diagnostic test for schistosomiasis japonica which involves the incubation of a selected batch of S. japonicum eggs with sera from patients and examination for.
AJEBAK 59 (Pt. 4) 503-514 (1981)
©MURINE HYBRIDOMA-DERIVED ANTIBODIES PRODUCING CIRCUMOVAL PRECIPITATION (COP) REACTIONS WITH EGGS OF SCHISTOSOMA JAPONICUM by KATHY M. CRUISE, GRAHAM F. MITCHELL. FE P. TAPALES, EDITO G. GARCIA AND HUANG, SUNG-RU (From the Laboratory of Tmmunoparasitology, The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia 3050. and Department of Parasitology, Institute of Public Health, University of the Philippines Health Sciences Center, Ermita 2801, Manila, Philippines.) (Accepted for publication May 13, 298}.) Summary. Of 7 hybridomas which secrete immii no globulins binding to crude extracts of Schi.itosoma japonicum adull worms and/or eggs in solid-phase radioimmunoassays (RTAs). 3 gave positive precipitation reactions in the circumoval precipitin test (COPT). The COPT is a simple and inexpensive immunodiagnostic test for schistosomiasis japonica which involves the incubation of a selected batch of S. japonicum eggs with sera from patients and examination for precipitates one or more days later. Using a competitive RTA with an egg antigen extract and a labelled COPT-positive hybridoma ascites fluid, PwF.41-1-3, the surprising observation was made that only one anti-egg antibody specificity appeared to be represented in the series of 3 antihodies (as ascites fluids). Using sera as inhibitors in the competitive RTA. inhibitory activity (presumably antibodies to the target antigenic determinant of PwF.41-1-3) was readily detected in sera from egg-immunized mice and was of relatively high tilre in a strain of mouse (C57BL/6) which can be readily sensitized for large graniiloma formation around entrapped eggs in the lungs. Negligible inhibitory activity was found in the sera from 5. japan hum-infected patients, even with sera from patients with prominent hepatosplenomegaly. The availability of COPT-positive hybridoma antibodies should facilitate isolation of at least one S. japonicum egg antigen involved in COP reactions and perhaps induction of immunopathological immune responses at least in mice.
INTRODUCTION The circumoval precipitin (COP) test (COPT) is currently the immunodiagnostic method of choice for schistosomiasis japonica in the Philippines (Garcia, 1976; Garcia, Cabrera, Cristi and Silan, 1968; Yogore, Lewert and Bias, 1978; Tanaka, 1976; Cabrera, Garcia and Silan, 1968). COP Abhreviatimu used in this paper: AWE, adult worm extract: COP, circumoval precipitin; COPT, circumoval precipitin test; EA. egg antigens (extract): ELISA, enzyme-linked immunosorbent assay: FCA, Freund's complete adjuvant; Tg, immunogiobulin: MSA-1. major serologic antigen number 1 of S. mansoni epas: FVC, polyvinylchloride: RIA, radioimmunoassay: T cell, thymus-derived (-influenced) cell.
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reactions were described many years ago and consist of segmented or bleb-like precipitates at localized sites of the schistosoma egg surface. These precipitates appear during incubation of eggs with sera from infected patients (Oliver-Gonzalez, 1954). For both sehistosomiasis japonica and schistosomiasis mansoni, the specificity and sensitivity of this test have been proven. Two studies have indicated that there is little relevant to the immunodiagnosis of sehistosomiasis which is added by more cotnplicated and expensive assays such as solid-phase ELISA or radioimmunoassays (RIAs) using isolated egg antigens and tagged anti-human immunoglobulins (Hillyer et al.., 1979; Tapales ct ai, 1981). Experiments with large numbers of sera from scbistosomiasis japonica patients have recently been performed to standardize the COPT in terms of duration of infection in rabbits used as egg donors (from liver), the number of eggs used and duration of incubation with seruin. These studies strongly suggest that egg maturity is an important variable to be considered in the development of a standardized COPT (Garcia et ai, unpublished data). A murine hybridoma antibody which binds to the soluble MSA-1 antigen of Schistosoma mansoni eggs has recently been described (Hillyer and Pelley, 1980). This antibody results in a COP reaction when incubated with S. mansoni eggs. MSA-1 has been reported to be a major species-specific and immunopathologically-active antigen in sehistosomiasis mansoni (e.g. Hamburger. Pelley and Warren. 1976). However, on the point of species-specificity, the anti MSA-1 hybridoma antibody does produce a COP reaction with 5. japonicutn eggs when the two reactants are incubated together for several days (Hillyer and Pelley. 1980). In the present paper we describe the identification of 3 murine hybridoma-derived antibodies which bind to isolated egg antigens in an RIA and which result in COP reactions with 5. japonicitm eggs. The availability of such hybridoma-derived antibodies will facilitate the analysis of COP antibody production in infected hosts and the nature of COP reactions. If antigens which lead to COP reactions are also involved in induction and expression of granulomatous sensitivity to 5. japonicutn eggs, then these hybridoma-derived antibodies will also be useful in the isolation and characterization of antigens responsible for immunopathologic anti-egg responses in sehistosomiasis japonica. MATERIALS AND METHODS Hyhridomas and radioimmunoassays (RIAs) The production and selection of hybridomas (Kohler and Milstein, 1975) producing antibodies to parasite antigens using BAl.B/c male mice as donors of cells for fusion with the modified myeloma ceil line NS-I have been described in detail (Mitchell et al.. 1979; Craig vt al., 1980; Mitchell et at.. ]98ia). Antigen preparations used for injection of mice or selection of hybridomas using solid-phase RIAs were as follows: lyophilized eggs from the livers of S. japoniciim-infected mice or rabbits were homogenized and sonicated to produce an S. japonicum egg extract (EA) (Tapales ef al, 1981) as were S. japonicum adults to produce an adult worm extract (AWE) (Mitchell et al., 1981a). The Parasonimus westcrmanii antigen preparation was also an AWE used as a skin testing reagent for paragonimiasis in the People's Republic of China. The Taenia taeniaeformis antigen preparation
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was an extract of the terminal proglotlids of adult tapeworms obtained from infected cats by Dr. G. R. Rajasekariah of the University of Melbourne Veterinary Clinical Centre, Werribee, Victoria. Extracts were prepared in horate buffer pH 9 5 or phosphate buffered saline, pH 7 3, All hybridomas used in these studies on schistosomiasis japonica were chosen because of evidence of binding of their products (as culture supernatants) to S. \aponivurn antigen preparations in solid-phase RIAs. Antigens (worms, eggs and AWEs) were injected intraperitoneally and subcutaneously in 1:1 Freiind's Complete Adjuvant (FCA) (Difco Laboratories, Detroit, Michigan. USA) and the mice boosted without adjuvant at least once prior to harvesting spleen cells for fusion. Details are as follows; S. japonicum adult worms—mice were injected with lhc equivalent of 800 ("g lyophilized worms as an extract in FCA on day 0 followed by 500, 300 and IOO ng at days 119, 149, and 180, respectively: S. japonicum eggs^mice were injected with 500 fig lyophilized eggs in FCA and boosted ]x; Taenia tueniaeformis AWE— mice received the crude extract in FCA on day 0 followed by ;i boost at day 17, and were given 500 eggs orally on day 42; Paragnnimu.v westernwnii AWE-—mice were injected with antigen (O.I ml of 1 ;50) in FCA on day 0 and were booked on days 28. 48 and 390. Mice were killed 4 days after boosting (which, other than for T. laeniaeformis oral administration of eggs, was always by intraperitonea! injeciion) and ! spleen equivalent fused with 10^ NS-1 cells. Screening of supernatanls was performed at 13-15 days post fusion and assayed by RIA using '-''I-sheep antimouse Ig. Cells in positive wells were cloned by limiting dilution and two positive clones from each original well selected and recloned. F'or production of ascites fluids, BALB/c male mice aged 8-10 weeks were injected intraperitoneally with 0 5 ml Pristane (2.6,10.14-tetramethylpcntadecane, Koch Light Laboratories, Colnbrook. Bucks.. U.K.) and injected 10 to 30 days later with 1 to 5 X 10" hyhridoma cells intraperitoneally. Ascites fluids were collected (as well as tumour cells for passaging) and centrifuged at 600 ,? for 10 min. They were stored overnight at 4°, any clots removed and portions of some pools inactivated by heating at 56° for 30 min. Pooled ascites fluids were centrifuged at 12,800 .? for 15 min in an Eppendorf centrifuge and stored at —20° until used. Full details of RIAs employed have been published (Craig et al. 1980; Mitchell et al., 1981a). Isotype analyses were performed using rabbit anti-mouse IgGi, igG-jn. TgGji,, and IgG;! (Litton Bionelics, Kensington, Maryland. USA) that had been eluted from protein A-Sepharose (Pharmacia, Uppsala. Sweden) columns to remove carrier proteins present. Sheep anti-mouse IgM was provided by Dr. Robin F. Anders of this Institute and was purified from an igM-Sepharose column. All antisera were jodinated using chloramine T by John Pye of this Institute. The COPT-positive hyhridoma protein PwF.41-1-3 was precipitated oul of ascites fluids by treatment with saturated ammonium sulphate (1:1) and was radioiodinated after dialysis against mouse tonicity phosphate buffered saline. pH 7.3 (PBS). Tn the competitive RIAs, EA-coated PVC plates were incubated with dilutions of ascites fluids in bovine serum albumin/Tween 20/PBS and 20.000 counts per minute of '--Mabelled PwF.41-I-3 in the same diluent (Mitchell et ai, 1981a). Details of sera from schistosomiasis japonica patients (Mitchell et al., 1981a) and 40 day-infected mice (Mitchell et a!., 1981b) have been provided. Murine anti-egg antisera were obtained from C57BL/6 and CBA/H mice derived from a specific pathogen-free facility and which had been injected subcutaneously and intraperitoneally with 2..'i()0 lyophilized eggs of S. japonicum in FCA or FCA alone on day 0, 2,500 eggs or PBS on day 12. 5,000 eggs intravenously on day 40 and bled 4 days later. Eggs used were obtained from the livers of 55 to 70 day infected rabbits. Such egg-immunized mice have been used for studies on lung granuloma formation using a radioisotopic assay (Mitchell el ai. 1981b). Circtmtoval precipitin test (COPT) Lyophilized eggs obtained from the lungs of 60 day-infected rabbits were incubated on cleaned slides with 2 drops of serum or ascites fluid, a cover-slip added and the chamber sealed with paraffin. After incubation at 37° for either 24 or 48 h, the presence of precipitates was determined using a bench microscope.
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RESULTS Details of hybridomas used Seven hybridoma-derived antibodies as ascites fluids or culture supernatants have been used in the studies to be reported. The 2 IPH hybridomas (IPH.134-18-6, an IgG:;« protein, and IPH.39-10-2, an IgM protein) were selected in an RIA using 5. japonicwn AWE, the 2 SEF hybridomas {SEF. 43-18-17, an IgM protein, and SEF.85-5-3, an I g d protein which binds only partially to protein A-Sepharose at pH8 0 on a single pass) using S. japonicum EA, the 2 PwF hybridomas (PwF.26-2-9 and PwF.41-I-3, both IgM proteins) using P. westermanii AWE, and the TTO hybridoma (TTO. 45-8-8, an IgM protein) using an extract of T. taeniaeformis tapeworms. IPH.134-18-6 has immunodiagnostic potential for sehistosomiasis japoniea in man and does not bind to 5. japonicum EA (Mitchell et ai, 1981a; Cruise. Mitchell, Garcia and Anders, 1981). The PwF hybridomas were chosen from a series of 10 because of detectable binding to 5. japonicutn AWE and EA. The TTO hybridoma (from a series of 6) was also found to bind to 5. japonicutn antigens (Table 1). Concerning the precise binding specificities of the hybridoma proteins, it must be cautioned that since 5 of the 7 are of the IgM isotype (Table 1), specific binding activities are not easy to determine. IgM antibodies are more TABLE 1 Results of COPT u.'^inf' liyhridonia ascites fluids with lyophilized ei;i;s of Schisiosonia japonicum Reagent incubated with eses *T
A,
Ji
*.
^^^r
3 positive human sera Day 40 infected mouse serum pool IPH.134-18-6 1PH.39-I0-2 PwF.26-2-9 TTO.45-8-8 SEF.43-18-17 SEF.85-5-3 PwF.41-1-3
Isotype of hybridoma antibody*
Binding in RIA* versus S. japonicum: EA
AWE
—
—
—
—
Percentage of eggs positive! in the 24 h COPT
48 h COPT
14 1 i!: 5-U
19 1 ± 5 4
—
—
76
IgG:;. IgM"
0 0
+ +
IgM IgM IgM
+ —
+ +
0 0 0 0 23 7 18 8 17.5
IgG,
+ 4-
IgM
+
N.D.H Low +
±
15-6 0 3§ 0
0 0 32 2 29-0 26.8
.
* All isotype determinations and RIAs were performed with hyhridom:i cuUurc ;uipernatants rather than ascites fluids. t All positive COPT reactions with mouse reagents consisted of hieb precipitates, segmented precipitates (amounting to a mean of approximately 50% of segmented plus bleb reactions) occurring with the 3 known positive human sera. In ihe test, 103 to 350 eggs were used on each slide (mean of 230) and were incubated with 2 drops of each reagent (used undiluted). i Arithmetic mean ± SEM. § One very small but definite bleb in 350 eggs. ^ ND — not done, this cell line being no longer available because of failure to survive freezing in liquid nitrogen.
HYBRIDOMAS AND COP REACTIONS
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^ 50 O
5 ^5 o (J
K
35
I 30 I/I
O
°- 25
10"
D 2 10 3 iQ-A fg io~1 DILUTION OF HYBRIDOMA ASClTES FLUID
10-2
10-3
10
Fig. I. Titrations of 3 positive and I negative hybridoma asciies fluids with eggs of Schi.ttosoma japonicum in a 24 h COPT (panel A) and a 48 h COPT (panel B) and expressed as the percentage of eggs positive for precipitation. Dilution was performed in phosphate buffered saline pH 7 3. N = neat (undiluted) ascites lluid. (.\ x), PwF.41-1-3; (O O), SEF.85-5-3; {•• - • ) , SEF,43-18-17; (A * ) . IPH.134-18-6; ( A ) , a mixture of the three positive hybridoma ascites fluids in a 1:1:1 ralio.
difficult to purify from ascites fluids and culture supernatants than protein Abinding IgG iintibodies, and in solid-phase RIAs they create problems of interpretation because of the relatively high levels of non-specific binding to PVC plates. It is thus extremely difficult to discriminate confidently between low binding activity versus non-specific binding to the parasite antigens even when binding to a bovine serum albumin-coated control PVC plate is lower (this being the case with all culture supernatants of hybridomas used in this study). In Table 1 the ± designations refer to weak and questionable specific binding to the indicated parasite antigen preparation. Since the 5. japonicum AWE will contain EA (egg-laying female worms are contained in the worm preparation used for AWE), any hybridoma antibody which binds to EA should also bind to AWE unless the target antigen is a late differentiation antigen of eggs. This may well apply to the COPT-positive bybridoma antibodies (Table 1). COP reactions Ascites fluids from groups of BALB/c mice injected intraperitoneally with 7 supposedly different hybridomas were screened for COP positivity using conditions optimized for detection of COP reactions with infected human sera (Garcia et al., unpublished data). Similar conditions had been
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used previously to demonstrate COP antibodies to eggs in sera from 5. japoniCHm-infected mice (Mitchell et ai, 1981b). The data in Table 1 indicate that COP reactions were obtained with 3 hybridoma ascites fluids (SEF.43i8-17, SEF.85-5-3 and PwF.4I-l-3) but not with 4 others (IPH.134-18-6, IPH.39-10-2, PwF.26-2-9 and TTO.45-8-8). One egg in the 48 h COPT with IPH.134-18-6 had a very small bleb and this is unlikely to be a genuine positive reaction. Results of a titration of ascites fluids witb eggs are shown in Fig. I as are the results obtained with a mixture of the 3 COPT-positive ascites fluids. Allowing for the well-known vagaries of the COPT (e.g. differences between the small samples of eggs used on the slides), the titration curves for the positive ascites fluids appear similar. Al! COP reactions obtained with murine hybridoma ascites fluids were of the medium or large bleb type (Lewert et ai, 1980) rather than the segmented precipitates found with sera from infected humans (Table I footnote). A mixture of ascites fluids also failed to generate segmented precipitates, all observed precipitates being bleb-like reactions similar to those seen with sera from short-term infected mice, rabbits and humans versus long-term infected humans.
Competitive RIAs The PwF.41-I-3 protein was precipitated from ascites fluid using ammonium sulphate, the mixture labelled with ^-''I and used with S. japonicum egg antigens (EA) in a competitive solid-phase RIA. Titrations of the COPT-positive hybridoma ascites fluids (from a dilution of 1:10 or 1:100) and two COPT-negative hybridoma ascites fluids (TTO.45-8-8 and PwF. 26-2-9) were performed in this assay. These inhibition of binding studies demonstrated quite clearly the following unexpected results: complete inhibition of binding of '-•'I-PwF.4I-I-3. comparable to that obtained with unlabelled PwF.4I-l-3, was seen with both SEF.43-18-17 and SEF.85-5-3 although the inhibitory titre of the SEF.85 ascites fluid was lOx less than that of PwF.4l or SEF.43 (Fig. 2). More predictably, another PwF.41 hybridoma protein which was available as ascites fluid for testing, PwF.415-5, had comparable inhibitory activity to PwF.41-1-3. Also, the COPTnegative hybridoma ascites fluids. TTO.45-8-8 and PwF.26-2-9. were without any inhibitory activity in the i-"'I-PwF.41-l-3 plus EA assay. It is important to emphasise that antibody activity was shown to be contained in the COPTnegative hybridoma ascites fluids, a particularly relevant binding activity being that of the IgM antibody PwF.26-2-9 to EA. The conclusion seems warranted that 1 speciflcity is contained in this series of 3 COPT-positive hybridoma proteins, there being the remarkable coincidence that one IgM hybridoma antibody selected against a P. westermanii AWE apparently has the same specificity as 2 independent hybridoma antibodies (IgG and IgM) selected against S. japonicutn EA. Of course, we cannot discount the possibility that inhibition in the competitive RIA is by steric hindrance resulting from binding of antibody to a determinant in close proximity to the target determinant of the labelled PwF.41 antibody.
509
HYBRIDOMAS AND COP REACTIONS
20 iO 80 320 1280 5280 RECIPROCAL OF SERUM OR ASCiTES DILUTION
20^80
Fig. 2. Inhibition of binding of '-•'i-PwF.41-1-3 to an S. japonicum egg extract in a competitive RIA using dilutions of sera from C57BL/6 ( • ) or CBA/H mice (O) immunized with S. japonicum. eggs in adjuvant (solid lines) or adjuvant alone (broken lines) and injected intravenously with eggs 4 days prior to bleeding, or three ascitfs fluids PwF.41 (x x), SEF.43 (A A) and SEF,85 (A A).
To determine whether PwF.41-1-3 has immunodiagnostic potential for schistosomiasis japonica (i.e. it is directed to an antigenic determinant which is immunogenic in all infected patients or that subpopulation with prominent disease), titrations of sera from known infected humans were assayed in the competitive '-^'I-PwF.41/EA system. Only weak inhibition of binding at the lowest dilutions of sera (1:20 and 1:40), and with only some of the sera, was observed using 10 sera in the assay even when donors were chosen which were known to have prominent disease (hepatosplenomegaly). No inhibition of binding (at 1:20, the lowest dilution used) was observed using sera from known uninfected individuals. Moreover, no prominent inhibition of binding was found with sera from mice infected for 40 days, these same sera (Mitchell et ai, 1981b) having been shown previously to be strong inhibitors in an ^-•'I-lPH.134-18-6/5. japonicum AWE competitive binding assay (Cruise et ai, 1981). However, using sera from mice imtnunized with eggs in adjuvant and which show high (C57BL/6) or low (CBA 'H) responsiveness in terms of lung granuloma formation (Mitchell ct ai, 1981b), inhibition of binding of ^-•'''I-PwF.41 to EA was obtained. Although only one pool of serum from each type of mouse has been examined, it is of interest that serum from egg-immunized C57BL/6 mice has a higher inhibitory titre than pooled sera from egg-immunized CBA'H mice (Fig. 2). Using this same inhibitory binding assay, it was further shown that a serum
-'^lO
K. M. CRUISE ET
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poot from tlie mice immunized with P. westermanii AWE, and used as cell donors for fusion in the generation of the PwF hybridomas, was inhibitory. This assay was performed on the off-chance that a mix-up had occurred at some stage during production or storage of PwF.41 and that it was actually an SEF or IPH hybridoma. The fact that the serum from the immunized cell donors inhibited the binding of ^-••'I-PwF.41 to EA increases confidence that P. westermanii AWE and 5. japonicum EA share at least one antigenic determinant recognized by the mouse and that PwF.41-1-3 (and PwF,41-5-5) are genuine anti-paragonimus hybridomas. DISCUSSION No published information is available on the spectrum of egg-derived antigens responsible for formation of precipitates associated with 5. japonicum eggs when incubated with infected host sera in the COPT, Considerable information is available on the antigens of S. mansoni eggs (Brown et al., 1977; Carter and Colley, 1979: Harrison. Carter and Colley. 1979: Hamburger etal.. 1976: Pelley cro/.. 1976, and reviewed in Mitchell and Anders, 1981). A hybridoma has been produced which secretes an antibody directed against a major serologic antigen (designated MSA-1) of 5. mansoni eggs and which results in a COP reaction (Hiltyer and Pelley, 1980). As in the 5. japonicum/ human system, it is not known how many antigens of S. mansoni eggs can result in a COP reaction with human sera, a reaction which is of major importance in tfie immunodiagnosis of schistosomiasis. The appearance of precipitates seen in association with the egg in the S. japonicum COPT is of some interest. Segmented precipitates are seen with some human sera and some egg batches. Antigens are perhaps extruded in pulses and this sporadic export of antigens may lead to the appearance of segmented precipitates with sera. However, lyophilized eggs are routinely used for the COPT and a periodic release of antigens seems unlikely from a non-viable source. It is more likely that numerous antigen-antibody reactions are involved such that 'precipitin arcs' in segmented precipitates represent different optimal concentrations of reactants for precipitation. If this is so, then small bleb-like precipitates reflect a limited number of precipitins in the serum, whereas segments reflect the presence of a complex mixture of antibodies of different avidities, amounts and possibly isotypes. In a recent and important study Lewert et al. (1980) provided evidence that bleb-like reactions are a feature of sera from individuals infected for a limited period of time. In another study bleb-like reactions predominated when lyophilized eggs from livers of rabbits infected for > 70 days (rather than the optimal 55-65 days of infection) were used in the COPT even with strongly positive sera from chronically-infected individuals (Garcia et al., unpublished data). Bleb-like precipitates are seen with sera from mice or rabbits infected for approximately 6-8 weeks (Tapales. Valdez and Garcia, unpublished data). Tn the present experiments only bleb-like precipitates were obtained with anti-egg hybridoma ascites fluids. Two of the 3 COPT-positive
HYBRIDOMAS AND COP REACTIONS
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antibodies were IgM proteins, the other an IgGi protein which binds poorly to protein A-Sepharose. It is of some interest that, when 50-60 day infected mouse serum is fractionated on protein A-Sepharose, very obvious bleb-like COP reactions have so far only been detected in the non-bound run-through fraction (which is depleted of IgG and enriched for IgM) and not with the pH 6 (IgGi-enriched) and pH 3 eluates (IgGj-enriched) (unpublished observations, c.f. Yogore, Ozcel and Lewert, 1976). The 7 hybridoma ascites fluids (plus PwF.41-5-5 which appears to be identical to PwF.41-1-3) used in Table 1 are currently being tested for any host-protective or disease-modulating activity in mice in Manila. These hybridomas all secrete antibodies which bind to S. japonicum AWE and/or EA in solid-phase RIAs. A total of 10 PwF hybridomas have been produced by one of us (H, S-R) using P. westermanii AWE for selection; two (PwF.41 and PwF.26) were found to secrete Ig (presumably antibodies) binding to S. japonicum EA in a routine screening for cross-reactions. Thus, a crossreaction exists between P. westermanii AWE and S. japonicitm EA, and also 5. japonicum COPT antigens, identified by the PwF.41 antibody, this antibody but not PwF.26 resulting in a COP reaction. The observation does not mean that sera from P. westermanii-infcctcd patients will necessarily lead to false positives and confuse the COPT for schistosomiasis japonica. Rather, it simply indicates that an antigen determinant recognized by the tnotise is shared between these two antigen mixtures. The use of hybridoma antibodies is siiowing up not only the expected cross-reactions between crude parasite antigen mixtures but a sharing of antigenic determinants which was not predicted from other more conventional serological tests (Hillyer and Pelley, 1980; Nussenzweig, personal communication). It was shown in a competitive RIA that the target antigenic determinant of PwF.41 in S. japonicum EA does not appear to be strongly immunogenic (with respect to antibody production) in schistosomiasis japonica patients. Mice immunized with S. japonicum eggs in adjuvant responded to this determinant by production of antibodies. Tt remains to be determined whether or not the antigen(s) containing this determinant, and isolated by PwF.41 antibody afiinity chromatography, are immunogenic in infected patients or whether the determinant (and antigen(s)) are capable of inducing immunopathologic T cell responses in ^uch individuals. Using i-''I-PwF.41-1-3 and the 3 COPT-positive hybridoma ascites fluids in competitive binding assays with 5. japonicum EA. the remarkable observation was made that the combining site specificities of all 3 appeared to be similar (identical?) or to be directed against determinants which sterically hinder in competitive assays. Tt has not been possible to check inhibitory activities of the 3 COPT-positive hybridoma antibodies in RTAs using the two SEF hybridoma proteins after labelling. SEF.43 is no longer available (Table 1 footnote) and no success has yet been obtained in several attempts to label SEF.85 and to retain adequate EA binding activity. Tf the combining site of this antibody is modified by radiolabelling (and we have not proven
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K. M. CRUISE ET AL.
this to be so), then it may be different from PwF.41. With the assumption that the 3 COPT-positive antibodies have identical specificity, then it is possible that there is a very limited number of major antigens of 5. japonicum eggs which induce COP antibodies, at least in mice. This might fit with the observation that a low COP responder strain of mouse (CBA/H) has been found (Mitchell et al., 1981b); the probability of finding a low responder must be higher when few antigens are involved in the antibody-detection system. The COPT-positive hybridoma antibodies will be used for several immunoparasitological investigations and purpo.ses. Firstly, they will be used to screen batches of lyophilized eggs to be used in a standardized COPT for routine immunodiagnosis of schistosomiasis japonica in the Manila laboratory. As mentioned above, recent studies have implicated egg maturity as the crucial variable in the COPT. It is also possible that egg maturity influences granuloma size in tissues of sensitized hosts and hybridoma antibodies will be useful in detecting differences in any egg batch which has a higher or lower capacity to induce granulomas in egg-sensitized mice. Secondly, they will be injected into egg-sensitized mice (and which are challenged intravenously with eggs) to determine their influence on lung granuloma formation in high and low responder mouse strains (e.g. C57BL 6 versus CBA/H) as assessed by a radioisotopic assay (Mitchell et al., 1981b). Tf inhibition of granuloma formation (or enhancement of granuloma formation) is found, then the hybridoma-derived antibodies will be used for isolation of antigens relevant to induction and/or modulation of immunopathology in the mouse. With isolated purified antigLMis. quantitation of immune responses is possible. Attempts at 'desensitization' will also be possible. Thirdly, the anti-egg hybridoma antibodies will be used to isolate COP antigens to be used in the development of an ELTSA assay for comparative field and laboratory studies with the COPT and other assays for schistosomiasis japonica based on the anti-worm hybridoma antibody. IPH.134-18-6 (Mitchell etal.. 1981a; Mitchell, 1981). Ackiiowledgrtients. This work was supported by the Rockefeller Foundation Great Neglected Diseases of Mankind Network, the Australian Naiionat Health and Medical Research Cotincil. the H. V. McKay Chariiahle Trust, and, for the M;inila laboratory, ihe Research Strenptheniiij: Component of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. We thank Robyn Hocking und Kuren Holder for excellent techniciil assistance in producing hybridomas and asciles fluids. Dr. Huang was a training Fellow of the World Health Organization in 1980, bis present address being the Tropical Medicine Research Institute. Beijing Friendship Hospital. Beijing. People's Republic of China.
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R. B. (1968): -Diagnosis of schistosomiasis japonie:i by cireumoval precipitin test using dried blood on filter paper.' Acta Med. Phil.. 4, 128.
HYBRIDOMAS AND COP REACTIONS , C. E., and COLLEY, D . G . (1979):
'Partial purification and characterization of Schisto.soma mansoni soluble egg antigen with Con A-Sepharose chromatography.' J. tmmunol., 122, 2204. CRAIG,
P. S., MITCHELL.
G . F., CRUISE,
K, M., and RICKARD, M . D . (1980): 'Hy-
bridoma antihody immunoassays for the detection of parasitic infection: attempts to produce an immunocilagnostic reagent for a taeniid larval cestode infection.' Aust. J. Exp. Biol. Med. Sci., 58. 339. CRUISB, K. M., MircHii.L. G. F,, GARCIA,
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