Proc. Nati. Acad. Sci. USAVol. 82, pp. 1771-1775, March 1985Immunology

Identification of a surface antigen on Theileria parva sporozoites bymonoclonal antibody

(East Coast fever/immunoelectron microscopy/immunoprecipitation/sporozoite surface antigen)

DIRK A. E. DOBBELAERE, STUART Z. SHAPIRO, AND PAUL WEBSTERInternational Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya

Communicated by Don W. Fawcett, November 5, 1984

ABSTRACT A mouse monoclonal antibody (mAbD1) thatneutralizes sporozoites of different stocks of the protozoanparasite Theikriaparva has been used to localize and identify asporozoite antigen. Protein A-colloidal gold was used to local-ize bound mAbD1 in immunoelectron microscopic studies.mAbD1 bound to sporozoite antigen, which was evenly spreadover the surface of all sporozoites. Immune complexes wereobtained by incubation of sporozoite suspensions with mAbD1followed by Zwittergent 3-14 extraction and precipitation withprotein A-Sepharose. One- and two-dimensional NaDodSO4/polyacrylamide gel electrophoretic analyses were performedon these complexes, and a major protein antigen with a molec-ular size of 68 kDa was identified. Other related componentsof 52 kDa, 47 kDa, and 28 kDa were also detected. Since anti-body to this antigen(s) neutralizes T. parva sporozoites fromdifferent stocks, the results could be of relevance to the devel-opment of a broad spectrum vaccine against the cattle diseaseEast Coast fever, which is caused by T. parva.

East Coast fever, a disease of cattle, is endemic in large ar-eas of East and Central Africa. The disease constitutes a ma-jor constraint to livestock development and is thus of greateconomic importance in the affected developing countries.

East Coast fever is caused by the protozoan parasite Thei-leria parva. This protozoan, which belongs to the sameclass, Sporozoea, as the malaria parasites, is transmitted bythe ixodid tick Rhipicephalus appendiculatus. When a T.parva-infected tick feeds on a susceptible host, sporozoitesmature in the tick salivary glands (1, 2) and are subsequentlyinjected with the saliva into the host, where they invade lym-phocytes. The sporozoites then undergo morphologicalchanges and nuclear division in the lymphocyte cytoplasmand transform into schizonts, which constitute the pathogen-ic stage of the parasite in cattle.The interactions of the sporozoite with the target lympho-

cyte have recently been described (3). Entry of sporozoitesinto the lymphocyte occurs by a phenomenon of passive en-docytosis, an energy-independent process that seems to in-volve a ligand-receptor interaction (3). A monoclonal anti-body (mAbD1) has been produced that blocks this entryprocess, thus neutralizing the infectivity of sporozoites fromseveral different T. parva stocks for lymphocytes in vitroand for cattle in vivo (4). This activity suggests that mAbD1detects a common sporozoite surface molecule that could beclosely associated with the entry process. The observationthat antibodies induced against sporozoites have neutralizingactivity (4-6) suggests that it might be possible to induce im-munity with antigens derived from this stage of the parasite.

In the present report, we demonstrate by immunoelectron-microscopy that the antigen recognized by mAbDl is indeedlocated on the surface of the sporozoite. We also present

some biochemical characterization of the sporozoite mole-cule(s) immunoprecipitated by mAbDi.

MATERIALS AND METHODSTicks. Nymphal R. appendiculatus ticks (reared in the IL-

RAD Tick Unit) were infected with T. parva by placing themin bags on the ears of cattle (7) infected with East Coast fe-ver. The cattle, which had been infected by inoculation withcryo-preserved sporozoite suspensions (8), were showing T.parva piroplasms in the blood at the time of tick application.The rate of infection with T. parva in the resultant adult tickswas determined (9), and appropriate batches of heavily in-fected ticks were selected for experimental use. Uninfectedadult ticks were raised under similar conditions but did notfeed on infected cattle. To induce development of maturesporozoites, adult ticks fed on rabbits (New Zealand White)for 4 days before processing.

Monoclonal Antibody mAbD1. The derivation of the mono-clonal antibody mAbD1 (IgG3) has been described else-where (4). Briefly, mice were immunized with crude freeze-thawed T. parva sporozoite preparations and their spleencells fused with X63-Ag8.653 myeloma cells (10). Antibody-producing cells were cloned by limiting dilution (11) and cul-ture supernatants were screened for anti-sporozoite activityby immunoautoradiography on Feulgen-stained cryostat sec-tions of infected salivary glands (4). For use in immuno-precipitation, mAbD1 IgG3 was first purified from ascitesfluid by adsorption on protein A-Sepharose (Pharmacia)(12).Immunoelectron Microscopy. Sporozoites were obtained

from salivary glands of infected ticks fed on rabbits for 4days (4) and incubated at 220C for 90 min with 107 bovineperipheral blood lymphocytes (PBL) (4), separated on Fi-coll-Hypaque (13). The sporozoite lymphocyte preparationswere then fixed in suspension for 30 min with formaldehyde[freshly prepared from paraformaldehyde, final concentra-tion 4% (wt/vol); Electron Microscopy Sciences] in Dulbec-co's phosphate-buffered saline (P1/NaCl) (pH 7.4). After fix-ation, the cells were washed by pelleting and resuspending inthree changes of P1/NaCl and one change of Pi/NaCl con-taining 50 mM NH4Cl. The cells were then resuspended inPi/NaCl containing 0.2% gelatin (PINaCl/gelatin), left for 10min and then pelleted. They were thereafter resuspended inmAbD1 (ascites fluid, diluted 1:300 in P1/NaCl/gelatin) andincubated for 30 min at 22°C. After incubation, cells werewashed 3 times in Pi/NaCl and once in Pi/NaCl/gelatin andresuspended in protein A labeled with colloidal gold (5-nmparticle size), in PINaCl/gelatin, and further incubated at22°C for 15 min. Cells were then pelleted (10,000 x g for 5min), washed 3 times in Pi/NaCl followed by a final wash in100 mM sodium cacodylate buffer (Sigma) (pH 7.3) contain-

Abbreviations: mAb, monoclonal antibody; PBL, peripheral bloodlymphocytes.

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ing 2% (vol/vol) glutaraldehyde (Electron Microscopy Sci-ences) and 2% (wt/vol) sucrose. The pellet was then proc-essed for electron microscopy, with a post-fixation in 1%(wt/vol) osmium tetroxide, en bloc staining in 1% (wt/vol)uranylacetate in 50 mM maleate buffer, dehydration in etha-nol, and embedding in an Epon/Araldite mixture. Sections,cut on a Sorvall MT.2B ultramicrotome, were mounted onuncoated copper grids, stained with uranylacetate and leadcitrate (14), and examined with a Zeiss EM.10A electron mi-croscope.

Immunoprecipitation and Autoradiography. One hundredT. parva-infected and 100 uninfected ticks fed on rabbits for4 days were dissected, and the salivary glands were removedaseptically and collected in ice-cold P1/NaCl. Glands werecentrifuged (50 x g for 5 min) in a 1-ml glass tissue grinderand the supernatant fluid was replaced by 0.5 ml of ice-coldPi/NaCl containing the protease inhibitors iodoacetamide (5mM) and phenylmethylsulfonyl fluoride (0.5 mM) (subse-quently referred to as Pi/NaCl/PI). To each preparation, 25/4 of purified mAbD1 (1 mg/ml) was added, after whichglands were homogenized and kept on ice for 1 hr. Prepara-tions were washed twice by resuspension in 12 ml of Pi/NaCl/PI and centrifugation at 2500 x g for 25 min. After thesecond wash, 0.5 ml of an extraction buffer (20 mMTris HCl, pH 7.4/1 mM EDTA/100 mM NaCl/5 mM iodoa-cetamide/0.5 mM phenylmethylsulfonyl fluoride) was addedand the pellet was freeze-thawed 3 times (-196 and +37°C).Zwittergent 3-14 (Calbiochem-Behring) was added to a finalconcentration of0.5% (wt/vol). Preparations were incubatedat room temperature for 1 hr. Pj/NaCl/PI (0.5 ml) was thenadded and preparations were centrifuged at 10,000 x g for 15min. The pellet was discarded and the supernatant extractwas added to a 0.2-ml pellet of protein A-Sepharose, mixedend over end for 1 hr at room temperature and then washed 3times with PsINaCl/PI and twice with P1/NaCl. The immunecomplexes adsorbed to protein A-Sepharose were radioiodi-nated with 125I-labeled Bolton and Hunter reagent (Amer-sham) (15) and washed 15 times with Pi/NaCl (iodinationwas carried out at this point in the procedure as attempts tosurface-label or metabolically label the parasite antigen inpreliminary experiments had failed). After the final wash, 80,u of triple strength NaDodSO4/polyacrylamide gel samplebuffer [186 mM Tris HCl, pH 6.8/6% (wt/vol) NaDod-S04/22.5% (vol/vol) glycerol/15% (vol/vol) 2-mercap-toethanol] was added and the preparations were heated for 5min in boiling water. Electrophoresis of the samples was car-ried out on a 7.5%-17.5% NaDodSO4/polyacrylamide gradi-ent gel (16), with 3% stacking gel. Autoradiography of thedried gels was performed by exposing the gel to Kodak X-Omat AR film for 20 hr.

Immunoprecipitation and Two-Dimensional Gel Electro-phoresis. One hundred and eighty T. parva-infected and 90uninfected ticks were dissected and homogenized in 1 ml and0.5 ml of PsINaCI/PI, respectively. The infected gland ho-mogenate was divided into two aliquots of 0.5 ml. To onealiquot, 10 ,l4 of purified mAbD1 was added, to the otheraliquot 10 ,lI of a control monoclonal IgG3 (anti-AMP, 1mg/ml; Bionetics) was added. To the uninfected gland ho-mogenate, 5 ,lI of mAbDi and 5 Al of control IgG3 wereadded. The three preparations were incubated for 1 hr onice, freeze-thawed 3 times, and gently sonicated in a bathsonicator (Bransonic 220). Thereafter, 25 ,lI of Zwittergent3-14 was added to each preparation. Preparations were fur-ther incubated at room temperature for 30 min, spun at10,000 x g for 15 min, and each supernatant was added to 0.5ml of 5% (vol/vol) protein A-Sepharose in P,/NaCl/PI.Tubes were mixed end over end for 1 hr at room tempera-ture, and then protein A-Sepharose pellets were washed 8times with PjINaCl/PI and 3 times with Pi/NaCl. After thefinal wash, 25 41 of two-dimensional gel electrophoresis

sample buffer (17), 5 ul of a mixture of 3% (wt/vol) Na-DodSO4 and 10% (wt/vol) 2-mercaptoethanol, and 5 ul ofglycerol were added to each pellet. Isoelectric focusing wasperformed in 1.3-mm diameter glass tubes with pH 3.5-10ampholytes (LKB) according to the method of Anderson andAnderson (18), but with the upper and lower reservoir buff-ers containing 200 mM NaOH and 70 mM H3PO4, respec-tively. After focusing, tube gels were incubated for 20 min atroom temperature in equilibration buffer (17) without dithio-threitol and stored at -200C until use. For the second dimen-sion, tube gels were thawed and laid on top of 7.5%-17.5%NaDodSO4/polyacrylamide gradient slab gels, overlaid with0.8% (wt/vol) agarose containing 2% (wt/vol) NaDodSO4,and electrophoresis was carried out (19). The resulting gelswere stained with silver nitrate (20). Two-dimensional gelelectrophoresis was also performed on a mixture of 5 /kl ofmAbD1 and 5 ul of control IgG3 to locate spots on the silver-stained gels originating from the antibodies.

RESULTSmAbDi Binds to a T. parva Sporozoite Surface Antigen.

Immunoelectron microscopy clearly demonstrated thatmAbD1 detected a sporozoite antigen that was evenlyspread over the entire surface of the T. parva sporozoite(Fig. 1). All sporozoites observed showed labeling of theirsurface; also, the occasional sporoblast (earlier stage of spo-rogony) or residual body (remnant found after completion ofsporogony), encountered in some preparations, showed sur-face labeling (not shown). The protein A-colloidal gold parti-cles were located in the sporozoite surface coat rather thanon the sporozoite membrane. Binding to components otherthan those of sporozoite origin was not observed.

Nature of Sporozoite Surface Coat Antigen(s). In numerousattempts to immunoprecipitate the antigen recognized bymAbD1 from soluble protein extracts of infected glands, nospecifically immunoprecipitated protein was detectable.However, when antibody was incubated with infected glandhomogenates prior to detergent solubilization, it was possi-ble to detect specifically immunoprecipitated proteins. InFig. 2 are shown the results of NaDodSO4/polyacrylamidegel electrophoresis of Bolton and Hunter reagent-labeled im-mune complexes that had adsorbed to protein A-Sepharose.A molecule with an apparent size of 68 kDa was isolatedfrom infected glands (lane C) but not from uninfected glands(lane B). The major bands observed in lanes B and C origi-nated from the antibodies that were also iodinated during ra-diolabeling and were present in excess compared to immuno-precipitated antigen. Minor bands, probably of tick salivarygland origin, were also observed in both lanes.The results of two-dimensional NaDodSO4/polyacryla-

mide gel electrophoretic analysis of immune complexes iso-lated from infected or uninfected salivary glands withmAbD1 or control IgG3 are presented in Fig. 3. The immu-noprecipitation products of infected salivary gland homoge-nate incubated with mAbDl, extracted and isolated on pro-tein A-Sepharose, are shown (Fig. 3A). Four componentswere immunoprecipitated that could not be precipitated fromthe same preparation incubated with control IgG3 (Fig. 3B).These molecules were also not isolated from uninfected ticksalivary gland homogenate, incubated with both mAbD1 andcontrol IgG3 (Fig. 3C). The major immunoprecipitated com-ponent observed had a molecular size of 68 kDa and an iso-electric point (pI) of 7-7.5. Two lesser components of 52 kDa(pI1, -7.8) and 47 kDa (pI, -8) were precipitated as well as aminor component of 28 kDa (pI, -7.3). The 68-kDa and 28-kDa components were consistent features of repeated immu-noprecipitation experiments. The 52- and 47-kDa compo-nents were only observed when the 68-kDa component wasisolated in abundant quantities.

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B

-aNV B AS.'*,ns

A-N

AS

~~~~~~~~~~~~~~~~~~~~~I !E'.tss >, ............,;P-1.^,,,E,.

|I- ; Z Z i t e * # ;,

FIG. 1. (A) Electron micrograph of a surface-labeled T. parva sporozoite and part of a bovine lymphocyte. Protein A-colloidal gold label (5-nm particle size), bound to mAbD1, is uniformly distributed over the sporozoite surface. mAbD1 was unable to penetrate where binding ofparasite to host cell had already occurred. (Bar = 0.1 ,um.) (B) High magnification micrograph of a sporozoite surface membrane, showingmAbD1 (visualized by protein A-colloidal gold) bound to antigens in the sporozoite surface coat. (Bar = 0.1 gam.)

DISCUSSION

Antibodies in antisera from East Coast fever-immune cattle(5, 21) or from rabbits inoculated with T. parva sporozoites(6) have been shown to abolish sporozoite infectivity for bo-vine lymphocytes in vitro. In previous work, it was shownthat mAbD1, a mouse monoclonal antibody against the spo-rozoite stage of T. parva, abolished sporozoite infectivity forPBL in vitro and for cattle in vivo, while blocking sporozoiteentry into the lymphocyte (4). This suggested that mAbDirecognizes a determinant on a sporozoite surface antigen. Inthe present work, we have demonstrated by immunoelec-tronmicroscopy that mAbDi recognizes a sporozoite antigenthat forms part of the sporozoite surface coat. Similar anti-gens have been detected on the surface of malarial sporozo-ites (22). The surface coat of the T. parva sporozoite hasbeen implicated in the recognition phase of sporozoite entryinto the target cell (3). During the initial phase, sporozoitesadhere loosely to the lymphocyte; subsequently, a veryclose contact is established between the membranes of thesporozoite and the lymphocyte at the site of adherence. Thisclose apposition spreads laterally and the membranes "zip-up" progressively around the entire circumference of thesporozoite. It is conceivable that mAbD1 binding to the spo-rozoite surface coat could interfere either with recognition,the close membrane apposition, or the "zipping up" process,thus preventing entry of the sporozoite into the lymphocyte.

Preliminary observations, based on immunofluorescence,suggested that the antigen recognized by mAbD1 is proteinin nature: it is susceptible to proteolytic digestion but not tophospholipases and nine exoglycosidases (data not shown).The labeling of the antigen by Bolton and Hunter reagentsupports this suggestion. By immunoprecipitation and auto-radiography, mAbD1 identified one antigen with a Mr of -68kDa. Immunoprecipitation with mAbDi and two-dimension-al gel electrophoretic analysis confirmed the existence ofthis antigen and also revealed other related components ofsmaller size, also recognized by mAbD1, that were not de-tected on one-dimensional gels.Other investigators have used antiserum from rabbits im-

munized with salivary glands from infected ticks and by im-munoblotting detected three proteins of -105 kDa, =70kDa, and -20 kDa, that were not detected with antisera fromrabbits immunized with uninfected tick salivary glands (D.Grab, personal communication). The 68-kDa protein detect-ed by mAbD1 could correspond to the 70-kDa protein de-tected by the rabbit antiserum.The significance of the smaller components is not clear.

They may be cleavage products of the 68-kDa protein,caused by proteolysis during extraction and immunoprecip-itation. Alternatively, they may be the products of a biologi-cally significant cleavage by an endogenous parasite en-zyme. Such specific proteolytic cleavage has been demon-strated for a Plasmodium falciparum merozoite surface coat

A

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1774 Immunology: Dobbelaere et aL

A B C

kDa

200 -

92.5 -69 --

46 -

30-'.

14.3-

FIG. 2. Autoradiogram of radiolabeled protein A-Sepharose-mAbD1-antigen immune complexes, analyzed by 7.5%-17.5% Na-DodSO4/polyacrylamide gradient gel electrophoresis under reduc-ing conditions. Lane A contains radiolabeled molecular size markers(Amersham). Uninfected (lane B) and T. parva-infected (lane C) ticksalivary gland homogenates were incubated with Zwittergent 3-14,adsorbed to protein A-Sepharose, and radioiodinated with Boltonand Hunter reagent. Arrow indicates a 68-kDa sporozoite surfaceprotein specifically immunoprecipitated from the infected tick sali-vary gland homogenate.

antigen, the cleavage of which may be required to expose ahigh-affinity binding site involved in the entry of the mero-zoite into the erythrocyte (23). T. parva sporozoite surfacecoat antigen(s), recognized by mAbD1, appear closely asso-ciated with recognition and/or entry of the T. parva sporozo-ite into the target lymphocyte. Release of mAbDl-specificsporozoite surface coat material and shedding of sporozoitesurface antigens reacting with mAbD1 at the surface of theinfected cell have been demonstrated in other immunoelec-tron microscopic studies (24) and may reflect a specificcleavage similar to that in P. falciparum.Attempts to immunoprecipitate the antigen with mAbD1

after detergent extraction failed. It was essential to incubatesporozoites from infected salivary gland homogenates withmAbD1 prior to extraction with detergent. The choice of de-tergent was also important: extraction of the antigen-mAbD1 complex from the sporozoite membrane was suc-cessful with Zwittergent 3-14 but not with Nonidet P-40,Zwittergent 3-10, or Zwittergent 3-12 (unpublished data).From other work, Zwittergent 3-14 appears to be superior in

solubilizing membrane proteins (25) but sometimes less suit-able for immunoprecipitation of proteins after extraction(26). Under the present experimental conditions, Zwitter-gent 3-14 would not have had adverse effects on antigen-mAbD1 binding, as this had already occurred by the timedetergent was added, but it still exhibited its powerful mem-brane protein solubilizing capacity. The difficulty encoun-tered in immunoprecipitating this antigen from soluble anti-gen extracts suggests that the determinant recognized bymAbD1 is a conformational determinant, with antibodybinding critically dependent on the conformation assumedby the antigen on the surface of the sporozoite. The proce-dure we have used here, of binding antibody prior to deter-gent extraction, should be useful to other investigators usingmonoclonal antibodies that recognize conformational deter-minants.Endocytosis of the sporozoite into the target cell is a rapid

process: some sporozoites can be found within lymphocytesas early as 10 min after being introduced to PBL in vitro (3).

FIG. 3. Two-dimensional NaDodSO4/polyacrylamide gel elec-trophoretic analysis of protein A-Sepharose-adsorbed immune com-plexes, isolated from T. parva-infected or uninfected tick salivarygland homogenates incubated with mAbD1 (anti-sporozoite) or con-trol IgG3 antibody prior to extraction with Zwittergent 3-14. (A) In-fected glands incubated with mAbD1. (B) Infected glands incubatedwith control IgG3. (C) Uninfected glands incubated with bothmAbD1 and control IgG3 antibodies. The approximate sizes of thecomponents indicated by arrows are as follows: 68 kDa, arrow 1; 52kDa, arrow 2; 47 kDa, arrow 3; and 28 kDa, arrow 4. The basic endof the gels is at the right.

While this observation has led some investigators to suggestthat a humoral response may not be able to protect cattleeffectively against sporozoite invasion (27), it has beenshown that sporozoites introduced to PBL in culture medi-um containing mAbD1 are inhibited from infecting cells (4).Thus, it is possible that such anti-sporozoite surface antibod-ies may also be protective in vivo. Even if such antibodies donot neutralize all of the sporozoites, they could reduce theseverity of the disease, which is dose dependent with respectto the sporozoite inoculum (28).There is evidence that genetically restricted (29) cell-medi-

ated immune responses to the T. parva schizont-infectedlymphocyte, and not a humoral response to the sporozoitestage, are responsible for the acquired protection of cattleobserved by others (30). This protection is often, however,restricted to specific T. parva stocks. Cattle that recoverfrom East Coast fever are resistant to infection with a stockof T. parva homologous to that which induced initial infec-tion, but they may react severely or die if challenged with aheterologous stock (31). There also appears to be good cor-relation between cross-resistance patterns in vivo and para-site antigen differences detected in vitro by monoclonal anti-bodies raised against the schizont stage (32). However,mAbD1 neutralized sporozoites from different theilerialstocks that were not cross-protective (4). mAbDi must,therefore, detect a determinant that is common to the sur-face of T. parva sporozoites from different stocks. We haveidentified the sporozoite surface coat antigen(s) carrying this

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determinant. These results could contribute to the develop-ment of a different approach to vaccination against EastCoast fever that could give more widespread protection thanthe immunity observed against the schizont stage of the par-asite.

We thank Dr. W. Voigt for provision of tick material and Mr. J.Kiarie for technical assistance. We also thank Dr. D. Grab for sup-ply and information on detergents as well as for communication ofresults before publication and Dr. J. Lonsdale-Eccles for helpful dis-cussions and technical help. D.A.E.D. is a staff member of the De-partment of Development Cooperation of the Belgian Government,seconded to the International Laboratory for Research on AnimalDiseases.

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