technical convenience}

an

incubatio4 period

_of

_{24 hours u¡as also} FT¡rther incubation step

with

LFC was also

assayed

_and

_{most patisfactory results were} oþtained

_for

g0 _minutes

_at

_{BTC. fr¡fluence of}

pH

_{was studied, best results being obtained}

by

diluting sera

in

pBS

_{at pI{}

_{8.0 and LfrC}

at

plf

7.5, whereas washing

of

slide pÌepara-tion

in

PBS at

pI{

?.2.

As indicated

_in

_Table

_f,

_{onl¡r le,ntil lectin}

fluorescent conjugate (LcF€), _{at the maximum} reactivit¡r, _{detected specificalþ IgM arrtibodies}

to

T. gondü

_in

_a

_{similar fashion}

_to

_{the refer_} ence .AIffgMFC.

For

other lectins, although Sivturg similar titers

for

IgM antibodies, much weaker fluorescpnce staining wa,s obsdrved, as well as non-s¡recific reactions with negative se.

ra

diluted

at

1:16.

356

TABLEI

Coñpelstive titrations with atiffe!éDt tectins conJugates ot

sera from: a) recer¡t T. gondlt infection, patterq I; b) old

T. gondll lnfection, pattem _IU; and c) Don-i¡fected i¡dividu¿t

Fluorescent

ConJugate ar¡d dilutioo us€d

LeDs tl¡tinaris (l:20)..r 2ffi 0 _{O I} Iæns cull¡r¡ris (I) (1:S0) _{2,56 0 0} O

Canavaüa elrstformts (l:5) _{256 16 16 o} Canavalla enslfonnis (I) (l:f0) 256 256 l0 _O Glyclne max (1:10) _{zE6 256 16 O}

Arâchis\ypoges(1:1) _{O 0 O 0}

Stand¿rd serum titel'

(') = Staridard serum wlth refelencþ titer of ZSO vttn

(*.) ₌ PES _- 0,01M phos¡¡t¡ate _{buffered saline} solution, at pH 8.0, as diluêr¡t.

(***) : Dilution used

(I) _- Lectins prepsred in _our laboratory

Table

II

_{depicts comparative results}

_in

_the

IgM-IF test with Lens cr rinaris and. with anti.

IgM

antibodies,

for

1?9 serum samples frorn patients

_with

acute and chronic

toxoplasmo-sis, other diseases and clinicalþ _normal indi-viduals.

fn

comparison

_with

_{the anti_IgM} fluo¡es-cent conjugate _{test, the test carried out rwith} lentil conjugate furnished. a cu-positivity index

of

0.969,

a

co-negativity index

_sf

1.000 and a

relative efficiencXr index

_of

0.gg9.

fn

64 cases

total

agrepment

of

_titers

_{wa.s seen}

_in

₄₂ $5.6%>, differences

_l2Û.l%)

of

one dilution

in

lg

and

of

two

dilutions

ìn

4,

cases

G.ïVo).

The

geometric

_mean

_titers

_for AHIgMFC ,was 8.8? and

for

I.lsF(.,1.1g. A pos.

itive

correlation,

r =

_0.g00,

_was

_obse¡ved,

between the IgM antibody titers obtained by both fluorescent _{corqiugates and, accordinglg}

an equation

_of

regression line could be

esti-mateif:

Y -

_1.1940

₊

_0.8894

_(fig.

_1).

_The significanpe

_of

_this

_{value Ìüas confirmed by} the finding

of

t-test value

of

12.66,

in

compar-ison

to

the

critÍcal value

of

2.66, (62

¿.f

at

I'Vo ls¡el).

Three different batches of LcFC

(I,

II

and

IIf)

were prepared

_in

_{the laboratory}

_for

_tlte study

_of

the

reproducibility

_of

_the

_reagents. They showed respectiveþ

_the

_{hemagglutinat_}

ing

titers

of

1,024, 2,0449 and 4,096 and.

dilu-tion

of

use

in

the

IF

test

of

1:40, l:g0 and. a b c PBS'/i

NOJIMOTO, _detection I. _of T. _rgM I.; HOSII{NO.SEIMIZU, S.; NAGASS .SIIGAEAR.â, T. K. & CA¡|¿ÍARC|O, M. E. _ I€c'Ds for _rhe antibodies tg T. gorrdx t¡ the disgûosls of a.cute toxoplasnosis by i¡uu'ofluorescence t€st. a€v.

I¡rsú. Med. Írop. Sãó prulo, 29:954_860, l9g?.

coEparativs résults of f'e¡¡ culrn¡rls ao¿ anti-tr¡maÏ

i*" år.i"ll,

_{conjugates rn rgM-rr tests, psrfoimed tn u9} sefl¡m

sa,mples

DtsgDosls

Acute toxoplas¡nosis CbÍonic toxoptasmosls

Chagas'disease

[ta,laÉa

Scbistosomlasls manso¡l

Mucocutaneous leishnaniasis

Rheumatoid a¡thrifis

Clidcally nor:oal individusls

Np of lg¡u.m

^to

(J E J

68 U

E

t--6 g

(9

o

J{

y' l. 134+O.839x

M

2fì

t0

10

11

l0 I

40

ffi

0 0 0 0 0 0

0

Fluorescent conjugste

Negative

XlLo€r lgt TITER AHbrFC,

FiC. _{1 -} R€gtession of IgM antibody titers detected btr

lentil lectin fluorescent _{conjugate (LCFC) against ttrers}

obtained by ariti-human IgM fluorescent coqlqgsIe í.ôIIIgMIC.). Numbers along dots correspond to the

fr€-quency of titers.

1:160. These results demonstrate possibility

of producing fluoresôent reagents of good qual_

ity

with

easy

Ín

relation

to

the conventional AHIgMFIC.

DISCUSSION

R€sults here prêsented show that lentil

lec-tins

la-beled

with

fluoresoent isothiocyanate

Ga¡r corNtitute a satisfa,ctory alternative reagent

for

the anti-IgM antibody conjugate,

in IF

test

for

the detection

of

anti-toxdplasma fgM

anti-bodies.

As sensitive as

the

antibody co4ju. gate, lectin conjugates have the advantage of maintaining closely reproducible reactive char

2 26

10 10

lt

10

I

,410

racteristics' among diîferent _{batches and, of}

beeing free

of

contaminating _{anti_toxoplasma}

a¡rtibodies.

Isola'tion

of

lentil

lectins here proeessed bry _Sel filtration chromatography on Sephadex

G-100,

as

described,

by

HOTVARD

et

âl r+,ls, gives

_a

_mixture

_of

_{two active components, A} a,rrd B,:r¡¡Ith similar biologicat p"op""ti"" :r4,]54s.

Both

show

a

molecular wbight about 49.000,

their hemagglutinating _{activities can be}

equal-ly

inhibited

by

d-mannose and d-glucose and,

in

gel precipitating _systems,

_a

_line

_of

immu-nochemical identity

is

presented _{by the anti_.A} a¡rd the anti-B immunosêra raised

in

a¡rimals. Because of this slose relationship, Ín the pre+

ent

_'tüofl<

the

A

and

B

lectin

eomponents w€re not fufther separated in a carbox¡rmethyl cèllulose _{chromatôgraphy as recomhtended. o.}

Activities

of

the fluorescent lectin conju. gates differed somewhat

from

those reported

for

native

lectins.

For instance, Spgf.fbf,pn

&

lry,EBERæ have found,

a

selective binding

of

phytohemagglutinin (pHA),

_a

_{lectin from} Phaseolus vulgaris,

_to

IgM

of

huma¡r normal

serum.

I{dn¡ever, w-e had poor _{results with}

this

lectin, possibly _because

_we

_{have dealt}

with IgM antibody combined

with

antigerr. rn

consequence of modiïications in molesular

con-fi,guration

_of

the imrnunoglobutin ¡trhen

form-ing

immunocomplø<es sorne

sugar

compo-nents could becorne less ex¡rosed.

Carbohydrates were estimated as l2.g7o oL

the IgM

molecules, comprising ?.6

parts

of nut.rmose and glucose, l.4 parts,of fucose and

sialig acid, and 1.0 pa,rt

of

galactose,

in

con-Positive

A¡ti.hulla¡r¡ IgM

er

0 0 0

q

0 0 (,

Negêtlvo

0

aì

10 10 11

NOJrMClrO, r. T. I.; HOSHING.SHIMI.TO, S.; NAGìÊSSE-SUGÁEARÂ, T. K. & CAMARC.O, M. t. - I'€ctÌas for tüe detection of IgM antibodies to T. gondti i.n the dlagnosts of aÉute toxoplasmosis bJ¡ itmunofluorescence t€st. R¿v.

IDst. Med. úrop. São P¡uIo, 29:354-360, 198?.

trast to 2.9% of. saccharides

in

the IgG

mole-svJstL,t2,2I.

It

should then be ex¡lected that lectins

with

affinity

to

mannose and glucose such as Con A,

a

lectin from Canavalia,

ensi-formis and

lentil

lectins, could provide good

reagents. Ilowever, Con A showed

to

be }ess effective than

lentil

lectins, probably because

of

factors as molecular size, spacial

configu-ration,

pH

and temBerature, which might be limiting its interaction with the IgM molecules

bound

to

antigen fixed. on microscope slides. For instance, HUET 1ó _{observed maximal}

reac-tivity

of Con A at pH ?.0 and ST"C, conditions fhat differed from those standardized

for

our

tests.

A

previous addition

of

mannose

or

glu-cose to the LcFC inhibited its activity in the IF

test.

Also, specifici.ty

of

the conjugate could

be

indicated

by

negative results when test:

ing

serum samples presenting serologic

pat-terns

II

oi

IIf,

correspo¡ding

to

chronic or pregressive toxoplasma infections,

and

sam-ples

from

clinically healthy individuals oI

cases

of

non-related diseases. Moreover,

posi-tive sera from patients with toxoplasmosis be-came completly negative

aftef

serum

treat-ment

with

2-mercaptoethanol.

To

obtain

sensitive results

with

lentil

lectin conjugates, different factors had

to

be carefully ascertained, such as

plf,

temperatu-res and periods

of

incubation.

For

diluents,

better results we¡e seen

for

pII

values above

72.

Incubation periods required were

in

gen-eral

longer :bhan

for

_the

reference system,

and 24 hours

at

room temperatwe lr¡as cho.

seù

for

incubating sera and, 90 minute¡ at 37"C

for

the conjugate. Besides different pt¡rs"

isocheryiical properties,

lentil

lectin

conju gates int€ract with.f,ewer sugar receptors found

on

IgM molecules than the number

of

epiüo-pes reacting

with

labeled anti-IgM antibodies.

By

lengthening the time

of

serum incubation more residual antibodies coul.d

bind

on

the antigens, thus renderÍng a large nu¡rrber of sug-ar receptors to react with the lentil conjugate.

ReliabiliQy

of

the lentil

lectin

conjugate

ùr the test

for

anti-toxoplasma a¡rtibodies twas indicated

_þ

the

relåtive efficiency index ot

0.989, derived

from

high co-negativity (1.000) and co-¡rositiviþ (0.969) indioes ür relation to

the

classical

IF test.

Similarity

of

results

358

between both

kind

of

conjugates was shown

by

the close geo,metric r4ean

titers

(8.3? for' AHIgMFC a¡rd ?.?9

for

LeF€) and

by a

posi-tive

titer

correlation

of

r =

0.800.

It

is

interesting

to

note also the similar'

ity

between staining patterns

of

both conju' gates, including even the non-specific polar stain ing eventually observed

for

negative sera.

Irl

this

way _{'we conclude}

on

the

advan-tages

in

using lentil lectins conjugates

in

the

IgM-IF test

for

toxoplasmosis, instead

of

ani'

mal immune sera derived coniugates,

in

view

of

the

economical aspects, technical ease in obtaining

quite

reproducible fluorescent con

jugate batches and relia.bility

of

test results.

Our preliminary results also indicate the pos

sibility

of

appþing the le0tins

in

simtar

fash-ion

for

enqlrme irnmunoassays

for

the

diag-nostic purposes.

RESTTMO

Lectinas pañÐ

a

detecção

de

anticorpos IgM anti-T. gondü no diagnóstico da toxoplasmose

aguda pela reagão de imunofluorescência

Lectinas foram marcadas com

fluoresceÍ-na e testadas na reação de imunofluorescência

paÍa

a

detecçáo de anticorpos anti-T. gondü, no diagnóstico da toxoplasmose aguda. Neste estudo tentou-se encontrar reagentes altern¿ti-vos para conjugados fluorescentes anti-IgM hu-mano (CFAIgMH), que com freqüência

con-tém anticorpos antitoxoplasma, como

sontami-nantes, devido às'infecções naturais

por

T.

gondü, entre os animais utilizados para

imu-irizacão.

Conjugados fluorescentes de lectina de

len-tiiha

@FT,) (Iens culinaris) fornece.ram

resul-tados satisfatóÌios.

Avaliação do CFT, efetuada em total de 179

amostraos de soros de pacientes com toxoplas"

mose aguda

e

erônica, com infecções.não re. lacionadas e de indivíduos sadios, mostrou va.

lores altos .nos Índices de eficiê'nsia relativa,

co-positividade

e

co-negatividade, respectiva,

mente

de

0,989, 0,969

e

1,0fi), em relação ao

IIOJIMOTO, I. _{T. I,; HosIflNGsHIT,,''u, s.;}

N.AGA*SE-'T,GIIEARÂ, _{T. K. &} ",*^*M

*itl?:r:t"'"iT

"#ti:iï

n,;;

r"r"r",1å*î tiu ¿tuso* or acute to*oprÀmãsis bv iemunoruorescencê tesr. ßey. de se empregar _o LFC _{como reagente alterna_}

tivo no diagnóstico _{sorológico da toxoplasmo_}

æ aguda consiste _{nos aspectos práticos da sua} pleparação.

ACX(NOWI¡EDGEMEI\[TS

We are grate$ul

_for

_{the sup¡rort received}

from Flrnda4ão dd emparo à pesquisa _{do Esta.}

do de São par¡Io (FApESp) _{and, from Consetho}

de

Desenvolvimento Científieo

_e

_Tecnológico

(CNPq-PIDE

_V).

_{Also, we tha¡¡k to Miss Maria} Inês Ca¡dillo and

to

Alrnir

Robson Ferreira

for

helping us

to

prepare the manuscript.

REFEBENCES

I. AGRAWAI,, B. B. L. & GoIÐSTEIN, I. J. _- ProIeID carbohydrate inteÉction. VI _ Isolation of ooncanan valin'.â, _{þ specific absorption on crôss_lirÍked dex¡raD}

qels. Biochim. biophys- _{Acta. (Amsú.), t4Z; 262_27r.}

1967.

i. ARDOIN, P.; COUZINNAU, P. & BAII!,IIr.E.DUCROCA, H. _- Su¡ I'utilization du sarcome TG_1g0 pour

I,oÞ-tèntion d,r¡ne suspension r¡che en ToxoptÀsm.t gondlt extÌacollulailes. C.R. Soc. Btol. (paris), fOf: fi1-rrs.

1967.

3. C.åMARGO, M. _E.; LESER, p. _{G. &} ROCCA, _A. _

Rheumatoid _{factots as a} can¡se for false positive IgM anti-toxoplasma fluorescent _{tests. a bchnique tor}

specific results. ßev. I¡st. Med. úrop. S. Þ¿ulo, 14: 310-313, 1972.

4. CAMATRC¡O, M. E. & _LESffi, p. c. _ _Diagnostic

jn-formation from serological tests in human

toxoptas-mosis. lI - .Evolutive _{study of antibodies and sero_}

logical pattem in acquired _{toxoplas¡osis, as detected}

by hemagglutination, _{compleEent fixation, IgG and}

IgM iDmunofluorescenæ _{tests. Rev. I¡st. U"e. top.}

s. P¿uIo, tE 227-238, rVI6.

5. CAIVIART|O, _{M. .8.; LESER,} p. _{c. f¿ LESER, 'lr. S. p.}

- Ilefinigão de perfis sorológicos ne toxoplasrncse.

Importância diagnóstica e epidemiológicå, nàv. br"s.

Pat. ctín., tB: l1B-12?, t9??.

6. CAMARCTO, M. Et.; LF.SER, p. c. & _{hoccA. á,. _} Detection _{of IgM anti-toxoplasDa antibodies in acute}

. acqutted a¡rd congenital _{toxoplaÐosis ¿fter protein A} treøtment _{of serun. Rev. Iût. Msrt. ttop. S. patlo,}

2ã: 201-æ6, lg8:t.

7. CH.ATEIIUN, C.; LUvrOtNE, A.; DUGOIIYON, J. ttr.;

BLANC, M. & ROUGÉ, p. _ Intelaction of polyctonal

and monoclonal human immunoglobutins _{G with} væ_

ious lectins (Concanavalin .á,, lentil and pea lectins).

In BOG-IIâNSEN, T. _{C., ed. Iæcüns, blotoß¡, bto.}

cheEistry, cllnicsl btochemtstry. Netp york, Itrs$er ato

cruyter, tgg?. a. Z, p. 3SB{02,

8. CLARK, _{que for} H. F. _prepa,ring & SIE?ÁRD, O. C. _ A diat¡sis recbnt

fluo¡escent antibody. Vfuology,

Z0: UZ-6M, t969.

9. DEIIrSCII, _{H. &} MORTON, _{J. I. _ Dissociatioû of}

huma¡¡ sen¡m eacroglobulins. _{S€lence, 12¡: æO.OOI.}

1957.

10. GÁLEN, _{maliúy. Ihe} R. S. & GÁMBINO, _{S. R.} _ BeSrond nor.

predictive value and, efflciencl¡ _-"Oi*f

diagnosis. New york, _{Johx Wil€y, 19?5.}

1I. HEIMBERGER, _{N.; HEIDE, II.; EAI,PT, II. &}

SCHIILTZE, _{rJ, t. _ Baustein anatysen o* ¡*ouo}

serûn proteinen. cüñ. Cblm, Acúe, 10: 298_80?, 1964.

12. IIICKMAN, S.; KORNEEIÐ, _R.; OSTERLåND, _{c. K.}

& I(OnNFEIÐ, S. _ The structure ot tfre

gtyco-IJeptides of r human _{ô M -} ¡mmunoglobulin, _J. hiot. frem., ?AZ¿ ZtS6-Zt6A, fgÏZ.

13. EOSTITNO.SHIMIZu, _{S.; MINEp, J. n, &}

C.âMARCTO¡

y. E. - Iéctin used in tho purificatiou _O"""Ë-oi Tbxoplana gondil taohyzoites. ¡. fa¡as¡t.,- O*:

Seg-991, 1980.

14. HOWARD, I. K. _{d¿ SAGE, II. J. _ Jsolstion} anal

cha,racterization _{of a} pMo_hemaggtutinl¡ _{from the lec_}

tin. Btochemistr¡¡, _{8: Z4eSZl4l, 1969.}

r5. IIOTWARD, _{N. M.;} I. _K.; SAGE, _{rL J.;} STETN, M. D.; yOIrNc,

r,EON, M. A. & DYCKES, D. F. _ g¡u¿;€s on a phytohemaggtutinin _{froE the lentil. II _}

MuI-tiple foñms of læns Gl¡Ifn¡rfs hemagglutiniu. _J. D¡ol. Chem., 246: 1590-1595, r9?1.

16. HIIET, M. _- Faactors affecting the molecula,r sEuc-ture and agglutinating ability of concanavalin .6, an¿

other lecti¡s, Euop. J. Blochem., _{59: 62?-&92, t9?5.}

l?. LOIÃ/RY, O. H.; ROSEBROUGH, _{N. J.; FARR, A. L.} & RANDALL, R. J. R. _ protein _{meesur€nont witb}

the folin phenol reagent, J. blol. Chm,, fg3: 26ü 27t, 795r.

18. LIJ¡IZ, _{mr¡nological} W. _- St¿tistical Eethods as alÐlied to

im-data. In, WEIn, D. M,, ed. IlÐdboo&

of exlEr¡mertal inmrrn6l6ry. _{trOtd, BlacJrweu} Scien-tific publications, _{l9?A. p. 1168_1202.}

t9. PEREIRA, IW _{E. A. _ Iæcti¡s. In: .ATAßSI, M.}

z. A.; V.{N OSS, C. J. & .AEÉiOI,oN, _{O. n.,}

"O.

Molecula¡ immunology. Ner ìrork, l¿arcef Oek¡<er, 1984. p. 319-935.

20. SpÞlVcLER, G. Â. & 1{EBEn, &. M. InteractioDs of PHA with human nonnal serum proteins. Ito:

BOCTIIÂNSEN, T. _{C., ed., Lect¡ns, biology,} blochem-isúrN¡, cl¡Dtc¿l b¡ocbFmjstry. New yorkr _{Weitor de} cruyter, l98l; p. 231-2{0.

21. \IEINSTEIN, Y.; GIvo!, D. & STR¡AIISBAI,cE, P.

insofu-NOJIMOIIO, I. T. I.; E0ffiINGsrrrr,nt{t, S.; NAcAf¡SlEgIcáEåEA, _{T. K. &} Cåt[ARfþ, _{M. t. -} I€ctÍDE lc, tbo

deüsctfon 94 _.Igì[ onH.bodf€s to T. gudlf ln _{tüo rflrgno{r ot aßutg toroplssosfs by lmmuaoûuoresceoce tnet. BE?i} Lnst. I[Ed. üAù.:São.pauto; 29¡854-360, 198?.

blllzed co¡cenayatl¡ A. t. rñ'mol., lO0! l4mA-14(N, 19f12.

28; IffEffE,. C, _- StsHsdcef u¡sthods t¡ sæt¡Da *-""".

i¡: PÂIIL, J. R. & WEnt, C. _- Sctrolostost eplden-lolo¡S¡. Nffi YorE, Acad€@ic press, l9?:1. p. 19A2.

23. YOI'NG, N. M.; r.EON, M. A, & TÄKAEASEI, t

-Sh¡dies on ¡ phytoh€rú¡gglutlDi¡ _l¡om th¡ taüI. ¡. brol. Cü€m., t{S! 1596-160t, lgtt.

Ræ€udo po¡B Inú¡fce[8o

w,

U0lgt.