SCREENING OF MURINE MONOCLONAL ANTIBODIES AGAINST LIVING SCHISTOSOMULA OF SCHr~~USU~A ~A~~~~r BY RADI~IMMUNOASSAY PETERM.WIEST,*~-ADAMV.WISNEWSKI,*JOAN H.JOHNSON,* BERNADETTE RAMIREZ,~ THOMASF.KRESINA*~~~G.RICHARDOLDS* *Program in Geographic Medicine, Intemational Health Institute, Brown University and Department of Medicine, The Miriam Hospital, f64 Summit Avenue, Providence, RI 02906, U.S.A. SResearch Institute of Tropical Medicine, Manila, Philippines (Received 5 July 1990; accepted 25 November 1990) Abstract-WIEST P. M., WISNEWSKI A. V., JOHNSON J. H., RAMIREZ B., KRESINA T. F. and OLDS G. R. 1991. Screening of murine monoclonal antibodies against living schistosomula of Sehjstosoma rnunso~~ by radioimmunoas~y. Znternufi~~ul JuurnulJbr PQrusitology 21:449454. A radioimmunoas~y was developed to screen supematants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stagespecific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. munsoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.

INDEX KEY WORDS: Schistosomn mansoni: trematode; monoclonal immunoblotting.

INTRODUCTION HUMAN schistosomiasis is a major health problem infecting over 200 million people throughout developing countries (Mahmoud, 1988). A major effort to control this multicellular parasite has been directed towards development of a vaccine based on immunologi~ly mediated resistance to infection. In this regard, monoclonal antibody technology has greatly assisted in the generation of candidate vaccine molecules which identify parasite antigens (Olds, 1988). In schistosomiasis, these reagents have been used for the purification of worm antigens, the screening of cDNA expression libraries as well as the basis for the development of idiotype based vaccine strategies (Capron, Dessaint, Capron, Ouma Br Butterworth, 1987; Colley, Cook, Freeman, Bartholomew & Jordan, 1977; Mitchell & Cruise, 1987; James, 1987; Kresina & Olds, 1989). A specific parasite stage for generation of monoclonal antibodies to schistosome antigens is the schistosomula. During infection of man, free-Iiving cercariae penetrate intact skin and transform into parasitic schistosomula (Mahmoud, 1988). During

antibody; radioimmunoassay;

this transition the trilaminate surface membrane of cercariae matures into a double unit bilayer that covers schistosomula (Hockley & McLaren, 1973; Wiest, Tartakoff, Aikawa & Mahmoud, 1988; Wiest, Kossman & Tartakoff, 1989). This surface membrane contains stage-specific glycoproteins which may represent targets for the host’s immune response (James, 1987). Monoclonal antibodies that recognize surface antigens of schistosomula have, therefore, been sought. In the present study a radioimmunoassay was developed to identify monoclonal antibodies that bind the surface of living schistosomula of S. m~soni. Four monoclonal antibodies were selected for characterization with regard to antigen binding and stage specificity. Each monoclonal antibody recognized distinct surface antigens of S. mansoni. Our data indicate that radioimmunoassay is an effective alternative method to identify monoclonal antibodies that recognize surface antigens of S. rn~n~on~. MATERIALSANDMETHODS Parasites. The Puerto Rican strain of S. mansoni was

maintained in CD-1 female mice (Charles River Laboratory, Wilmington, DE) and Biomphaluriu glubratu (albino) snails.

tTo whom all correspondence should be addressed 449

450

P. M. Wtnsr et ai

Snails were shed for 1 h in spring water and cercariae collected. Mechanical schistosomula were prepared as a modification of the methods of Ramalho-Pinto,-Gazzinelli, Howells, Mota-Santos. Figuerado & Pellenrino (1974). Briefly, cercariae were con&&rated in spring-water on ice. The supematant was removed and the organisms were agitated for t min. The bodies were then separated from the tails using a 60% (v/v) Percoll gradient centrifuged at 600 x g for 10 min at 4°C (Lazdins, Stein, David & Shet. 1982). Cercarial bodies were contaminated with less than 1% tails. Parasites were washed and incubated in RPMI-1640 medium (Whittaker M.A. Bioproducts, Walkerville, MD) supplemented with 2 mM+glutamine, 100 i.u.mll’ penicillin and 100 pg ml-’ streptomycin in a 6% CC&/air incubator at 37°C for 3 h. These parasites were considered 3-h-old schistosomula. Previous studies have indicated that these parasites have typical characteristics of schistosomula such as water intolerance, surface binding of concanavalin A and the presence of a double unit surface bilayer (Wiest et al., 1988). Twenty-four-hour and 4-day-old schistosomula were cultured under identical conditions for the appropriate time periods with addition of 10% heat-inactivated fetal calf serum to the culture medium. Lung stage schistosomula were prepared by infecting mice percutaneously with SO00 cercariae each (McLaren & Terry, 1982). Seven days later, the lungs were removed, minced; and incubated in Earle’s lactalbumin (ELAC) at 37°C for 3 h. The extract was nassed through a 1Ii mm nylon mesh to remove large pieces of lung tissue. Parasites were then washed with ELAC and solubilized in boiling electrophoresis buffer (0.125 M-TrisHCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol) for 5 min. Lung extracts from uninfected animals were prepared in a similar manner to serve as negative controls. Preparation ofmonoclonai antibodies. Female 6-week-old CD-l mice were each immunized intraperitoneally with an emulsion of So00 freeze/thawed 3-h-old schistosomula in phosphate-buffe~d saline, pH 7.4 (PBS) mixed I:1 (v:v) with Freund’s complete adjuvant (Difco, Detroit, Ml). Mice were immunized 3 weeks later with 5000 freeze/thawed organisms mixed 1:1 (v:v) with incomplete Freund’s adjuvant. Animals were then boosted twice at 3 week intervals with 5000 freeze/ thawed parasites each in PBS. After resting for 3 weeks, mice were injected again with 5000 freeze/thawed schistosomula in PBS and spleen cells were used for somatic cell fusion 4 days later. Hybridoma clones were generated by fusing splenocytes to P3NSI murine myeloma cells (ATCC No. TlBl8) (Olds SCKresina, 1990). Supernatants of growing clones were screened 14 - 21 days after fusion for binding activity as described below. Large quantities of monoclonal antibodies were produced as ascites fluid in BALB/C mice which had been injected intra~ritoneaily with 0.5 ml pristane (2,6,1~,14_tettamethyI~ntad~ne, Aldrich Chemical Co., Milwaukee, WI). Isotypes of monoclonal antibodies were determined by Ouchterlony double diffusion using isotype specific antisera (Cappel Laboratories). Radioimmunoassay. Polystyrene 96-well culture plates with ‘V-shaped bottoms (Gibco, Grand Island, NY) were blocked with 200 ~11of 5% bovine serum albumin-PBS (BSA-PBS) for 30 min at 37°C. Thereafter, 300 organisms were added to each well and blocked with 150~1 of 5% BSAPBS for 30 min at 4°C. Parasites were washed three times with 1% BSA-PBS and incubated with 100 ~1 of culture supernatant for 2 h at 4°C with continuous agitation on a microplate shaker (Dynatech Laboratories, Chantilly, VA). Parasites were washed three times and incubated with

50,00&100,000 c.p.m. of affinity-purified ‘“‘I-labeled F(ab’), fragments of sheep anti-mouse immunoglobulin (specific activity: 74-370 Bq pg.‘; New England Nuclear, Boston, MA) in 1% BSA-PBS for I hat 4°C. Organisms were washed five times and counted on a Gamma-counter. A I:100 dilution of serum in PBS from mice infected with 400 cetcariae for 6 weeks was used as a positive control. Negative control samples included spent culture medium, i/i00 dilution of normal mouse serum (NMS) in PBS, and an irrelevant monoclonal antibody (isotype IgG). Significant binding was defined as samples with radioactivity at least two to three times over background organisms in PBS-BSA alone. At the completion of this assay, schistosomula were viable as determined by motility and trypan blue dye exclusion (Ellner, Olds, Lee & Edmonds, 1982). Enzyme-linked immunosorbent assay. Soluble worm antigen preparation (SWAP) and soluble egg antigen (SEA) were prepared as previously described (Colley et al., 1977). Wells of 96well plates (Falcon 3911, Becton Dickinson, Oxnard, CA) were individually coated with 100 ~1 of l&20 pg ml -’ SWAPor I~~grnl-‘ S~AinPBSat~Covernight. Unbound sites were blocked with 150 ~1 of 5% BSA-PBS for 1 h at 37°C. Plates were washed three times with 1% BSA-PBS containing 0.1% Tween-20 and then incubated with 100~1 of supematant for 2 h at 37°C. After washing five times, plates were incubated with 75 ~1 of a l/400 dilution of alkaline phosphatase-conjugated anti-mouse IgG immunoglobulin (NEN, Boston, MA) overnight at 4°C. Finally, 60 ~1 of 1:10 dilution of the substrate p-nitrophenyl phosphate (Sigma) was added to each well. Plates were read within 3 h on an automated 2550 ELISA reader (BioRad, Richmond, CA) at 40.5nm. Jmmunoblot analysik Antigens were separated by 10% SDS-polyacrylamide electrophotesis (SDS-PAGE) and transferred to nitrocellulose (Schleicher and Schuell, Woburn, MA). Strips were blocked with 5% dry milk in Trisbuffered saline (20 mM-Tris-HCI, pH 7.4, 500 rnM-NaC1, TBS) overnight at 4°C. Strips were then washed three times with I % dry milk, 0.5% Tween-20 and incubated with either a I:25 dilution of ascites in TBSor undiluted supernatant for 3 h at 37°C. Washed strips were incubated with a I:500 dilution of horseradish peroxide-conjugated goat anti-mouse immunoglobulin (Organoh Teknika, West Chester, PA) for 1.5 h at 37°C and developed with 4-chloto-l-napthol (Bio Rad. Richmond, CA), Antigen preparation. Paramyosin was puritied from 6week-old S. mansoni using the procedure of Harris & Epstein (1977). Purified 68 kDa S. mansoni glycoprotein was a generous gift from Dr Charles King, Case Western Reserve University, Cleveland, Ohio (King, Lett, Nanduri, El Ibiathy, Peters, Olds & Mahmoud, 1987). RESULTS Mice were immunized with freeze/thawed schistosomula and somatic cell fusions performed. This immunization protocol not only induces significant resistance against infection with S. mansoni, but also results in a significant polyclonal antibody response to the surface of living schistosomula (Kresina & Olds, 1989). Hybridoma supernatants were screened by radioimmunoassay for binding to 3-h-old schistosomula. Of 196 clones evaluated, 20 clones demonstrated significant binding activity by radioimmunoassay. When the clones were subsequently re-screened by

Radioimmunoassay TABLE I-BINDING

451

for S. mansoni

OFMONOCLONAL ANTIBODIES TODWERENT DEVELOPMENTAL STAGES OF

Schisiosoma mansoni Monoclonal antibody 204-3E4 204-2C5 204-3Fl 204-4E3

Antigen binding* Cercariae (c.p.m. f S.D.)

Schistosomula (c.p.m. f S.D)

615&50 382f 55 610+ 126 NDt

446zt74 447f37 193zk-31 102f22

SWAP

SEA

COD)

COD)

0.807 0.805 0.027 1.163

0.788 0.687

NDt

1.047

*For cercariae and schistosomula radioimmunoassay was performed with hydridoma supernatant with results expressed in c.p.m. f standard deviation, N=6. Background was 290f 16 c.p.m. for cercariae and 50* 13 c.p.m. for schistosomula. Infective mouse serum (IMS, 1:lOO) was 688f 145 c.p.m. ELISA was used to analyze antibody binding of hybridoma supematant to SWAP and SEA. Ascites (1:50) were used for 204-3E4. Optical densities (OD) for IMS (1:lOO)and NMS (1:lOO)for SWAP were 1.413 and 0, respectively. For SEA, OD for IMS (1:lOO)and NMS (1:lOO)were 1.66 and 0.136, respectively. tNot done.

radioimmunoassay, 19 remained positive for binding to 3-h-old schistosomula. Hybridoma supernatants that bound schistosomula by radioimmunoassay were tested for binding to SWAP and SEA by ELISA. Of the 19 clones, five

FIG. 1

Immunoblot analysis with monoclonal antibody 2043E4 against S. mansoni. A 68 kDa antigen (arrow) was recognized in 3-h-old schistosomula (lane 1), SWAP (lane 2) and purified 68 kDa 31-3B6 glycoprotein (lane 3). No proteins were identified when normal ascites fluid was reacted with purified 68 kDa antigen (lane 4).

clones bound to SWAP and four clones recognized SEA. All monoclonal antibodies which recognized SWAP also bound SEA. With regard to parasite stage specificity, 74% of the monoclonal antibodies recog nized surface antigens of schistosomula but not adult worms. Of all monoclonal antibodies identified, four were selected for antigen characterization based on rapid and stable growth of hybridoma cell lines. Monoclonal antibody 204-3E4 (IgG,,) recognized 3-h-old schistosomula and cercariae by radioimmunoassay as well as SWAP and SEA by ELISA (Table 1). When analyzed by immunoblotting, 2043E4 reacted with a 68 kDa parasite antigen present in schistosomula and adult worms (Fig. 1). 204-3E4 also bound purified 68 kDa glycoprotein (Fig. 1). Previous studies have demonstrated that this 68 kDa glycoprotein from S. mansoni induces significant protection (28-66%) against cercarial challenge in mice (King et al., 1987). Monoclonal antibody 204-2C5 (IgG,,) bound cercariae and schistosomula by radioimmunoassay and SWAP and SEA by ELISA (Table 1). Immunoblot analysis demonstrated that 204-2C5 also recognized a 68 kDa protein in schistosomula and SWAP (data not shown). Binding of the monoclonal antibody 204-3Fl (IgG, subclass indeterminant) to surface antigens of S. mansoni was stage specific. This antibody reacted with cercariae and schistosomula by radioimmunoassay but not SWAP or SEA as determined by ELISA (Table 1). Immunoblot analysis demonstrated that 204-3Fl recognized a single 18 kDa parasite antigen (Fig. 2). The epitope was expressed in cercariae, 3- and

P. M. WRESTet al.

452

I

234

5

6

FIG. 2 Immunoblot analysis with monoclonal antibody 204-3Fl against S. mansoni. A 18 kDa antigen (arrow) was recognized in cercariae (lane I), 3-h-old schistosomula (lane 2) and 24-h-old schistosomula (lane 3), but not 4-day cultured schistosomula (lane 4) 7-day lung stage schistosomula (lane 5) or SWAP (lane 6). 24-h-old schistosomula, but not in 4-day-old cultured parasites, 7-day lung stage schistosomula or SWAP (Fig. 2). Monoclonal antibody 204-4E3 (IgM) bound the surface of schistosomula by radioimmunoassay but binding was not intense. In contrast, the antibody was highly reactive against SWAP and SEA by ELISA (Table 1). Immunoblot analysis demonstrated that the antibody reacted with a 97 kDa antigen in SWAP. A candidate molecule for this 97 kDa antigen is paramyosin, a myofibrillar protein in invertebrates (Lanar, Pearce, James & Sher, 1986; Pearce, James, Hieny, Lanar & Sher, 1988). To determine if this 97 kDa antigen was paramyosin, this protein was purified from adult worms (Fig. 3). Immunoblot analysis demonstrated that the antibody did indeed react with paramyosin (Fig. 3). DISCUSSION Various screening methods have been used to identify monoclonal antibodies that recognize surface antigens of schistosomula of S. mansoni. These techniques include ELISA, indirect immunofluorescence assay (IFA), solid-phase radioimmunoassay and functional assays (King er al., 1987; Gregoire, Shi, Rekrosh & Loverde, 1987; Harn, Mitsuyama & David, 1984; Bickle, Andrews & Taylor, 1986; Dissous, Grzych & Capron, 1982; Smith, Clegg, Snary & Trejdosiewicz, 1982; Strand, McMillan &

Pan, 1982; Taylor & Butterworth, 1982; Zodda, Abdel-Hafez & Phillips, 1983). In the present report a radioimmunoassay was developed to screen murine monoclonal antibodies that bind the surface of living schistosomula. The advantage of this method is that large numbers of supematants can be rapidly screened. Furthermore, the use of living organisms allows maintenance of surface epitopes similar to those expressed on the parasites in vivo. The possibility that potential epitopes may be denatured by fixatives or destroyed by endogenous proteases during antigen preparation is therefore lessened. An alternative screening method IFA also uses living organisms as target antigens. This procedure is however laborious and semiquantitive unless photometry is used (Samuelson & Caufield, 1985). Previous workers have used living schistosomula as targets for radioimmunoassay to quantify the amount of IgG bound to the parasite’s surface (Omer-Ali, Smithers, Bickle, Phillips, Ham & Simpson, 1988). This is the first report for the use of radioimmunoassay to screen hybridoma-derived monoclonal antibodies for binding to schistosomula. Four monoclonal antibodies were selected for characterization. One antibody reacted with a 68 kDa antigen found in cercariae, schistosomula and adult worms. Upon further investigation, this monoclonal antibody bound to immunoaffinity-purified 68 kDa glycoprotein (King et al., 1987). This antigen of S.

Radioimmunoassay

:

z.7

39-

453

for S. mansoni

kDa protein in 3-h- and 72-h-old schistosomula but not cercariae, 5-day lung stage or IO-day liver parasites (Bickle et al., 1986). A monoclonal antibody that reacts with a single 18 kDa surface-labelled polypeptide in schistosomula and lung-stage parasites has been described (Dalton, Tom & Strand, 1987). The present monoclonal antibody appears to react with an 18 kDa antigen different from the previously reported antibodies since our antibody does bind cercarial antigen but not lung-stage organisms. Lastly, a monoclonal antibody that reacted with paramyosin was identified. This 97 kDa myofibrillar protein, which can induce significant protection (2453%) in mice against cercarial challenge, is found in muscle and elongate bodies of the tegument but is not exposed on the parasite’s surface (Lanar et al., 1986; Pearce et al., 1988; Matsumato, Perry, Levine, Blanton, Mahmoud & Aikawa, 1988; Flanigan, King, Lett, Nanduri & Mahmoud, 1989). Tegumental turnover or exocytosis of elongate bodies have been suggested as a means to explain release of paramyosin epitopes for stimulation of the host’s immune system (Sher, James, Correa-Oliveira, Hieny & Pearce, 1989). Furthermore, this monoclonal antibody bound SEA, suggesting that paramyosin may be localized to eggs and/or miracidia. In summary, we utilized a radioimmunoassay using living schistosomula to screen hybridoma cell lines that produce monoclonal antibodies against the parasite’s surface. Characterization of four monoclonal antibodies demonstrates that this methodology allows selection for surface antigens of S. mansoni. These data suggest that radioimmunoassay will be useful to screen hybridoma cell lines to identify anti-schistosome monoclonal antibodies.

FIG. 3 Immunoblot analysis of 204-4E3 against S. mansoni. The monoclonal antibody reacted with purified paramyosin from S. mansoni (lane 1). Lane 2 demonstrates purified paramyosin by amido black staining. No binding was found when an irrelevant monoclonal antibody (IgM) was reacted with paramyosin (lane 3).

Acknowledgements-The authors thank Valerie J. Sears for her assistance in the preparation of this manuscript. The research was supported by the National Institute of Health grants AI00780 (P.M.W) and A125167 (G.R.O.), and a grant from the Rhode Island Foundation (P.M.W).

is expressed in adult worms, schistosomula and the head glands of cercariae. Furthermore, immunization of mice with this antigen induces significant protection (28-66%) against cercarial challenge. Monoclonal antibody 204-2C5 also recognized a 68 kDa antigen which may be similar to the protective 68 kDa protein by King et al. (1987). A third monoclonal antibody bound a 18 kDa protein in cercariae, 3- and 24-h schistosomula but not in 4-day, lung stage or adult worms. Several laboratories have identified stage-specific surface antigens with similar molecular weights in S. mansoni by radiolabelling the surface of schistosomula (Simpson, James & Sher, 1983; Liberti, Festucci, Ruppel, Gigante & Cioli, 1986; Simpson, Payares, Walker, Knight & Smithers, 1984). A stage-specific monoclonal antibody recognized a predominant 16

BICKLE Q. D., ANDREWS B. J. & TAYLOR M. G. 1986. Schistosoma mansoni: characterization of 2 protective monoclonal antibodies. Parasite Immunology 8: 95-107. CAPRON A., DESSAIN~ J. P., CAPRON M., OUMA J. H. & BUTTERWORTHA. E. 1987. Immunity to schistosomes: progress toward vaccines. Science 238: 1065-1072. COLLEYD. G., COOK J. A., FREEMANG. L., BARTHOLOMEWR. K. & JORDAN P. 1977. Immune responses during human schistosomiasis mansoni. 1. In vitro lymphocyte blastogenic response and heterogeneous antigen preparation from schistosome eggs, worms and c&cariae: International Archives of Allergy and Applied Immunoloav 53: 42Cb430. DALTON J. P., TOM-T. D. &-STRAND M. 1987. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vaccinated mice. Proceedings of the National Academy of Science of the United States of America 84: 4268-4272. DISSOUS C., GRZYCH J. & CAPRON A. 1982. Schistosoma mansoni surface antigen defined by a rat monoclonal IgG,,.

REFERENCES mansoni

Journal of Immunology 129: 2232-2254

ELLNERJ. J., OLDSG. R., LEEC. W. & EDMONDS K. L. 1982. Destruction of a multicellular parasite Schistosoma mansoni by cytotoxic T lymphocytes. Journal of Clinical Investigation 70: 369-378.

FLANIGANT. P., KING C. H., LETT R. R., NANDURIJ. & MAHMOUDA. A. F. 1989. Induction of resistance to Schistosoma mansoni infection in mice by purified parasite paramyosin. Journal of Clinical Investigation 83: lOlO1014. GREGOIRE R. J., SHI M., REKROSHD. M. & LOVERDEP. T. 1987. Protective monoclonal antibodies from mice vaccinated or chronically infected with Schistosoma mansoni that recognizes the same antigens. Jaumaf of Immunology 139: 3792-380 1. HARND.A., MITSUYAMA M. &DAVIDJ. R. 1984. Schistosoma mansoni anti-egg monoclonal antibodies protect against cercarial challenge in viva. Journal of Experimental Medicine 159: 1371-1387.

HARRISE. H. & EPSTEIN I-I. F. 1977. Myosin and paramyosin of Caenorhu~d~tis elegans: biochemical and structural properties of wild type and mutant proteins. Celf 1D:709719. NOCKLEYD.

J. & MCLAREND. J. 1973. Schistosoma mansoni changes in the outer membrane of the tegument during development from cercariae to adult worms. International

Journal for Parasitology

3: 13-27.

JAAIESS. -L. 1987. Sch&sama spp. : progress toward a defined vaccine. Experimental Parasitoloav 63: 247-252. KINGC. H., LET-T R. i., NANDURI J., EL IB&HY S., PETERS P. A. S., OLDS G. R. & MAHMOUD A. A. F. 1987. Isolation and characterization of a protective antigen for adjuvant-free immunization against Schistosoma mansoni. Journal of Immunology 139: 4218-4224.

KRESINAT. F. & OLDS G. R. 1989. Anti-idiotypic antibody vaccine in mutine schistosomiasis mansoni: comprising the internal image of antigen. Journal of Ciinicat Znvestjgation 83: 912-917.

LANARD. E., PEARCEE. J., JAMESS. L. & SHERA. 1986. Identification of paramyosin as schistosome antigens by intradermally vaccinated mice. Science 234: 593-596. LAZDINSJ. K., STEINM. J., DAVIDJ. R. & SHERA. 1982. Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by ~nt~fugation on discontinuous density gradients of Percoll. Experimental Parasitology 53: 39-47. LIB~RTIP., FESTUCCI A., RUPPELA., GIGANTES. & Cro~r D. 1986. A surface labeled 18 kilodalton antigen in Schistosoma Parasitology

mansoni. 18: 55-61.

Molecular

and

Biochemical

MAHMOUD A. A. F. 1988. Schistosomiasis. In: Trot&al and Geographica/ Medicine (Edited by WARRENK: S. and MAH~OUDA. A. F.). McGmw-Hill, New York. MA~IJMATOY., PERRYG., LEVINE R. J. C., BLANTON R,, MAHMOUD A. A. F. & AIKAWA M. 1988.Paramyosinandactin in schistosomal teguments. Nature (London) 333: 75-78. MCLAREND. J. & TERRYR. J. 1982. The protective role of acquired host antigens during schistosome maturation. Parasitology Immunology 4: 12%134. MITCHELL G. F. & CRUISE K. M. 1987. Schistosomiasis: antigens and host-parasite interactions, In: Par~itjc Antigens, Vol. 7 (Edited by PEARSON T. W.), pp. 275-316.

Marcel Dekker, New York. 0~0s G. R. 1988. Immunopathology and resistance in helminth infections. In: Parasite Infections (Edited by

LEECHJ. H., SANDE M. A. and RENTSR. K.t. Churchill Livingston, New York. 0~0s G. R. & KRE~INAT. F. 1990. Characterization of a family of monoclonal antibodies which bind Schistosoma japonicum egg antigens and express an interstrain strain cross-reactive idiotvpe. Parasite Immunolonv 12: 199-211. OMER-ALIP., SEAIT&&S. R., BICKLE Q., PHILLIPS S. AI., HARN D. & SKMP~ON A. J. G. 1988. Analvsis of the antiSchistosoma mansoni surface antibody response during murine infection and its potential contribution to protective immunity. Journalof Immunology la 258-264. PEARCEE. J., JAMESS. L., HIENYS., LANARD. E. & SHERA. 1988. Induction of protective immunity against S~h~tosama rn~on~ by vaccination with schistosome paramyosin (Sm97), a nonsurface parasite antigen. Proceedings of the National Academy of Science of the United States of America 85: 5678-5682.

RAMALHO-PINTO F. J., GAZZINELLI G., HOWELLS R. F., MOTASANTOST. A., FIOUERADO E. A. & PELLEGRINO J. 1974. Schistosoma mansoni: a defined system for the stepwise transformation of cercariae into schistosomula in vitro. ~xperj~nta~

Parasjtology

36: 360-369.

SAMUELSON J. C. & CAUFIELDJ. P. 1985. The cercarial glycocalyx of Schistosoma mansoni. Journal of Ceil Biology 100: 1423-1434. SHER A., JAMESS. L., CORREA-OLIVEIRA A., HIENY S. & PEARCEE. 1989. Schistosome vaccines: current progress and future. Parasitology 98 (suppl.): S61-S68. _ SIMPSONA. J. G., JAMESS. L. & SHERA. 1983. Identification of surface antigens of s~histo~mula of Sch~tasoma mansoni recognized by antibodies from mice immunized by chronic infection and by exposure to highly irand Immunity 41: radiated cercariae. Infection 591-597. SIMPSONA. J. G., PAYARES G., WALKERT., KNIGHT M. & SMITHERS S. R. 1984. The modulation of expression of polypeptide surface antigens of developing schistosomula of Schistosoma mansoni. Journal of Zymology 133: 272% 2730. SMITHM.

A., CLEGCJ. A., SNARYD. & TREJDOSIEWICZ A. J. 1982. Passive immunization of mice against Schistosoma mansoni with IgM monoclonal antibody. Parasitology 84: 83-94.

STRANDM.,

MCMILLANA. & PAN X-Q. 1982. Schistosoma reactivity with infected human sera and monoclonal antibody characterization of a giycoprotein in different developmental stages. Experimental Parasitology mansoni:

SQ: 145-156.

TAYLORD. W. & BUTTERWORTH A. E. 1982. Monoclonal antibodies against surface antigens of schistosomula of Schistosoma mansoni. Parasitology

84: 65-82.

WZEST P. M., TARTAKOFF A. M., AII(AWAM. & MAHMOUD A. A. F. 1988. Inhibition of surface membrane maturation in schistosomula of Schistosoma mansoni. Proceedings of the National Academy of Science of the United States of America 85: 3825-3829. WEST P. M., KOSSMAN R. J. & TARTAK~FF A. M. 1989. Determinants of surface membrance maturation during the cercarial-schistosomula transformation of Schistosoma mansoni. American Journot of Tropical Medicine and Hygiene 41: 70-79.

ZODDAD. M., ABDEL-HAFEZ S. K. & PHILLIPSS. M. 1983. Characterization of monoclonal antibodies against Schistosoma mansoni. American Medicine and Hygiene 32: 69-77.

Journal

of

Tropical

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay.

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosom...
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