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Toxoplasma gondii: Characterization of monoclonal antibodies that recognize rhoptries

Toxoplasma gondii: Characterization of monoclonal antibodies that recognize rhoptries

EXPERIMENTALPARASlTOLOGY68,7‘l-82(1989) Toxoplasma gondii: Characterization of Monoclonal Recognize Rhoptries JOSEPH D. SCHWARTZMAN Department of...

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Characterization of Monoclonal Recognize Rhoptries


of Pathology,


of Virginia


of Medicine,



C. KRUG’ Charlottesville,



SCHWARTZMAN, J. D., AND KRUG, E. C. 1989. Toxoplasma gondii: Characterization of monoclonal antibodies that recognize rhoptries. Experimental Parasitology 68, 74-82. We have previously reported on a series of monoclonal antibodies that recognize the rhoptries of Toxoplasma gondii and that interfere with the action of penetration enhancing factor. The antibodies immunoprecipitate several related antigens from [35S]methionine-labeled parasites that range in size from 60 to 43 kDa. By immunoblot, one of the antibodies reacts with the 60 kDa protein in the presence of protease inhibitors. Trypsin digestion of the antigen destroyed antigenic reactivity indicating that the 60 kDa antigen is a protein. The antigen was stable to periodate oxidation and failed to react with Schifl’s reagent, indicating that the antigen contains little or no carbohydrate. Two-dimensional gel electrophoresis followed by immunoblot showed that the antigen recognized by Tg 49 was an acidic protein with an approximate pZ of 5.8. o 1989 Academic RCSS, IIIC. INDEX DESCRIPTORS AND ABBREVIATIONS: Toxoplasma gondii; Coccidia; Cells, cultured; Antibodies, monoclonal; Antibody specificity; Polyacrylamide gel electrophoresis; Immunoenzyme technics; Immunoprecipitation; Immunoblot; Trypsin (EC; Penetration enhancing factor (PEF); Phenylmethylsulfonyl fluoride (PMSF); Ethylenediaminetetraacetic acid (EDTA); Sodium dodecyl sulfate (SDS).

asites per cell without affecting uptake of unrelated particles (Norrby 1971). We have previously reported the production of a series of monoclonal antibodies that identify rhoptries immunocytologicaUy and that interfere with PEF activity in a plaquereduction assay using fibroblast monolayers (Schwartzman 1986). We present here immunoelectronmicroscopical confirmation that one of these antibodies recognizes rhoptries, and we further characterize the relationship of these antigens to previously described T. gondii antigens.


The parasite Toxoplasma gondii has the ability, unique among the coccidia, to invade a wide variety of host species and host cell types. The anterior pole of the asexual stages of this parasite has a characteristic apical complex which resembles that of other members of the phylum apicomplexa. The specialized organelles of this complex are thought to function in host cell invasion (reviewed by Werk 1985). In particular, the rhoptries appear to discharge their contents in the process of entering new host cells (Nichols et al. 1983; Porchet-Hennere and Nicolas 1983). It has been suggested that rhoptries secrete a substance referred to as PEF, that facilitates entry of T. gondii into host cells (Norrby and Lycke 1967; Lycke et al. 1975). PEF increases the number of infected cells and the number of par-


1 Present address: University of Colorado Health Science Center, Campus Box 168,42OO E. Ninth Ave., Denver CO 80262. 74 0014-4894189 $3.00 Copyright 0 1989 by Academx Press, Inc. All rights of reproduction in any farm reserved.


The RH strain of T. gondii was grown in human fibroblast monolayers as previously described (Pfefferkorn and Pfefferkom 1976). For large scale culture, 150-cm2 plastic culture flasks (Coming Glass Works, Coming, NY, U.S.A.) or 6OOcm’ NUNC cell factory dishes (Vanguard Intemational, Neptune, NJ, U.S.A.) were used. Infected cell cultures were allowed to lyse and the extracellular parasites were separated from cell debris by passage through approximately 1.5 cm3 of CF-11 cellulose (Whatman Ltd., Kent, England, UK) (Tanabe et al. Parasite





1977). equilibrated in Hanks balanced salt solution (GIBCO Laboratories, Grand Island, NY, U.S.A.) with 3% (voVvo1) fetal bovine serum (Sterile Systems Inc., Logan, UT, U.S.A.). In some experiments, PMSF, leupeptin, or pepstatin (Sigma Chemical Co., St. Louis, MO, U.S.A.) were added to all solutions at the time of harvest to a final concentration of 2-20 m&f to inhibit protease activity. Immunoelectronmicroscopy. For immunoelectronmicroscopy, T. gondii growing in HF monolayers were fured in 1% glutaraldehyde, 1% formaldehyde in 0.1 M phosphate buffer, pH 7.2, for 1.5 hr at room temperature, and then rinsed in the same buffer. The monolayer was scraped from the plastic flask, collected by low-speed centrifugation, and dehydrated through graded steps to 70% methanol at room temperature. The pelleted specimen was stained in 2% uranyl acetate for 15 min at room temperature and the dehydration was continued at -20 C to 90% methanol. The specimen was infiltrated in LR gold resin (London Resin Co. Ltd., Surrey, England, UK) and the blocks were polymerized for 24 hr at - 20 C and 24 hr at room temperature under ultraviolet irradiation using a model XX-15 Blackray lamp (UVP Inc., San Gabriel, CA, U.S.A.). Sections (60-80 nm thick) were cut and collected on nickel grids. Immunostaining was carried out using Tg 49 antibody at a concentration of 5 p,g protein/ml of phosphate-buffered saline, pH 7.8, for 3 hr at 37 C. After extensive washing in phosphatebuffered saline, the grids were exposed to 3 t&ml biotinylated horse anti-mouse IgG specific for heavy and light chains (Vector Laboratories, Burlingame, CA, U.S.A.) for 1 hr at 37 C. After washing, the grids were exposed to 50 &ml of ferritin-conjugated avidin D (Vector) in phosphate-buffered saline, washed in phosphate-buffered saline and then in distilled water, and dried. Grids were examined in a Hitachi 12-A electron microscope. Negative controls consisted of substitution of the first or second antibodies with 1% normal horse serum in phosphate-buffered saline. Biosynthetic labeling and immunoprecipitation. Biosynthetically labeled parasites for immunoprecipitation were produced in heavily infected fibroblast monolayers that were rinsed 6 hr after infection with Eagles MEM lacking methionine (Selectamine, GIBCO) which was supplemented with 3% dialyzed fetal bovine serum (Sterile Systems). After a 30-min incubation at 37 C in methionine-free medium, 133 &i/ml of sterile aqueous [35Slmethionine (Amersham, Arlington Heights, IL, U.S.A.) was added and the cultures were incubated for 8 hr or until lysis occurred. Free parasites were separated using CF-11 cellulose, washed thrice by centritiigation in phosphate-buffered saline, pH 7.2, counted in a hemocytometer, and frozen at -20 C until used. Labeled T. gondii suspended in phosphate-buffered saline (approximately 10’ organisms per microliter)



were disrupted in a water-cooled horn sonicator (Ulrasonic Systems) for 7.5 min at maximum setting. The resulting mixture was made 0.05% in Nonidet-P40 (Sigma) and centrifuged for 15 min at 13,OOOg. The supematant was reacted with 0.1 mg of monoclonal antibody complexed to protein A Sepharose beads (Sigma) and incubated at 23 C for 3 hr with shaking. The beads were washed by centrifugation and resuspension: thrice in 0.05% NP-40, once in 1.0 M NaCl, and twice in distilled water. The pellet was boiled 4 min in electrophoresis buffer (final concentrations: 0.05 M Tris, pH 6.8, 2% SDS, 178 mM 2-mercaptoethanol, 5% glycerol, and 0.001% bromphenyl blue) and run on a 10% polyacrylamide gel in the discontinuous buffer system of Laemmli (1970). The gel was processed in Enhance scintillant (New England Nuclear, Arlington, MA, U.S.A.) according to manufacturer’s instructions, dried onto filter paper, and exposed to Kodak X-Omat AR film at -70 C for l-2 weeks and developed. Immunoblotting. Polyacrylamide gels run with prestained molecular weight standards (Bethesda Research Laboratories, Gaithersburg, MD, U.S.A.) were sandwiched with nitrocellulose paper (pore size 0.1 pm, Schleicher and Schuell, Keene, NH, U.S.A.) and antigens were electrophoretically transferred in an electroblot apparatus (Biorad, Richmond, CA, U.S.A.) at 130 mA overnight under conditions described by Towbin et al. (1979) and modified by King et al. (1985). Strips of the nitrocellulose paper were cut and stained with 0.1% napthol blue black (Sigma) in 50% methanol, 10% acetic acid in water, for determination of the pattern of protein transfer, or were blocked with 5.0% nonfat dry milk (Johnson et al. 1984) before incubation with primary antibody that had been diluted 1:500 in phosphate-buffered saline containing 1 mg/ml bovine serum albumin. After washing, localization of monoclonal antibody was detected by sheep anti-mouse IgG conjugated to horseradish peroxidase (specific for heavy and light chains; Sigma). Positive bands were developed in 50 mM Tris, pH 7.2, with 0.25 mg 3,3’-diaminobenzidinelml (Sigma) and 0.05% H,O,, dried in the dark, and photographed. To test antigen stability to protease digestion nitrocellulose strips with transferred T. gondii, antigens were treated with trypsin (0.25% in Hanks balanced salt solution with 0.02% EDTA, and without calcium and magnesium; GIBCO) or 0.1% pronase E type XXV from Streptomyces griseus (Sigma) in Hanks balanced salt solution for 35 min at 37 C. The enzymes were inhibited by soaking in 20% fetal bovine serum and then were processed identically to immunoblots, with matched controls treated identically save for omission of the enzyme. A similar approach was used to determine the sensitivity of T. gondii antigens to mild periodate oxidation (Woodward et al. 198.5).



Strips were rinsed in 50 mM sodium acetate buffer, pH 4.5, incubated 1 hr at 25 C in 10 m&Z sodium periodate in the same buffer, rinsed, and soaked in 50 mM sodium borohydride in phosphate-buffered saline for 30 min. These strips, along with controls in which only the periodate step had been omitted, were processed as above. Zsoelectric focusing. For two-dimensional immunoblots, isoelectric focusing was conducted according to the procedure of O’Farrell(l975). Tube gels were cast with 4.2% deionized acrylamide containing 4% NP-40 detergent, 9.5 M urea, and a one-twentieth dilution of pH 4-8 ampholytes (Isolab, Akron, OH, U.S.A.). Antigen in 8 M urea was loaded and prefocused for 80 min at 200-400 V, then isoelectric focusing to equilibrium was carried out for 15.5 hr at 800-900 V. Ampholytes were allowed to diffuse out of the gels for 2 hr, and the

gel was layered over a 10% SDS-PAGE and electrophoresed at 20 mA until the tracking dye reached the end of the gel. The slab gel was then processed as an immunoblot. Two companion tube gels were cut in 5-mm sections and the pH was determined by electrode for graphical interpolation of pZ. RESULTS

Specific localization of avidin-ferritin occurred over the bodies of rhoptries in sections of T. go&i treated with antibody Tg 49 (Figs. lA, B). Control preparations in which normal horse serum was substituted for specific antibody showed little nonspecific adherence of ferritin (Fig. IC). The

FIG. 1. Electron micrographs of T. gondii treated with Tg 49 or control serum, anti-mouse IgG biotin, and avidin-fenitin. (A, B) Tg 49. (C) Normal horse serum, r, rhoptries; er, endoplasmic reticulum. Arrowhead: extracellular antigen. Bars = 0.1 pm.

T. gondiiRHOF'TRY

most anterior portions of the rhoptries tended not to show ferritin (Fig. lB), and other electron-dense bodies resembling micronemes were also not labeled. Ferritin was also seen over endoplasmic reticulum and in the extracellular space, where it overlay patches of amorphous electrondense material representing excreted antigen (Fig. 1A). The pattern of antigens recognized by three monoclonal antibodies is shown in Fig. 2. Tg 13 precipitated three major [35S]methionine-labeled antigens of M, 60,000, 55,000 and 43,000. Tg 31 precipitated two antigens at M, 51,000 and 43,000, while Tg 49 precipitated proteins at M, 51,000 and 60,000. Each antibody recognized one antigen in common, but the three ABC





FIG. 2. Autofluorograph of SDS-polyacrylamide gel electrophoresis of immunoprecipitates of various monoclonal antibodies. (A) Tg 13, (B) Tg 31, (C) Tg 49, (D). Control (anti-Leishmaniu antibody) MW standards x~O-~.



did not give identical patterns. Tg 13recognized a unique antigen in this analysis: the band at M, 55,000 was not shared with any of the other antibodies. The immunoprecipitates performed with protease protection showed the same number of shared bands among the three antibodies as seen in the absence of PMSF (data not shown). The control for nonspecific absorption (an antiLeishmania donovani monoclonal antibody, prepared in our laboratory, that recognizes a surface epitope of culture promastigotes) was used at equal antibody concentrations and precipitated no labeled antigens. Only one of the three antibodies recognized antigens under fully denaturing conditions of immunoblotting. Tg 49 (Fig. 3) detected a protein of 60 kDa in the presence of inhibitors. When protease inhibitors were not added to infected cultures at the start of the process of purification, the 60 kDa protein appeared to be degraded to a number of smaller proteins (data not shown). Pepstatin, leupeptin, or PMSF all inhibited this proteolysis (Fig. 3). We also used immunoblots to test the stability of the antigen recognized by Tg 49 to enzymatic digestion and mild periodate oxidation (Fig. 4). Trypsin destroyed the epitope recognized by Tg 49; pronase E had a similar effect (data not shown). The antigen was stable to periodate oxidation (Fig. 4, lanes E-H), and did not change in mobility when oxidized prior to being electrophoresed. In addition, no bands were visualized when gels of T. gondii were stained with SchitYs reagent. Two-dimensional electrophoresis of solubilized T. gondii antigens immunoblotted with Tg 49 showed a single spot with M, 60,000 and an approximate isoelectric point of 5.8 (Fig. 5). DISCUSSION

Monoclonal antibodies reactive with T. gondii have been produced in a number of laboratories with specificities to a variety of






FIG. 3. Immunoblot of T. gondii antigens with Tg 49 antibody (A, D, I) MW standards, from the top down, corresponding to MW of 200, 91, 68, 43, 25, and 18 x 10w3. (B, C) Control (no protease protection). (E) 2 m&f PMSF. (F) 20 m&f Leupeptin. (G) 20 mM Pepstatin. (F) Combined PMSF, leupeptin, and pepstatin.

different antigens (Handman and Remington 1980; Johnson et al. 1981; Kasper et al. 1983, 1984; Kimata and Tanabe 1987; Naot and Remington 1981; Ogata et al. 1984; Sadak et al. 1988; Sethi et al. 1980; Sethi and Brandis 1981). Most of these have recognized membrane epitopes (Johnson et al. 1983; Johnson 1985; Kasper et al. 1983, 1984); some have recognized cytoplasmic or intraparasite epitopes (Johnson et al. 1981, 1983; Kimata and Tanabe 1987; Ogata et al. 1984; Sadak et al. 1988; Schwartzman 1986). Our antibodies were originally characterized functionally and morphologically (Schwartzman 1986), the immunochemical data presented here allows us to compare them to others reported in the literature.


Our antibodies recognize a family of antigens of M, 60,000-43,000 by immunoprecipitation and M, 60,000 by immunoblot. These antigens appear to be related to one another as shown by the similarity of their immunoprecipitation pattern and by our previous demonstration of partial competition of the antibodies in solid-phase immunoassay (Schwartzman 1986). However, the pattern of the immunoblot and the immunoprecipitates are not identical; this may be explained by differences in preparation of the antigens. For immunoprecipitations antigens were extracted in low concentrations of detergent at physiological pH, while for immunoblots the antibody was exposed to higher concentrations of antigen which had been denatured and solubilized in SDS. There is evidence (manuscript in preparation) that only a small fraction of the antigen recognized by Tg 49 is soluble in physiological buffers, as most is in an insoluble form associated with tachyzoites. We hypothesize, therefore, that processing of antigen observed in immunoprecipitates may be important for secretion and biologic activity. The immunoprecipitates show that the three antibodies recognize molecules with related epitopes, although each antibody seems to recognize a unique pattern of fragments with cross-reaction between antibodies on individual fragments. Although PMSF was capable of preventing proteolysis of the antigen recognized by Tg 49 in immunoblots, a similar protocol did not change the pattern of the immunoprecipitates. The radiolabeled antigen may have been cleaved prior to the addition of the protease inhibitor, or other proteases not inhibited by PMSF may be responsible for the fragments seen in the immunoprecipitates. These fragments may represent minor components of PEF in comparison to those demonstrated on immunoblots. That the antigen recognized by Tg 49 is a protein is evidenced by its sensitivity to trypsin. We presume that the related anti-





79 FG



FIG. 4. Immunoblot of T. go&ii antigens with Tg 49 antibody (A) Amido black stain of transferred antigens. (IS) MW standards, from the top down, corresponding to MW of ZOO,97, 68,43,25, and 18 x 10p3. (C) Control (no trypsin). (D) Trypsin treatment. (E) Amido black. (F) Control (no periodate). (G) MW standards as in lane B. (H) Periodate treatment.

gens recognized by Tg 13 and 31 are also protein. The epitope recognized by Tg 49 is resistant to periodate oxidation, the antigen does not change its mobility after oxidation, and none of the antigens are detected by Schiff’s reagent; this indicates that the antigen is probably not composed of carbohydrate. Ogata et al. (1984) have described antigens from the insoluble fraction of T. gondii which show a similar pattern on immunoblots and similar immunolocalization to our antibodies. The most prominent proteins in their preparations seemed to be at M, 43,000, but the pattern was otherwise very similar. Their monoclonal antibodies were also partially cross-reactive, which resem-

bles those described here. The functional characterization of antigens was not reported by Ogata et al. Kimata and Tanabe (1987) describe an antigen which is located in the anterior organelles, which appears to be secreted upon host cell entry and which is found associated with the parasitophorous vacuole (but not the tubulo-vesicular network) of infected cells. The antigen was localized prominently near the anterior tips of the rhoptries or micronemes, and was less concentrated in the bodies of rhoptries. Their antibody recognized a single M, 63,000 band on reduced and nonreduced immunoblots. This antigen may be related to those described here, but the higher molecular









FIG . 5. Immunoblot of two-dimensional electrophoretogram of 7’. gondii antige Ins, devr :Ioped u rith Tg 49 antibody. Arrow indicates single positive reaction. MW standards x 10m3.

mass and the differences in distribution within the anterior organelles leave the relationship somewhat in question. Sadak et al. (1988) describe a series of monoclonal antibodies which recognize rhoptry antigens which very much resemble those described here, both in their morphological and immunoblot findings. The antigen recognized by their antibody 4A7 was shown in pulse-labeling experiments to appear first as M, 66,000 and 68,000 moieties, and later to be processed to M, 60,000 and 55,000 bands. The immunoelectronmicroscopy showed a pattern very similar to that seen with Tg 49. It appears that there may be a family of immunogenic molecules in the anterior organelles, some of which may share antigenie similarity. The functional roles of these molecules remain to be sorted out. A “circulating antigen” of T. gondii has been described (Hughes 1981; Hughes and Van Knapen 1982; Ise et al. 1985). This antigen has been characterized as having a

higher molecular weight than the antigens characterized here, and we have no other indication of possible relationship. The results of two-dimensional electrophoresis and immunoblotting indicate that the rhoptry antigen recognized by Tg 49 is an acidic protein, which is in contrast to findings that the contents of rhoptries are basic by histochemical criteria (de Souza, and Souto-Padron 1978), and that basic polypeptides have penetration-enhancing activity (Werk et al. 1984). Our results do not preclude the existence of such basic proteins, but our antibodies interfere with the biological activity of a penetrationenhancing factor which is acidic, which is in agreement with an earlier characterization by Norrby (1971). It is possible that these rhoptry antigens represent PEF, or that biologically active PEF may be released as a proteolytic fragment of the antigen; we have not yet been able to demonstrate the biological activity of various antigen fractions prepared with

T. gondii



gondii. Australian Journal for Experimental Biology the aid of our antibodies. We have evidence and Medical Sciences 59, 303-306. from previous immunolocalization that JOHNSON, D. A., GAUTSCH, J. W., SPORTSMAN, J. R., rhoptry antigens are not expressed on the AND ELDER, J. H. 1984.Improvedtechniqueutihzparasite surface (Schwartzman 1986), and ing nonfatdry milkfor analysisof proteinsandnuare therefore unlikely to be parasite memcleicacidstransferredto nitrocellulose. Gene Analysis Techniques 1, 3-8. brane components. Immunolocalization reL. H., BRADLEY, M. S.,ANDPFEFFERKORN, ported here suggests that the rhoptry anti- KASPER, E. R. 1984. Identification of stage-specific sporozogen is secreted, which would be consistent ite antigensof Toxoplasma gondii by monoclonal with PEF. We believe that the antigens deantibodies.Journal of Immunology 132,443-449. scribed here correspond to PEF, but the KASPER, L. H., CRABB,J. H., AND PFEFFERKORN, biologic activity of purilied antigen has not E. R. 1983.Puriticationof a majormembrane protein of toxoplasmagondii by immunoabsorption yet been shown.

ACKNOWLEDGMENT This work wassupportedby a grantfrom the U.S. PublicHealthService,NIAID (AI 23074). REFERENCES DE SOUZA, W., ANDSOUTO-PADRON, T. 1978.Ultrastructural localization of basic proteins on the conoid, rhoptriesand micronemes of Toxoplasma gondii. Zeitschrif fur Parasitenkunde 56, 123-129. HANDMAN, E., ANDREMINGTON, J. S. 1980.Serological andimmunochemical characterization of monoclonalantibodiesto Toxoplasma gondii. Immunology 40, 579-588. HUGHES, H. P. A. 1981.Characterizationof the circulatingantigenof Toxoplasma gondii. Immunology Letters 3, 99-102.

HUGHES, H. P. A., ANDVAN KNAPEN, F. 1982.Characterizationof a secretoryantigenfromToxoplasma gondii andits rolein circulatingantigenproduction. International Journal of Parasitology 12,433-437. ISE,Y., IIDA, T., SATO,K., SUZUKI,T., SHIMADA, K., AND NISHIOKA, K. 1985.Detectionof circulating antigensin seraof rabbitsinfectedwith Toxoplasma gondii. Infection and Immunity 48,269-272.

with a monoclonalantibody. Journal of Immunology 130,2407-2412. KIMATA, I., ANDTANABE,K. 1987.Secretionby Toxoplasma gondii of an antigenthat appearsto becomeassociated with the parasitophorous vacuolemembrane uponinvasionof the hostcell. Journal of Cell Science 88, 231-239.

KING,S. M., OTTER,T., ANDWITMAN,G. B. 1985. Characterizationof monoclonalantibodiesagainst Chlamydomonas flagellar dyneins by high-resolution protein blotting. Proceedings of the National Academy of Sciences USA 82, 4717-4721. LAEMMLI,U. K. 1970.Cleavage of structuralproteins duringthe assemblyof the headof bacteriophage T4. Nature (London) 227, 680-685. LYCKE,E., CARLBERG, K., ANDNonany, R. 1975. InteractionsbetweenToxoplasma gondii and its host cells: Functionof the penetration-enhancing factor of toxoplasma.Infection and Immunity 11, 853-861. NAOT,Y., AND REMINGTON, J. S. 1981.Useof enzyme-likedimmunosorbent assays(ELISA) for detectionof monoclonalantibodies:Experiencewith antigens of Toxoplasma gondii. Journal of Immunological Methods 43, 333-341. NICHOLS, B. A., CHIAPPINO, M. L., AND O’CONNOR,. G. R. 1983.Secretionfrom the rhoptriesof Toxoplasma gondii duringhost-cellinvasion.Journal of

A. M. 1985.Theantigenicstructureof toxoplasmagondii:A review. Pathology (Australia) 17, Ultrastructure Research 83, 85-98. 9-19. NORRBY, R. 1971.Immunological studyof the hostcell JOHNSON, D. A., GAUTSCH, J. W., SPORTSMAN, J. R., penetration factor of Toxoplasma gondii. Infection ANDELDER,J. H. 1984.Improvedtechniqueutilizand Immunity 3, 278-286. ing nonfatdry milk for analysisof proteinsandnuNORRBY, R., AND LYCKE, E. 1%7.Factorsenhancing cleicacidstransferredto nitrocellulose. Gene Analthe host-cellpenetrationof Toxoplasma gondii. ysis Technique 1, 3-8. Journal of Bacteriology 93, 53-58. JOHNSON, A. M., HAYNES,W. D., LEPPARD, P. J., MCDONALD, P. J., ANDNEOH,S. H. 1983.Ultra- O’FARREL,P. H. 1975.High resolutiontwo dimenof proteins.Journal of Biolstructuralandbiochemicalstudieson the immuno- sionalelectrophoresis histochemistry of Toxoplasma gondii antigensusing ogy and Chemistry 250,4007Xl21. monoclonal antibodies. Histochemistry 77,209-215. OGATA,K., KASAHARA, T., SHIOIRI-NAKANO, K., IGJOHNSON, A. M., MCNAMARA,P. J., NEOH,S. H., ARA~HI,I., ANDSuzurcr, M. 1984.ImmunoenzyMCDONALD,P. J., ANDZOLA, H. 1981.Hybridmatic detectionof three kinds of 43,000-molecomassecretingmonoclonal antibodyto Toxoplasma ular-weightantigensby monoclonalantibodiesin JOHNSON,



the insoluble fraction of Toxoplasma gondii. Infection and Immunity 43, 1047-1053. PFEFFERKORN, E. R., AND PFEFFERKORN, L. C. 1976. Toxoplasma gondii: Isolation and preliminary characterization of temperature-sensitive mutants. Experimental


39, 365-376.

PORCHET-HENNERE, E., AND NICOLAS, G. 1983. Are rhoptries of coccidia really extrusomes? Journal of Ultrastructure


84, 194-203.

SADAK, A., TAGHY, Z., FORTIER, B., AND DuBREMETZ, J. 1988. Characterization of a family of rhoptry proteins of Toxoplasma gondii. Molecular and Biochemical Parasitology 29, 203-2 11. SCHWARTZMAN, J. D. 1986. Inhibition of a penetration-enhancing factor of Toxoplasma gondii by monoclonal antibodies specific for rhoptries. Znfection and Immunity

51, 76&764.

SETHI, K. K., AND BRANDIS, H. 1981. Generation of hybridoma cell lines producing monoclonal antibodies against Toxoplasma gondii or rabies virus following fusion of in vitro-immunized spleen cells with myeloma cells. Annales d’lmmunologie (Paris) 132, 2!&41. SETHI, K. K., ENDO, T., AND BRANDIS, H. 1980. Hybridomas secreting monoclonal antibody with spec-



iticity for Toxoplasma gondii. Journal of Parasitol66, 192-l%. TANABE, K., KIMATA, I., ISEKI, M., AND TAKADA, S. 1977. Separation of Toxoplasma tachyzoites by filtrating peritoneal exudate of infected mice through cellulose powder. Japanese Journal of Parasitology 26, 113-115. TOWBIN, H., STAEHELIN, T., AND GORDON, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National ogy


of Sciences


76, 435W354.

WERK, R. 1985. How does Toxoplasma gondii enter host cells? Reviews of Infectious Diseases 7, 449457. WERK, R., DUNKER, R., AND FISCHER, S. 1984. Polycationic polypeptides: A possible model for the penetration-enhancing factor in the invasion of host cells by Toxoplasma gondii. Journal of General Microbiology 130, 927-933. WOODWARD, M. P., YOUNG, W. M., JR. AND BLOODGOOD, R. A. 1985. Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. Journal of Immunological Methods 78, 143-153. Received 13 June 1988; accepted 26 September 1988