Rev. Inst. Med. trop. São Paulo24(6) :327-336, rtoventbro-dezembro, 1982

PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST SCHISTOSOMAMANSONI AND A TRYPANOSOMATID: A METHODOLOGICAL REPORT (.)

J. Dânlel LOPES, Célia R. W. CARNEIRo, Adriana A. VILELA and E. Plessmann CAMARGO

SUMMARY

Monoclonal antibodies were raised against two different parasites: the trypa-nosomatid Herpetomonas muscarurn muscarum and Schistosoma mansoni. Theproduction of monoclonal antibodies agâinst these organisms was found to de-pend on variables such as immunization schedules, molecular weight and sourceof polyethylenegfycol, batches or fetal bovine serum, macrophage feeder-layers,cell lines and culture conditions of the mouse myeloma cells. Adopting a selectedprotocol, herein described, a total of 35 stable monoclonal antibodies against H.m.fiiuscarum anrl 10 against S. m¿nsoni were obtained. Concerning H.m. muscarum,a schedule ,with shorter intervals between immunizing inoculations yielded anti-bodies of the IgM class; contrarywise, longer intervals gave a predominance ofIgG ,antibodies. With S. mansoni, different time schedules of infection did notalter the predominance of IgG antibodies. Indirect immunofluorescence sho'weddifferent reaction patterns with both parasites: some antibodies reacted predo-minantly against the surface and some against the flagellum of the trypanoso-matid. .Monoclonal antibodies against S. mansoni reacted either against the tegu-ment or against the gut cell layer in sections of whole worms. The biological im-portance of both kinds of monoclonal antibodies is now under investigation,

UDC 616.995.1.22593.161.13

Since its original description by I(ôHLER& MILSTEIN zz, the hybridoma technology forproduction of monoclonal antibodies opened anew era in the use of antibodies as diagnosticand investigative tools. Such potential uses havealready been applied to several parasitic disea-ses and are helping to clarify aspects of hostparasite relationships. Monoclonal antibodiesallowed the identification of specific antigenicdeterminants of various parasites ls 2s,2e, thebetter understanding of the immune responsesin some parasitic diseases 17,31,44, as well as theidentification of species and strains of parasi-

INTRODUCTION

(.) This investigation received financial suppolt from the UNDP/World Bank/WHO Special Programme for Reseârchand Training in Tropical Diseases, from the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico -CNPq, and from Fundação de Amparo à Pesquisa do Estado de São paulo

- FApESpDisciplina de Parasitologia, Departamento de Microbiologia, Imunologia e Parasitologia. Escola Paulista de Medicina,São Paulo, Brazil

327

tes3,32,'42. They may also become standard rea-gents for diagnostic purposes r0.2i.

The importance of developing the trybrido-ma technology in Brazil, a country with manyendemic parasitic diseases, cannot be overem-phazised. However, since begginers might beoverwhelmed by difficulties other than thoserepresented by the hybridoma technology itself,including selection of reagents and culture me-dia as well as methods and protocols to befollo¡¡¡ed, a description of successful procedu-res seems useful.

LOPES, J. D.; CARNEIRO, C. R.1V.; VILELA, A. Á. & C^MARGO,Schisúosoma mensoni and a trypanosomatid: .fr methodological

Monoclonal antibodies were preparedagainst two different organisms in order tostandardize the methodology: an insect trypa,nosomatid, Herpetornonas muscarum musca-rum and Schistosoma mansoni. Insect trypano-somatids are known to induce partial protec-tion against lethal challenge by Trypanosomactuziao. It is also kno,wn that sera from micèimmunized'with insect trypanosomatids cross-react with T, cruzi extracts 3s. Cross-reactionsbetween several trypanosomatid species and T.cruzi have been demonstrated by indirect im-munofluorescence (IIF) with either sera fromChagas' disease patients or rabbit anti-trypano-somatid hyperimmune sera24. Moreover, Iderpe-tomonas muscarum musoarurn proved its suita-bility as alternative antigen for the diagnosis ofChagas' diseâse by IIF 2't. These results suggestthe existence of common antigenic determinantsbetween insect trypanosomatids and T. cruzi.The biological significance of such antigens is,however, unclear.

Concerning schistosomiasis, it is estimatedthat this disease affects more than ten millionpeople in Brazil. The frequency of severe formsof schistosomiasis mansoni varies from 4% toIzqo ß. These numbers make clear that any kindof epidemiological control of the disease v¡ill behighiy wellcome, and that any immunologicalapproach to achieve this aim should be triedrl.In spite of the several in viúro models of immu-nologic killing of schistosomul'à6,7,8.1233, and thedata on the concomitant immunity in animalsand possibly in man37,38, little is known aboutthe antigens involved in those mechanisms orin some immunopathologic sequels of schisto-somiasis such as renal lesions due to depositionof immune cornplexes, which have been describ-ed in experimental models 2,s and in man 1,36. Inaddition, a serologic test has yet to be develop-ed to measure worm burden or to be used. asa criterion f91' ç1¡¡g 11,38.

The present report describes a method forthe derivation of monoclonal antibodies againstH.m. muscarum and against antigens shed byS. mansoni in infected mice. \ile hope that ourpresent results might be useful to those tryingto establish the hybridoma technique, since ma-ny variations and modifications have been ad.d.-ed by several Authors to the original method e.

22,30 4l ¿¡¡d some by us to overcome difficultiesimposed by local limitations of reagents andsupplies.

328

E. P. - Production of monoclonal antibodies againstreport. Rev. Inst. Med. trop. São Paulo 24:327-336, 1982.

MATERIALS AND METHODS

Mice: inbred BALB/c and outþred Swiss al'bino mice were used. BALB/c mice, infected ornot with S. mansoni were kindly provided byProf. Tomás A. Mota Santos (UFIVIG) and Dr.J. Gilberto Vieira (SP).

.

Cell lines and media: parental myeloma celllines were provided by The Salk Institute, CeIlDistribution Center, San Diego, California. SP2,/0-,A'914 (SPz) 3s, P3.X63-AgB-6.5.3 (X63) ts and P3-

NS1,¡1-4g4.1 (NS1)2r were tested. All lines areB Azaguanine resistants and do not secrete im-munoglobulins.

Dulbecco's modified Eagle's (DMEM -Gibco *) and RPMI 1640 (Gibco, Difco $, Flowf)

medium, enriched with 10 to 20Yo of fetal bo-vine (FIow, Laborciin*, MicrobiológicaV), hu-man or horse sera were used as growth me-dium and compared. Media were sterilized byfiltration. Arginine (200 mgll). asparagine (36

mg/I), folic acid (I2 mSlD,2-mercaptoethanol(50 pM), N-2-hydroxyethil-piperazine-N-2-ethanesulfonic acid - HEPES (25 mM), Na,IICO, (2

g/1) and gentamicín (40 mg/l) v¡ere added toall media. Once a'week, L-glutamine (200'mM)was added to stored media. Hybrids were select-ed in the HAT medium (growth medium con-taíning 207o (v,/v) fetal bovine serum (FBS)plus hypoxanthine (400 pM), aminopterin (0.4

¡iM) and thymidine (16 pM)æ). Fourteen daysafter fusion, aminopterin was no longer addedto the HAT medium (HT medium). Propaga-tion of clones 'was made in growth mediumplus 20Vo FBS. Cell hybrid lines were grbwnin 80 ml plastic culture flasks (Costar ** No.3025) in a humidified incubator in an atmosphe-re of 5lo COr. It should be observed that wecouldn't find a reliable CO, incubator made inBtazil. '\Ã/e have been able, however, to adapta compressor to the incubator from FANEM(Model 051,/E) in order to obtain a continuousflow of air/CO.. to achieve the desired. atmosphe-re.

* Grand Island Biological Co., Grand Island, N.y., U.S.A.$ Difco Laboratories, Detroit, Mich., U.S..A..f Flow Labotatories Inc., Mclean, Virginia, U.S.A.+ Laborclin Produtos de Laboratório, Ltda, pinhais, pr.,

BrazilV Microbiológica Consultoria, Análises e Prods. Biol. Ltda.,

R.J., Brazil.** Costar, Camþridge, Mass., U.S.A.

LOPES, J. D.; CARNEIRO, C. R. ltr.; VILELA, A. ¿,. & CAMARGO,Schistosoma mansoni and ¿ trypanosomatid: A method.ological

Macrophage feeder-layers: selection, cloningand expansion of hybrids were always madeover a macrophage feeder-layer. Macrophageswere obtained by washing the peritoneal câvityof either inbred or outbred mice with cold su-crose 0.34M4t. Cells were kept at 0"C in an icebath, counted and distributed into plates andculture flasks as follows: 2x10 s macrophagesdivided in 96 aliquots of 50 pl per 96,well plate(Costar No. 3596), 5x10s divided in 24 aliquotsof 50 pl per 24-well plate (Costar No. 3524) and5x10s per 30-ml culture flask. Feeder-layers wereprepared at periods ranging from 4 hours to 5

days before use in HAT, HT or growth medium,depending on the cells to be plated.

Organisms: If.m. muscarum (ATCC 30260)was cultured in a trypticase-sucrose medium(FYTS) described by ROITMAN et al.:¿. Viabtecells, at the logarithmic phase of growth, weretwice washed in PBS, p}f7.2, adjusted to 2x10rcellVml for either intraperitoneal (IP) or in-travenous (IV) injection. Cells of the same sus-pension were fixed in 2Vo f.ormaldehyde in PBS,pH 7.2 (v,/v), washed twice and used as anti-gens for IIF.

Cercariae of S. mansoni were obtained fromBiomphalaria glabrata snails infected in the la-boratory. Adult worms for IIF reactions wereobtained by liver perfusion t: of infected hams-ters. Worm sections were prepared accordingto \MILSON et al. ¿3.

Immunization of mice: two different sche-dules of immunization with H. rn. muscarumwere used: a) 102 viable culture cells were in-jected IP, without adjuvant, followed by 102

cells injected IV 30 days later; b) two IV in-jections of 102 cells were made with an inter-val of 70 days. In both cases, mice were sacri-ficed and spleen cells were removed and usedfor fusion at the 4th day after the last injection.

Concerning S. mansoni, mice were infectedby subcutaneous injection of 20 living cerca-riae. Splenic cells from infected mice, withviable schistosome eggs in stools, were usedfor fusion after periods of either 7 weeks or 4months.

CeIl fusion and selection of hybrids: myelo-ma cells, at the logarithmic phase of growthand over 90% viability, as scored under phasemicroscopy, were washed once in the growth

E. P. - Production of monoclonal antibodies againstreport. Rev. Inst. Med. trop. São paulo 24:A2?_886, 1982.

medium, counted and transferred to a 50-mI,sterile, plastic centrifuge tube (Corning * No.25330). Spleens,were asseptically removed,minced in 5 ml of serum-free medium, filtered.through a nylon mesh and washed twice ingrowth medium by centrifugation. Cells werecounted, adjusted to the desired concentrationand mixed with the myeloma cells in the sametube. Fusion and selection of hybrids were per-formed as described by St. GROTH and SCHEI_DEGGER,4I, using variable amounts of rrryeto_ma and spleen cells (Table I) and polyethyle-neglycol (PEG), MW 4,000 (Sigma ** or Merck!)at 507o (w,/v) in distilled water, plus 50lo DMSO(Merck), at pH 7.5. Other sources or differentMW of PEG gave poorer results. After fusion,the cell suspension was distributed as 4g ali-quots of 50 pl to two 24-wel1 Costar plates, onmacrophage feeder-layers in HAT med.ium. Theculture medium was changed every other dayafter the ?th day of fusion, and screening ofsupernatants for the presence of antibodiesbegan as soon as macroscopically visible growthwas detected.

Detection of antibodies: two methods wereused to screen for the presence of antibodiesanti-H,m. muscarum: IIF and a modified ra-dioimmunoassay (RIA). IIF was performed. withfixed culture cells of II.m. muscarum as describ_edzc. It was employed when small numbers ofsupernatants were to loe tested, as in the caseof the 24-well plates or culture flasks. It wasalso used after previous screening by RIA oflarge numbers of supernatants from cloning p1a-tes in order to characterize the immunoglobu-lin involved and the pattern of the reaction(surface, diffuse, flagellum, etc.).

For screening large numbers of superna-tants of cloning plates, a RIA test was d.evelop-ed as follows: 10ó fixed II.m. muscarum cellswere dispensed, in 50 pl volumes, in a g6 U-shap-ed wells Takatsy plate. After fan-drying, theplates were filled with a solution of 1% BovineSerum Albumin (BSA) (w/v), incubated for 60minutes at room temperature, and washed B

times with PBS, pH 7 2. Supernatants ,were col-lected with a sterile B-tip multichannel pipette(Titertek - Flow) and dispensed direcily onto

* Corning Glass 'Works, Corning, N.Y., U.S.A-** Sigma Chemical Co., Saint Louis, Mis., U.S.A! E. Merck, Darmstadt, Germany

329

LOPES, J. D.; CARNEIRO, C. R. W.; VILELA, A. Á. & CAMÁRGO, E. P.Schistosoma mansoni and a trypanosomatid: A methodological report.

the antigen layer. After 60 minutes of incubationat 37"C, the plates were washed at least 5 timeswith PBS. Radioiodinated rabbit IgG anti-mou-:se gammaglobulin, prepared in our laboratoryby DEAE cellulose purification and 131 I labeledby the chloramine-T method, was dispensed ineach well (50,000 cpm per well). After 60 minu-tes of incubation at 37"C and 5 'washes withPBS, the plates were again fan-dried, coveredwith X-ray film (Sakura) in a dark chamberand left overnight. After development, the filmshowed dark (positive) and clear (negative)spots, the former being selected for IIF testingand clone expansion.

Supernatants were screened for antibodiesanti-S. mansoni by IIF against sections of wholeadult worms, according to WILSON et al.4:.

For the determination of subclasses of lgs,immunodiffusion in agarose gel (ID) againstspecific antisera (Meloy'*) was employed.

Cloning of positive colonies: cloning of se-lected hybrids was always made by the dilu-tion method in 96-well plates, on macrophagefeeder-layers. Final concentrations of 1 to 10

cellVwell were used. Supernatants were screen-ed for the presence of antibodies by RIA assoon âs macroscopically visible colonies wereseen. Of the positive spots shown by autoradio-graphy, those with single colonies, detected ma-cro-and microscopically, were selected for IIFand expansion..Antibodies so obtained were con-sidered monoclonal, but were later checked byID. When positive wells showed more than asingle colony, a recloning with less cells perwell was performed; this procedure was repeat-ed until positive wells contained only one co.lony.

Preparation of ascites fluids: B,A,LB,/c mice,previously injected IP with 0.5 mt of Pristaner(2, 6, 10, l4-tetramethyl pentadecane), received100 10r viable hybrid cells IP. After ascitic tu-mours had grown (usually within ?-14 days),mice were killed and ascites collected and cen-trifuged for removal of cells. The supernatants,to which 10 mM sodium azide was added, werestored Ìn 1 ml aliquots àt -20'C

RESULTS

Imrnunization of mice and cell fusion

The t\¡/o different protocols tested for im-munization with H.m. rnuscarum yielded quitedifferent results. Previous IP injection of anti-gen followed by an IV booster one month laterresulted in lower frequenqy of antibody-produc-ing clones (14 out of 48), more labile clones(only 6 out of 14 could be maintained) andantibodies predominantly of IgM.class. More-over, all six surviving clones were IgM secretors.On the other hand, IV injections and longer in-tervals before boosting produced higher fre-quency of growing hybrids (48 out of 48). higherfrequency of antibody-producing hybrids (30 outof 48) and antibodies mostly of IgG class (30

out of 48; 5 initial cultures were also positivefor IgM - Table I.! .

The two protocols employed to producedrnonoclonal antibcciies reacting against S. man-soni gave fairly comparable results. In the firstcase, using outbred albino mice after 16 weeksof infection, 21 out of 48 cultures showed grow-ing hybrids, B were antibody secreting and 6

coulci be maintained (5 IgG and I IgM). In thesecond experiment, with BALB/c mice after 7

weeks of infection, 20 out of 48 cultures showedgrowing hybrids, 14 were antibody-producersand 4 IgG secretors could be maintained (Ta-

ble I).

Myeloma lines ¿nd cell fusions

SP2 was the only cell line to grow wellunder our conditions, with a doubling time of15 to 20 hours. Different sources of FBS andRPMI 1640 were tested and well tolerated. SP2grew over plastic or glass, with or without fee.der-layers and was used in all fusion experi-ments. Poorer results were obtained with eitherNS1 or X63.

No growth was ever observed with horse,human or bovine serum. Concerning FBS, opti-mal results were obtained with most batchesfrom Flow Laboratories. It must be eynphazised,ho'wever, that different batches from the samesources may produce different results.

CelI fusions were successful when the pro-cedure described by St. GROTH & SCHEIDEG-GER 4r was followed and PEG, MW 4,000, from

- Production of monoclonal antibodÍes againstRev. fnst. Med. trop. São Paulo 24t32'l-336, 7982.

Meloy Labs. Inc., Biological p¡ods. Div., Springfield,Virginia, U.S..{.,{.ldrich Chemical Co., Mitwaukee, Wis., U.S.,{.

330

LOPES, J. D.; CARNEIRO, C. R,. W.; VILELA,.q,..4. & CAMARGO, E. p.Schistosom¿ mansoni and a trypanosomatid: A methodotogical repoú.

MU,

TABLE Isummary of four hybridoma experiments against H,m.muscarum and s. mansoni

Protocols ofimmunizations

MU*

SM,

IP-IV/3O days

IV-IV/?0 days

SÀ,{" 7 weeks ofinfection

16 weeks ofinfection

* Five initial cultures were positive also for

Cells per fusionMyeloma - Spleen

Sigma or Merck, was used at bO% (w/v) with5% (v/v) DMSO, at pH ?.5. No hybrids wereobtâ,ined with PEG from other sources or byfollOwirtg other protocols for cell fusione,22,30.Some PEG showed high toxicity to the celis,and subsequent absence of growth. Table I sum-marize the results of four successful fusionexperiments.

Cloning and expansion of hybrids

Cloning of hybrids was always made by thedilutÍon method. Cloning efficiency was muchinfluenced by the presence of macrophage fee-der-layers which, in addition to stimulatinggrowth also cleared cell debris from cultures.Preliminary experiments have shown that in theabsence of macrophages no gro'wth was obtain-ed. Satisfactory cloning resulted when indivi-dual wells received 1 to 2 cells, with an effici-ency ranging from 5 to 35%. When more than2 cells per well were added, the efficiency im-proved but fewer wells contained a single co-lony and therefore recloning was necessary.

Fewer clones were lost during expansionwhen propagation was made in 2 steps, al",vaysover a macrophage feeder-layer: colonies werecollected from 96 well plates and cultured in24well plates before being transferred to plas-tic culture flasks for expansion. The presenceof macrophages greatly prevented cell death;when the transference was made in the absenceof macrophages, large numbers of d.ead cellswere seen aftet 2448 hours, and many cloneswere lost.

Production of ascites

Some hybrids did not produce ascitic ru-mours at all while few others yielded ascites

1.2x10r

3x10?

1.6x10r 2.5x107

2x10r

Wells \trithgrowing hybrids

3x107

- Production of monoclonal antibodies ¿gainstRev. Inst. Med. trop. São paulo 242927-396, IgB2.

1.2xI0?

48/48

No. positive No. stable clonesclones

2L/48

IgM

74/48

8/48

fluids of low antibody content as judged by IIFtiters. The majority of hybrids, however, produc-ed ascites ,with high titer of antibod.ies after?-14 days. We have also tried to induce ascitesin (Cs?81/10 x BALB,z c) F, mice. Despite theneed for longer periods to develop (B weeks inâverage), ascitic fluids were also obtained inthose F, mice.

Detection and characterization of antibodies

Supernatants of hybrid cultures gave posi_tive results only when clones were macrosco_pically visible and the medium turned acidic.IIF, initÍally used to screen cultures, could alsobe used to determine the class of immunoglo_bulin produced as well as the cellular localiza_tion of the antigens. IIF was the only test em_ployed for detecting antibody activity againstS. mandoni antigens. (Table II and Fig. l).

R,I.A, used when large numbers of superna-tants of cloning plates were tested for anti-bodies'agaÍnst II.m. muscarum, proved adequa-te as A screening assay for the selection of su-pernatants to be tested by IIF.

DISCUSSION

Protocols for the production of monoclonalantibodies ¿1's rn¿nv9, 16,20,22,28,30,4r. Although hav_ing similar outlines, they vary in details concern-ing immunization schedules, nature of antigens,choice of myêloma cell lines. growth media, che-micals and use of laboratory facilities. In Bra-zil, lack of proper equipment and many specialchemicals is a condition which may require mo-difications of the existing protocols. Reportingthese modifications may be usêful to other re-search groups involved in the production of mo-

331

5/48

being tested

6/48

Class of

IeM

IcG.

5 IgGI IgM

rgG

LOPES, J, D.; CARNEIRO, C. R. rt\¡.; VILELA, A. A. & CAMARGO, E. P. - Production of monoclonat antibodies againstSchistosoma mansoni and a trypanosomatid: r{ methodological report. Rev. Insú, Med, trop. São Paulo ?4:32?-336, 1982.

Fusion

MU,

TABLE IIPatterns of IIF and subclasses of lgs obtainsd in four fusion exþeÍiments

MU,

Number of MCantibodies

SM,

* rD performed with supernatânts âgâinst specific antisera: only positive ¡eactions are scored

sM,,

nocional antibodies against parasites. In thepresent pape|we describe the procedures adop-ted to circumvent local difficulties for the pro-duction of antibodies against S. mansoni andIf.m. muscarum.

We have tried, for fusion experiments, Bdifferent cell lines: NS12r, X6Brs and Sp2:s. Be_cause SPz produces l¡ybrids ,which are moresensÍtive to environmental conditions 16, NSLand X63 are the cell lines currently used in mostlaboratorÍes. We found, however, that Sp2 waseasier to maintain in culture, and thus the onlycell line to yield hybrids. This discrepancybetween our results and those reported. byothers30'41 is not entirely surprising because ourconditions are different from those prevailingin other laboratories, a fact "which is known tointerfere with cell's behavior in culture zo.

Our best results in hybridization experi_ments rMere obtained with the Sp2 line, at thelogarithmic phase of growth, after at least twopassages in fresh culture medium, when viability was greater than 9A%. Viability was easilymonitored by phase microscopy. In spite ofideal culture conditions, however, some colo_nies of hybrids did not thrive and./or stoppedproducing antibodies, a fact that occurs alsowith other kinds of cell lines 2s,30. This may bedue to overgrowth of non-secreting revertantsor to unknown regulatory mechanisms.

A serious handicap in the hybridization pro_cedure may be the source of pEG¿r. Satisfacto-ry growth ,was only obtained ,when cell fusions\¡¡ere promoted by pEG, MV/ 4,000, from Sigma(Lot No. 59C-00?0) or Merck (Lot No. 668?0)

332

IIF patterns

5 diffuse

16 diffusel0 membrâne4 flagellum

2 worm gut2 tegument2 diffuse

2 worm gut1 tegumentI diffuse

Ig subclasses *

5 IgM

but not when other commercial preparations ofPEG were used. This is in accordance'with pre-vious findings by St. GROTH & SCHEIDEG-GER,41. Therefore, it is recommended that celltoxicity by PEG be carefully checked prior toany crucial experiment. When hybrids fail togrow even when plated on macrophage feeder-layers, toxicity of PEG must be suspected.

Fetal bovine serum (FBS) is a very expen-sive requirement ,which is becoming scarceworldwide. Finding a readily available substitutefor FBS would be highly desirable. Although wetried other supplements in the growth medium,cells and hybrids failed to gro,w in the presenceof horse, human or bovine serum, despite theaddition of aminoacids and vitamins to RPMI1640 28. Different batches and sources of FBSIed to a great variabilily in the doubling timeand in the viability of cells. Best results werealways obtained with FBS from Flow Labs.(Lot No. 29111105).

Providing a CO, atmosphere for cell growthrepresented a serious problem from the start.As soon as flasks and plates became too nume-rous to be accomodated in candle jars, we hadto modify a FANEM incubator (Model 0b1lE)by coupling to it a stand.ard compressor (AURI,Model 50) in order to produce a continuousflow of Air/COr. Small variation in CO, concen-tration, f.rorn íVo to BYo, and the incubãtor tem-perature, from 35 to BB"C, did not criticallyaffect growth of either cells or hybrids. Thesame was true for different batches and sourcesof RPMI 1640 and for the amount of HEpESused, which could vary from 2b to g0 mM.

5 IgGII loêA

1 IgG;B

6 IgGr

3 IgGl

LOPES, J. D.; CARNEIRO, C. R. W.; VILELA, A. A. & CAM,{RGO, E. P. - Production of monoclonal antiþoalies againstSchistosoma mansoni and a trypanosomatid: ,{ methoCological report. Rev. Inst. Med. trop. São Paulo 24:327-336, 1982.

Hybrids failed to grow in the absence ofmacrophages. The addition of peritoneal macro-phages as feeder-l4ys¡s 20,28.30 supported growthof hybrids. Macrophages from inbred or out-bred mice or rats could be used as well.

Results on the production of monoclonalantibodies varied according to antigens andimmunization schedules used. It is assumedthat both antigens and immunization schedulesprobably modulate the number and classes ofIgs obtained26.

Concerning H.m. muscarum, an IV booster4 days before fusion greatly increased the num-ber of positive colonies. A longer interval bet-ween the first and last inoculations resultedin predominance of IgG antibodies. In reverse,shorter intervals yielded antÍbodies predominan-tly of the IgM class. 'Whether these antibodies,ïgM or IgG, react with different epitopes, wasnot pursued as yet, but the different patternsof IIF reactions observed (diffuse, surface-res-tricted and flagellar) strongly suggest that dif-ferent antigens are involved.

Concerning S. mansoni, infected rather thanimmunized mice were used as spleen donorsfor cell fusion. Under these conditions, circulat-ing antigens, either excreted or shed by livingworms, represent the major antigenic stimu-lus 3¡. This is in accordance with the IIF pat-terns which showed positive reactions exclusi-vely against the gut's cell layer or against thetegument of ,whole worm sections. These find-ings may also explain the poorer results obtain-ed by the RIA test with soluble proteic wormantigens q, 'which are prepared from homogena-tes of fresh adult worms. Monoclonal antibodiesagainst antigens shed by living worms may hetpto standardize an assay to measure circulatingworm antigen and to characterize antigens in-volved in immune complexes. Such investiga_tion is now under study in our laboratory.

RESUMO

Produção de anticorpos monoclonais contraSchistosoma mansoni e um Tripanosomaúídeo:

Descrição metodológica.

Anticorpos monoclonais foram prod.uzid.oscontrâ dois diferentes orgairismos: o tripanoso-matídeo Herpetomonas muscarum muscarum eSchistosoma mansoni. Verificou-se que a obten-

334

çáo desses anticorpos depende de variáveis co-mo esquemas de imunizaçáo, origem e peso mo-lecular do polietilenoglicol, lotes de soro fetalbovino, feeder-layers de macrófagos, linhagensde células e condições de cultura de células mie-Iomatosas de camundongo.

Aplicando um protocolo aqui descrito, fo-ram obtidos 35 anticorpos monoclonais estáveiscontra H.m. muscarum e 10 contra S. mansoni.Com relação ao tripanosomatídeo, um esquemade intervalos menores entre as imunizações re-sultou em anticorpos da classe IgM; ao con-trário, intervalos mais longos resultaram emanticorpos predomiriantemente da classe IgG.Com S. mansoni, infeoções mais ou menos lon-gas produziram igualrhente anticorpos predomi-nantemente da classê, IgG. Padrões diferentesde reação foram obsçrvados através de imuno-fluorescência indireta: alguns anticorpos mos-traram predominância de reaçáo contra a su-perfície e outros contra o flagelo do tripanoso-matídeo. Os anticorpos monoclonais anti-S.mansoni reagiram contra o tegumento ou con-tra o tubo digestivo em cortes de vermes adul-tos. A importância biológica dos dois tipos deanticorpos está agora sendo investigada.

ACKNOWLEDGEMENTS

The Authors are indebt to Sueli Tomaselida Encarnação, Maria Amélia Romano, Euni-ce José de Sant'Ana and Leny Aiach for thetechnical assistance, to Professor L.R. Travas-sos for reviewing the manuscript and to pro-fessor J. R,. Magalháes for the photographic eo-cumentation.

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2. ANDRADE, Z. A. & SUSIN, M. - Renal changes tnmice infected wiih S. mansoni. Am. J, Trop, Med.Hyg. 23: 400-403, 19?4.

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