EXPERIMENTAL
PARASITOLOGY
40,189-197
Schistosoma
mansoni: Antigens
A. M.
DEELDER,
( 1976)
Demonstration in Infected
of Two Circulating Hamsters
H. T. M. KLAPPE, G. J. M. J. VAN DEN AARDWEG, AND E.H.E.M. VANMEERBEKE
Laboratory of Parasitology, University of Leiden, Rapenburg 33, Leiden, The Netherlands (Accepted
for publication
27 October
1975)
DEELDER, A. M., KLAPPE, H. T. M., VAN DEN AARDWEG, G. J. M. J., AND VAN MEERBEKE, E. H. E. M. 1976. Schistosomu mansoni: Demonstration of two circulating antigens in infected hamsters. Experimental Parasitology 40, 189-197. In this study the presence of two circulating schistosome derived antigens, probably both polysaccharides, was demonstrated in hamsters heavily infected with Schistosoma mansoni. One antigen was an anodic, heat-stable, high molecular weight substance; it was demonstrated in serum, adult worm antigen and in the excretory and secretory products of adult worms. The antigen was demonstrated in the epithelial cells of the schistosome gut. A second antigen, cathodic, heat-stable and a low-molecular weight substance (MW < 30,000), was demonstrated in hamster serum, hamster urine, adult worm antigen, and in the excretory and secretory products of adult worms. Two additional schistosome derived antigens, both heat-labile, were demonstrated in hamster urine. INDEX DESCRIPTORS: Schistosoma munsoni; Immunoebctrophoresis; Immunodiffusion; IFA; Antigen; Antibodies; Hamsters; Mice; Rabbits.
INTRODUCTION
During the last few years, several studies have been concerned with the detection of circulating antigens in schistosomiasis. Berggren and Weller (1967) described a circulating schistosome derived antigen in the serum of mice and hamsters heavily infected with Schistosoma mansoni. Gold, Rosen, and Weller (1969) characterized this antigen further and demonstrated its presence in the urine of infected hamsters. They found the antigen to be anodic, heatstable and dialyzable. Nash, Prescott, and Neva (1974) reported that the circulating antigen was a large molecular weight substance, most likely a polysaccharide. It was demonstrated in S. mansoni, S. haemutobium and S. japonicum homogenates and in the serum of mice heavily infected with 189 Copylight All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
S. munsoni and S. japonicum. Recently, Nash (1974) and von Lichtenberg, Bawden, and Shealy (1974) showed the same antigen to be present in the epithelial cells of the schistosome gut by means of an indirect fluorescence reaction. Bawden and Weller (1974) developed a complement fixation reaction for the detection of circulating antigen in the sera of infected mice and hamsters. Our studies demonstrate the presence of two circulating antigens, which are probably polysaccharides. One of these antigens is an anodic (negatively charged at pH 8.2) large molecular weight substance, and corresponds to the antigen described by Nash et al. (1974). The other antigen is a cathodic (positively charged at pH 8.2) low molecular weight substance; it was
190
DEELDER ET AL.
demonstrated both in serum and in urine of S. munsoni infected hamsters. Both antigens were found in adult worm extracts and in the excretory and secretory products of adult worms. MATERIALS
AND METHODS
Preparation of Antigens Adult S. mansoni worms, serum, and urine were collected from golden hamsters 45 days after exposure to 1500 cercariae. Five thousand worms and an equal volume of Superbrite glass beads (type 120, 3M Company) were placed in a 400 ml vessel containing a 0.1% (w/v) saline solution and spun in a Sorvall Omni-mixer at 16,000 rpm for 20 min; the vessel temperature was kept at 04 C. The mixture was decanted and centrifuged at 25,000g (1 hr; 2 C) and subsequently the supernatant was freeze-dried and the adult worm antigen was designated AWA. The antigen was examined by immunoelectrophoresis as described previously (Deelder 1973); at least 20 different antigens could be demonstrated. A 10% (w/v) solution of antigen in distilled water contained 15 mg protein and 11 mg polysaccharidelml solution. Fractions of AWA were prepared in the following ways: (a) An AWA solution (10% w/v) was boiled at 100 C for 30 min, centrifuged at 2OOOgfor 30 min, freeze-dried and labeled AWA 100; (b) AWA (400 mg) dissolved in 4 ml distilled water was mixed with an equal volume of 10% (w/v) trichloroacetic acid (TCA); after centrifugation (ZOoOg, 30 min) the TCA insoluble precipitate was resuspended in 5% TCA and centrifuged again. The two TCA soluble supernates were desalted on a 1.6 x 40 cm column filled with Sephadex G-25 (Pharmacia), freeze-dried, and labeled AWA-TCA; (c) Fractionation of AWA was carried out by gel filtration with Sephadex G-200 in a 1.6 x 100 cm column, using a Tris buffer (pH 7.4; 0.05 M); the
resultant fractions were dialyzed against distilled water in the cold to remove Tris and freeze-dried; (d) AWA was separated in two fractions with a molecular weight higher and lower than 30,000 by means of ultrafiltration in a hollow fiber T-tube (80 T-tube, Bio-Rad); (e) A 10% (w/v) AWA solution was shaken with an equal volume of chloroform; both water- and chloroformsoluble fractions were tested for antigenic activity. Hamster urine was obtained from the pooled bladder punctures of infected hamsters; the urine was centrifuged (ZOOOg,10 min), desalted on a 1.6 x 40 cm column filled with Sephadex G-25 and freeze-dried. Urine from infected hamsters (UrHi) was fractionated as was described for AWA. Urine of uninfected hamsters of the same age was used as a control. Hamster serum was obtained from the pooled bleedings of infected hamsters; the serum was freeze-dried, labeled SeHi and fractionated as was described for AWA. In order to obtain excretory and secretory antigens (ESA), adult worms were rinsed five times in a sterile Ringers solution immediately after perfusion and subsequently incubated in sterile Ringers solution (37 C, 3 hr; abou’t 6000 worms in 30 ml Ringers). At the end of the incubation period the worms were still alive; they were removed from the incubation fluid, which was centrifuged (2OOOg, 10 min). The supernate was filter-sterilized and freeze-dried. After lyophilization the antigens were desalted on a 1.6 x 40 cm column filled with Sephadex G-25 and freezedried again. Antigens present in the hatching fluid of S. mansoni eggs were collected by incubating eggs (isolated from hamster livers by means of homogenization and trypsinization as described by Browne and Thomas 1963) in distilled Walter (37 C, 3 hr; about 1000 eggs/ml water). After removal of the eggs and miracidia the fluid was treated as described for ESA.
SCHISTOSOME
ANTIGENS
Antisera Immune mouse serum was obtained from the pooled bleedings of Swiss mice 19 weeks after exposure to 100 cercariae; the serum was freeze-dried. Immune rabbit sera were drawn from rabbits immunized with five weekly injeotions of 2 mg antigen dissolved in 0.5 ml distilled water; Freund’s complete adjuvant was emulsified with the antigen in the first injection. Antisera were produced against AWA, AWA-TCA, ESA, and the TCA soluble fraction of ESA. Furthermore, four rabbits were immunized with AWATCA mixed with methylated bovine serum albumin (Nash et al. 1974). An anti AWA-TCA/FITC conjugate was prepared by isolation of the IgG fraction of an immune rabbit serum and conjugation to fluorescein isothiocyanate (FITC) according to the technique described by The and Feltkamp ( 1970). When necessary for the experiment, 100 ~1 of 3X concentrated serum was adsorbed by addition of 10 mg antigen and incubated 1 hr at room temperature; the serum was then centrifuged and the supernate used. Immunodiffusion electrophoresis
( ID ) and Zmmuno(IE)
Object slides covered with 0.95% (w/v) agarose (pure agarose, Koch-Light) in Veronal buffer (pH 8.2, 0.08 M) were used for both techniques. Antigen solutions of 14% (w/v) in distilled water were used, while antisera were concentrated three times either by freeze-drying or with LyInstrument Company, phogel (Gelman U.S.A.). ID patterns were punched with a Gelman template No. 71692; 7 ~1 of antigen and serum were used; ID time was 48 hr. IE was carried out with a LKB 6800A IE apparatus. Patterns were made with a LKB template 681OA; well cutters were modified to give a diameter of 3 mm. Antigen solution (7 ~1) and 100 ~1 of serum were used. Electrophoresis was carried out for
IN
191
HAMSTERS
3 hr at 55 V and 3.5 mA per slide. After diffusion, washing and drying, the slides were stained with Amid0 black. Polyacylamide
Gel Electrophoresk
Electrophoresis was carried out on polyacrylamide gel slabs with a continuous gradient (3-26% ) with a Gradipore MultiCell Unit according to the technique described by Margolis and Kenrick (1968). Antigen samples (5 ~1) were separated during 23 hr at 100 V and 30 mA per gel slab; a Tris-borate-EDTA buffer (pH 8.28, 0.1 M) was used. Gels were stained with Amido black for protein and with Schiff reagent for polysaccharide. Electropherograms were scanned with a Vitatron TLD100 densitometer (Vitatron, The Netherlands ) . RESULTS
Immunoelectrophoresis of AWA against immune mouse serum revealed the presence of a sharp anodic precipitate (Fig. 1). This same antigenic fraction could not be demonstrated with the immune sera of rabbits immunized with AWA. Mouse immune serum showed two distinct precipitates, one anodic and one cathodic (i.e., negatively and positively charged alt pH 8.2, respectively), against AWA, when this had been heated to 100 C or had been treated with TCA (Fig. 2) ; a third vague precipitate could be observed occasionally. When a fraction with a molecular weight lower than 30,000 was prepared from this heated antigen by means of ultrafiltration, only the cathodic antigen could be demonstrated in this fraotion. Three cathodic precipitates could be demonstrated by means of immunoelectrophoresis of urine of infected hamsters against immune mouse serum (Fig. 2). After heating of the urine to 100 C or after treatment with TCA (Fig. 2) only one precipitate remained. Antibodies against all three antigens could be adsorbed by incubation of the immune mouse serum with AWA.
192
DEELDER
FIG. 1. Immunoelectropherogram well against mouse immune serum. contains antigen of the intermediate
AL.
of adult Schistosoma munsoni antigen (AWA) The arrow indicates the anodic antigen. The host, Biomphalaria glubrutu.
In the TCA-soluble fraction of the lyophilized infected hamster serum two antigens could be demonstrated wi’th immune mouse serum: one anodic and one cathodic fraction (Figs. 2 and 3). Occasionally a third vague precipitete could be observed near the well. One gram of lyophilized hamster
/OAA r
ET
I
FIG. 2. Diagrammatic summary of immunoelectropherograms of various antigens against mouse immune serum; AWA 100 = adult Schistosoma munsoni antigen heated to 100 C; ESA-TCA = the TCA-soluble fraction of excretory and secretory antigens; SeHi-TCA = the TCA-soluble fraction of infected hamster serum; UrHi = urine of infected hamsters; UrHi-TCA = the TCA-soluble fraction of UrHi; AA = anodic antigen; CA = cathodic antigen.
in upper lower well
serum produced 5 mg of TCA-soluble material. In the concentrated whole serum of infected hamsters no schistosome antigens could be demonstrated. No precipitates could be demonstrated in uninfected hamster serum (whole serum or TCA-fraction) with immunoelectrophoresis against sera of infected or uninfected mice. An anodic antigen similar to that observed in the serum of infected hamsters and a cathodic antigen similar to that observed in the serum and urine of infected hamsters could be demonstrated in ESA by means of immunoelectrophoresis against immune mouse serum. In ESA, heated ESA, and in the TCA-soluble fraction of ESA the same two antigens could be demonstrated. Rabbits immunized with ESA failed to develop antibodies against ‘these two antigens, although they produced antisera which showed good precipitates against the heat-labile (protein) antigens. By means of immunodiffusion an identical TCA-soluble and heat-stable antigen could be found in AWA, ESA, UrHi, and in egg hatching fluid (Fig. 4). Furthermore, the antigen could be found in SeHi and it proved to be chloroform-insoluble; the antigen had a molecular weight lower than 30,000 and was probably identical to the cathodic antigen demonstrated with immunoelectrophoresis. An identical antigen could be shown in
SCHISTOSOME
FIG. 3. Immunoelectropherogram fected hamster serum (both wells)
ANTIGENS
IN
of the TCA-soluble against mouse immune
AWA, ESA and SeHi (Fig. 5), but not in UrHi. The antigen was heat-stable, TCAsoluble, and chloroform-insoluble and had a molecular weight of at least 200,000, according to elution on a Sephadex G-200 column; it was probably identical to the anodic antigen demonstrated with immunoelectrophoresis. The polysaccharide nature of the anodic antigen could be shown, when pure anodic antigen (isolated as described below) proved to be stainable with Schiff reagent and not with Amido black after polyacrylamide gel pore-limit electrophoresis (Fig. 6). The position of the stained spot in the gel corresponded with the position of human az-macroglobulin (MW 800,000), which was used as a reference. Rapid isolation of the anodic antigen was performed by heating of an AWA-solution ( 100 C, 30 min), followed by TCA-treatment and subsequent desalting on a Sephadex G-25 column. The pure antigen eluted as a single sharp peak with the void volume. When the antigen was heated during a shorter period, no satisfactory separation could be obtained on a G-25 column. Probably, fractions containing contaminants diminished the sharpness of the peak. A further fractionation of the desalted antigen on a Sephadex G-200 column proved to be necessary in that case. Rabbits were immunized with anodic antigen after it had been mixed with
HAMSTERS
fraction serum.
of Schistosoma
mansoni
in-
methylated bovine serum albumin. After the IgG fraction of an immunized rabbit had been isolated and conjugated with FITC, the resultant conjugate was incubated with frozen sections of adult S. mansoni worms. In this way a bright fluorescence in ‘the epithelial cells around the lumen of the digestive tract could be shown, DISCUSSION
The findings reported in this study confirm the presence of a circulating antigen of schistosome origin in golden hamsters heavily infected with S. munsoni (Berggren and Weller 1967; Gold et al. 1969; Nash et al. 1974). In corrtrast to these authors, however, two polysaccharide antigens (one with an anodal and one with a cathodal electrophoretic mobility) were shown to exist. The properties of the anodic antigen correspond with those observed by Nash et al. ( 1974). It proved to be identical with an antigen designated as No. 16 in an earlier study ( Deelder 1973) ; antibodies against antigen No. 16 could be demonstrated in sera from experimentally infected Swiss mice by immunoelectrophoresis in this study. In highly concentrated, whole hamster serum anodic antigen could be demonstrated by immunodiffusion but not by imPossibly the concenmunoelectrophoresis.
194
DEELDER
ET
AL.
more pronounced than that of the cathodic antigen. The anodic antigen could not be demonstrated in hamster urine, but it was found in the excretory and secretory products of adult worms; when these antigens were fractionated on a column filled with Sephadex G-200 the anodic antigen eluted
tration of the antigen was too low, or irrterference with other substances in the concentrated serum prohibited formation of the precipitate. In the TCA-soluble fraction of hamster serum, however, the antigen could be demonstrated clearly by immunoelectrophoresis; its precipitate was always
UrHi
0 AWA
= AWA fraction with molecular For the explanation of further abbreviations see Fig. 4.
near ‘the void volume, indicating a molecular weight of at least 200,000; Nash et al. (1974) reported a molecular weight of at least 100,000. However, a molecular weight of about 800,000 is suggested by the position of the antigen after pore-limit electrophoresis in a polyacrylamide gel, although the position in the gel will not only be
FIG. 6. Polyacrylamide gel electrophoresis of anodic antigen after staining with Schiff reagent. The arrows indicate the position of the antigen in a 3-10s gel (left) and in a 3-26s gel (right).
OSeHi
schematic weight
diagram only the higher than 30,000.
determined by the molecular weight but also by the structure of the molecule. The antigen spot could be stained with Schiff reagent, again corroborating the evidence for the polysaccharide nature of the antigen. Rabbits could be immunized only against anodic antigen when the antigen was administered with methylated bovine serum albumin, as was described by N,ash et al. ( 1974). A specific FITC-conjugated IgG fraction was prepared from a rabbit immunized in ,this way; with this conjugate a bright fluorescence in the cells lining #the schistosome gut was demonstrated. This finding corresponds with the recent observations of Nash (1974) and von Lichtenberg et al. ( 1974). The cathodic antigen was demonstrated in the TCA-soluble fraction of hamster serum by means of immunoelectrophoresis against immune mouse serum. This same antigen could also be demonstrated in hamster urine, in the excretion and secretion products of adult worms and in the secretion products of schistosome eggs. The cathodic antigen was heat-stable, TCAsoluble, and insoluble in chloroform, in-
196
DEELDER ET AL.
dicating a polysaccharide. By means of ultrafiltration it was established that the antigen had a molecular weight lower than 30,000; this finding was confirmed by the observation that the antigen eluted in the last fractions, when excretion products of adult worms were separated on a Sephadex G-200 column. Further experiments to obtain more specific data about the molecular weight by means of pore-limit electrophoresis proved ‘to be unsuccessful. A third TCA-soluble and heat-stable antigen could occasionally be demonstrated in the TCA-soluble fraction of hamster serum by means of IE against immune mouse serum. The position of the precipitate was always near *the well. The antigen was not charaaterized further. In several experiments two further cathodic precipitates could be found with IE against urine of infected hamsters. Adsorption of the antiserum with adult worm antigen resulted in disappearance of the precipitates, indicating the schistosome origin of the antigens. In contrast to the other antigens described these two antigens were heat-labile; they might correspond with the heat-labile antigen found by Okabe and Tanaka ( 1958, 1961) in the urine of S. japonicum infected patients. It is interesting that our observations of the molecular weight of the anodic antigen agree exactly with those of Nash et al. (1974) but not with those of Gold et al. ( 1969), who reported a molecular weight lower than 10,000. Gold et al. also observed their anodic an,tigen in the urine of infected hamsters, whereas in the present study it was the cathodic antigen which was found in hamster urine. As Gold et al. used only immunodiffusion and not immunoelectrophoresis, it might be possible that [their urine antigen was cathodic. The urine they used was unconcentrated and undesalted, which gave unsatisfactory results with immunodiffusion in our experiments. The ultimate goal of our studies, the development of a sensitive method for the demonstration of circulating antigen in hu-
man serum, has so far been hampered by the difficulty of eliciting specific immune sera in rabbits. Our experiments to isolate the specific antibody fraction from immune mouse serum have so far proved unsuccessful, since it has not yet been possible to bind the antigen to a suitable support. Preparation of a specific antiserum against the cathodic antigen might enable the developmen’t of a sensitive test for the demonstration of schistosome antigen in urine. ACKNOWLEDGMENT We wish for carefully
to thank reading
Professor Dr. the manuscript.
A.
W.
Senft
REFERENCES M. P., ANU WELLER, T. H. 1974. Schistosoma mansoni circulating antigen: Detection by complement fixation in sera from infected hamsters and mice. The American Journal of Tropical Medicine and Hygiene 23, 1077-1084. BERGGREN, W. L., AND WELLEH, T. H. 1967. Immunoelectrophoretic demonstration of specific circulating antigen in animals infected with Schistosoma mansoni. The American Journal of Tropical Medicine and Hygiene 16, 606-612. BROWNE, H. G., AND THOMAS, J. I. 1963. A method for isolating pure, viable Schistosoma eggs from host tissues. Journal of Parasitology 49, 371374. DEELDER, A. M. 1973. Immunology of experimental infections with Schistosoma mansoni in the Swiss mouse and with Fasciola hepatica in the rabbit. Acta Leidensia 39, I-107. GOLD, R., ROSEN, F. S., AND WELLER, T. H. 1969. A specific circulating antigen in hamsters infected with Schistosoma mansoni. Detection of antigen in serum and urine, and correlation between antigenic concentration and worm burden. The American Journal of Tropical Medicine and Hygiene 18, 545551. LICHTENBEHG, F. v., BAWDEN, M. P., AND SHEALY, S. H. 1974. Origin of circulating antigen from the schistosome gut. An immunofluorescent study. The American Journal of Tropical Medicine and Hygiene 23, 1088-1091. MARGOLIS, J., A~TD KENRICK, K. G. 1968. Polyacrylamide gel electrophoresis in a continuous molecular sieve gradient. Analytical Biochemistry 25, 347-362. NASH, T. E. 1974. Localization of the circul’ating antigen within the gut of Schistosoma mansoni. The American Journal of Tropical Medicine and Hygiene 23, 1085-1087.
BAWDEN,
SCHISTOSOME
ANTIGENS
NASH, T. E., PRESCOTT, B., AND NEVA, F. A. 1974. The characteristics schistosomiasis. The 1500-1507.
of a circulating antigen in Journal of Immunology 112,
OKABE, K., AND TANAKA, T. 1958. precipitation reaction for ica, a preliminary report. ]ournal 5, 45-52.
A new urine schistosomiasis japonThe Kurume Medical
IN
HAMSTFB
OKABE, K., AND TANAKA, T. 1961.
197
Urine precipitin schistosomiasis japonica. The Kurume Medical Journal 8, 24-37. THE, T. H., AND FELTKAMP, T. E. W. 1970. Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation; II. A reproducible method. lmmunology 18, 865-873, 875-881. reaction
for
PARASITOLOGY
40,189-197
Schistosoma
mansoni: Antigens
A. M.
DEELDER,
( 1976)
Demonstration in Infected
of Two Circulating Hamsters
H. T. M. KLAPPE, G. J. M. J. VAN DEN AARDWEG, AND E.H.E.M. VANMEERBEKE
Laboratory of Parasitology, University of Leiden, Rapenburg 33, Leiden, The Netherlands (Accepted
for publication
27 October
1975)
DEELDER, A. M., KLAPPE, H. T. M., VAN DEN AARDWEG, G. J. M. J., AND VAN MEERBEKE, E. H. E. M. 1976. Schistosomu mansoni: Demonstration of two circulating antigens in infected hamsters. Experimental Parasitology 40, 189-197. In this study the presence of two circulating schistosome derived antigens, probably both polysaccharides, was demonstrated in hamsters heavily infected with Schistosoma mansoni. One antigen was an anodic, heat-stable, high molecular weight substance; it was demonstrated in serum, adult worm antigen and in the excretory and secretory products of adult worms. The antigen was demonstrated in the epithelial cells of the schistosome gut. A second antigen, cathodic, heat-stable and a low-molecular weight substance (MW < 30,000), was demonstrated in hamster serum, hamster urine, adult worm antigen, and in the excretory and secretory products of adult worms. Two additional schistosome derived antigens, both heat-labile, were demonstrated in hamster urine. INDEX DESCRIPTORS: Schistosoma munsoni; Immunoebctrophoresis; Immunodiffusion; IFA; Antigen; Antibodies; Hamsters; Mice; Rabbits.
INTRODUCTION
During the last few years, several studies have been concerned with the detection of circulating antigens in schistosomiasis. Berggren and Weller (1967) described a circulating schistosome derived antigen in the serum of mice and hamsters heavily infected with Schistosoma mansoni. Gold, Rosen, and Weller (1969) characterized this antigen further and demonstrated its presence in the urine of infected hamsters. They found the antigen to be anodic, heatstable and dialyzable. Nash, Prescott, and Neva (1974) reported that the circulating antigen was a large molecular weight substance, most likely a polysaccharide. It was demonstrated in S. mansoni, S. haemutobium and S. japonicum homogenates and in the serum of mice heavily infected with 189 Copylight All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
S. munsoni and S. japonicum. Recently, Nash (1974) and von Lichtenberg, Bawden, and Shealy (1974) showed the same antigen to be present in the epithelial cells of the schistosome gut by means of an indirect fluorescence reaction. Bawden and Weller (1974) developed a complement fixation reaction for the detection of circulating antigen in the sera of infected mice and hamsters. Our studies demonstrate the presence of two circulating antigens, which are probably polysaccharides. One of these antigens is an anodic (negatively charged at pH 8.2) large molecular weight substance, and corresponds to the antigen described by Nash et al. (1974). The other antigen is a cathodic (positively charged at pH 8.2) low molecular weight substance; it was
190
DEELDER ET AL.
demonstrated both in serum and in urine of S. munsoni infected hamsters. Both antigens were found in adult worm extracts and in the excretory and secretory products of adult worms. MATERIALS
AND METHODS
Preparation of Antigens Adult S. mansoni worms, serum, and urine were collected from golden hamsters 45 days after exposure to 1500 cercariae. Five thousand worms and an equal volume of Superbrite glass beads (type 120, 3M Company) were placed in a 400 ml vessel containing a 0.1% (w/v) saline solution and spun in a Sorvall Omni-mixer at 16,000 rpm for 20 min; the vessel temperature was kept at 04 C. The mixture was decanted and centrifuged at 25,000g (1 hr; 2 C) and subsequently the supernatant was freeze-dried and the adult worm antigen was designated AWA. The antigen was examined by immunoelectrophoresis as described previously (Deelder 1973); at least 20 different antigens could be demonstrated. A 10% (w/v) solution of antigen in distilled water contained 15 mg protein and 11 mg polysaccharidelml solution. Fractions of AWA were prepared in the following ways: (a) An AWA solution (10% w/v) was boiled at 100 C for 30 min, centrifuged at 2OOOgfor 30 min, freeze-dried and labeled AWA 100; (b) AWA (400 mg) dissolved in 4 ml distilled water was mixed with an equal volume of 10% (w/v) trichloroacetic acid (TCA); after centrifugation (ZOoOg, 30 min) the TCA insoluble precipitate was resuspended in 5% TCA and centrifuged again. The two TCA soluble supernates were desalted on a 1.6 x 40 cm column filled with Sephadex G-25 (Pharmacia), freeze-dried, and labeled AWA-TCA; (c) Fractionation of AWA was carried out by gel filtration with Sephadex G-200 in a 1.6 x 100 cm column, using a Tris buffer (pH 7.4; 0.05 M); the
resultant fractions were dialyzed against distilled water in the cold to remove Tris and freeze-dried; (d) AWA was separated in two fractions with a molecular weight higher and lower than 30,000 by means of ultrafiltration in a hollow fiber T-tube (80 T-tube, Bio-Rad); (e) A 10% (w/v) AWA solution was shaken with an equal volume of chloroform; both water- and chloroformsoluble fractions were tested for antigenic activity. Hamster urine was obtained from the pooled bladder punctures of infected hamsters; the urine was centrifuged (ZOOOg,10 min), desalted on a 1.6 x 40 cm column filled with Sephadex G-25 and freeze-dried. Urine from infected hamsters (UrHi) was fractionated as was described for AWA. Urine of uninfected hamsters of the same age was used as a control. Hamster serum was obtained from the pooled bleedings of infected hamsters; the serum was freeze-dried, labeled SeHi and fractionated as was described for AWA. In order to obtain excretory and secretory antigens (ESA), adult worms were rinsed five times in a sterile Ringers solution immediately after perfusion and subsequently incubated in sterile Ringers solution (37 C, 3 hr; abou’t 6000 worms in 30 ml Ringers). At the end of the incubation period the worms were still alive; they were removed from the incubation fluid, which was centrifuged (2OOOg, 10 min). The supernate was filter-sterilized and freeze-dried. After lyophilization the antigens were desalted on a 1.6 x 40 cm column filled with Sephadex G-25 and freezedried again. Antigens present in the hatching fluid of S. mansoni eggs were collected by incubating eggs (isolated from hamster livers by means of homogenization and trypsinization as described by Browne and Thomas 1963) in distilled Walter (37 C, 3 hr; about 1000 eggs/ml water). After removal of the eggs and miracidia the fluid was treated as described for ESA.
SCHISTOSOME
ANTIGENS
Antisera Immune mouse serum was obtained from the pooled bleedings of Swiss mice 19 weeks after exposure to 100 cercariae; the serum was freeze-dried. Immune rabbit sera were drawn from rabbits immunized with five weekly injeotions of 2 mg antigen dissolved in 0.5 ml distilled water; Freund’s complete adjuvant was emulsified with the antigen in the first injection. Antisera were produced against AWA, AWA-TCA, ESA, and the TCA soluble fraction of ESA. Furthermore, four rabbits were immunized with AWATCA mixed with methylated bovine serum albumin (Nash et al. 1974). An anti AWA-TCA/FITC conjugate was prepared by isolation of the IgG fraction of an immune rabbit serum and conjugation to fluorescein isothiocyanate (FITC) according to the technique described by The and Feltkamp ( 1970). When necessary for the experiment, 100 ~1 of 3X concentrated serum was adsorbed by addition of 10 mg antigen and incubated 1 hr at room temperature; the serum was then centrifuged and the supernate used. Immunodiffusion electrophoresis
( ID ) and Zmmuno(IE)
Object slides covered with 0.95% (w/v) agarose (pure agarose, Koch-Light) in Veronal buffer (pH 8.2, 0.08 M) were used for both techniques. Antigen solutions of 14% (w/v) in distilled water were used, while antisera were concentrated three times either by freeze-drying or with LyInstrument Company, phogel (Gelman U.S.A.). ID patterns were punched with a Gelman template No. 71692; 7 ~1 of antigen and serum were used; ID time was 48 hr. IE was carried out with a LKB 6800A IE apparatus. Patterns were made with a LKB template 681OA; well cutters were modified to give a diameter of 3 mm. Antigen solution (7 ~1) and 100 ~1 of serum were used. Electrophoresis was carried out for
IN
191
HAMSTERS
3 hr at 55 V and 3.5 mA per slide. After diffusion, washing and drying, the slides were stained with Amid0 black. Polyacylamide
Gel Electrophoresk
Electrophoresis was carried out on polyacrylamide gel slabs with a continuous gradient (3-26% ) with a Gradipore MultiCell Unit according to the technique described by Margolis and Kenrick (1968). Antigen samples (5 ~1) were separated during 23 hr at 100 V and 30 mA per gel slab; a Tris-borate-EDTA buffer (pH 8.28, 0.1 M) was used. Gels were stained with Amido black for protein and with Schiff reagent for polysaccharide. Electropherograms were scanned with a Vitatron TLD100 densitometer (Vitatron, The Netherlands ) . RESULTS
Immunoelectrophoresis of AWA against immune mouse serum revealed the presence of a sharp anodic precipitate (Fig. 1). This same antigenic fraction could not be demonstrated with the immune sera of rabbits immunized with AWA. Mouse immune serum showed two distinct precipitates, one anodic and one cathodic (i.e., negatively and positively charged alt pH 8.2, respectively), against AWA, when this had been heated to 100 C or had been treated with TCA (Fig. 2) ; a third vague precipitate could be observed occasionally. When a fraction with a molecular weight lower than 30,000 was prepared from this heated antigen by means of ultrafiltration, only the cathodic antigen could be demonstrated in this fraotion. Three cathodic precipitates could be demonstrated by means of immunoelectrophoresis of urine of infected hamsters against immune mouse serum (Fig. 2). After heating of the urine to 100 C or after treatment with TCA (Fig. 2) only one precipitate remained. Antibodies against all three antigens could be adsorbed by incubation of the immune mouse serum with AWA.
192
DEELDER
FIG. 1. Immunoelectropherogram well against mouse immune serum. contains antigen of the intermediate
AL.
of adult Schistosoma munsoni antigen (AWA) The arrow indicates the anodic antigen. The host, Biomphalaria glubrutu.
In the TCA-soluble fraction of the lyophilized infected hamster serum two antigens could be demonstrated wi’th immune mouse serum: one anodic and one cathodic fraction (Figs. 2 and 3). Occasionally a third vague precipitete could be observed near the well. One gram of lyophilized hamster
/OAA r
ET
I
FIG. 2. Diagrammatic summary of immunoelectropherograms of various antigens against mouse immune serum; AWA 100 = adult Schistosoma munsoni antigen heated to 100 C; ESA-TCA = the TCA-soluble fraction of excretory and secretory antigens; SeHi-TCA = the TCA-soluble fraction of infected hamster serum; UrHi = urine of infected hamsters; UrHi-TCA = the TCA-soluble fraction of UrHi; AA = anodic antigen; CA = cathodic antigen.
in upper lower well
serum produced 5 mg of TCA-soluble material. In the concentrated whole serum of infected hamsters no schistosome antigens could be demonstrated. No precipitates could be demonstrated in uninfected hamster serum (whole serum or TCA-fraction) with immunoelectrophoresis against sera of infected or uninfected mice. An anodic antigen similar to that observed in the serum of infected hamsters and a cathodic antigen similar to that observed in the serum and urine of infected hamsters could be demonstrated in ESA by means of immunoelectrophoresis against immune mouse serum. In ESA, heated ESA, and in the TCA-soluble fraction of ESA the same two antigens could be demonstrated. Rabbits immunized with ESA failed to develop antibodies against ‘these two antigens, although they produced antisera which showed good precipitates against the heat-labile (protein) antigens. By means of immunodiffusion an identical TCA-soluble and heat-stable antigen could be found in AWA, ESA, UrHi, and in egg hatching fluid (Fig. 4). Furthermore, the antigen could be found in SeHi and it proved to be chloroform-insoluble; the antigen had a molecular weight lower than 30,000 and was probably identical to the cathodic antigen demonstrated with immunoelectrophoresis. An identical antigen could be shown in
SCHISTOSOME
FIG. 3. Immunoelectropherogram fected hamster serum (both wells)
ANTIGENS
IN
of the TCA-soluble against mouse immune
AWA, ESA and SeHi (Fig. 5), but not in UrHi. The antigen was heat-stable, TCAsoluble, and chloroform-insoluble and had a molecular weight of at least 200,000, according to elution on a Sephadex G-200 column; it was probably identical to the anodic antigen demonstrated with immunoelectrophoresis. The polysaccharide nature of the anodic antigen could be shown, when pure anodic antigen (isolated as described below) proved to be stainable with Schiff reagent and not with Amido black after polyacrylamide gel pore-limit electrophoresis (Fig. 6). The position of the stained spot in the gel corresponded with the position of human az-macroglobulin (MW 800,000), which was used as a reference. Rapid isolation of the anodic antigen was performed by heating of an AWA-solution ( 100 C, 30 min), followed by TCA-treatment and subsequent desalting on a Sephadex G-25 column. The pure antigen eluted as a single sharp peak with the void volume. When the antigen was heated during a shorter period, no satisfactory separation could be obtained on a G-25 column. Probably, fractions containing contaminants diminished the sharpness of the peak. A further fractionation of the desalted antigen on a Sephadex G-200 column proved to be necessary in that case. Rabbits were immunized with anodic antigen after it had been mixed with
HAMSTERS
fraction serum.
of Schistosoma
mansoni
in-
methylated bovine serum albumin. After the IgG fraction of an immunized rabbit had been isolated and conjugated with FITC, the resultant conjugate was incubated with frozen sections of adult S. mansoni worms. In this way a bright fluorescence in ‘the epithelial cells around the lumen of the digestive tract could be shown, DISCUSSION
The findings reported in this study confirm the presence of a circulating antigen of schistosome origin in golden hamsters heavily infected with S. munsoni (Berggren and Weller 1967; Gold et al. 1969; Nash et al. 1974). In corrtrast to these authors, however, two polysaccharide antigens (one with an anodal and one with a cathodal electrophoretic mobility) were shown to exist. The properties of the anodic antigen correspond with those observed by Nash et al. ( 1974). It proved to be identical with an antigen designated as No. 16 in an earlier study ( Deelder 1973) ; antibodies against antigen No. 16 could be demonstrated in sera from experimentally infected Swiss mice by immunoelectrophoresis in this study. In highly concentrated, whole hamster serum anodic antigen could be demonstrated by immunodiffusion but not by imPossibly the concenmunoelectrophoresis.
194
DEELDER
ET
AL.
more pronounced than that of the cathodic antigen. The anodic antigen could not be demonstrated in hamster urine, but it was found in the excretory and secretory products of adult worms; when these antigens were fractionated on a column filled with Sephadex G-200 the anodic antigen eluted
tration of the antigen was too low, or irrterference with other substances in the concentrated serum prohibited formation of the precipitate. In the TCA-soluble fraction of hamster serum, however, the antigen could be demonstrated clearly by immunoelectrophoresis; its precipitate was always
UrHi
0 AWA
= AWA fraction with molecular For the explanation of further abbreviations see Fig. 4.
near ‘the void volume, indicating a molecular weight of at least 200,000; Nash et al. (1974) reported a molecular weight of at least 100,000. However, a molecular weight of about 800,000 is suggested by the position of the antigen after pore-limit electrophoresis in a polyacrylamide gel, although the position in the gel will not only be
FIG. 6. Polyacrylamide gel electrophoresis of anodic antigen after staining with Schiff reagent. The arrows indicate the position of the antigen in a 3-10s gel (left) and in a 3-26s gel (right).
OSeHi
schematic weight
diagram only the higher than 30,000.
determined by the molecular weight but also by the structure of the molecule. The antigen spot could be stained with Schiff reagent, again corroborating the evidence for the polysaccharide nature of the antigen. Rabbits could be immunized only against anodic antigen when the antigen was administered with methylated bovine serum albumin, as was described by N,ash et al. ( 1974). A specific FITC-conjugated IgG fraction was prepared from a rabbit immunized in ,this way; with this conjugate a bright fluorescence in the cells lining #the schistosome gut was demonstrated. This finding corresponds with the recent observations of Nash (1974) and von Lichtenberg et al. ( 1974). The cathodic antigen was demonstrated in the TCA-soluble fraction of hamster serum by means of immunoelectrophoresis against immune mouse serum. This same antigen could also be demonstrated in hamster urine, in the excretion and secretion products of adult worms and in the secretion products of schistosome eggs. The cathodic antigen was heat-stable, TCAsoluble, and insoluble in chloroform, in-
196
DEELDER ET AL.
dicating a polysaccharide. By means of ultrafiltration it was established that the antigen had a molecular weight lower than 30,000; this finding was confirmed by the observation that the antigen eluted in the last fractions, when excretion products of adult worms were separated on a Sephadex G-200 column. Further experiments to obtain more specific data about the molecular weight by means of pore-limit electrophoresis proved ‘to be unsuccessful. A third TCA-soluble and heat-stable antigen could occasionally be demonstrated in the TCA-soluble fraction of hamster serum by means of IE against immune mouse serum. The position of the precipitate was always near *the well. The antigen was not charaaterized further. In several experiments two further cathodic precipitates could be found with IE against urine of infected hamsters. Adsorption of the antiserum with adult worm antigen resulted in disappearance of the precipitates, indicating the schistosome origin of the antigens. In contrast to the other antigens described these two antigens were heat-labile; they might correspond with the heat-labile antigen found by Okabe and Tanaka ( 1958, 1961) in the urine of S. japonicum infected patients. It is interesting that our observations of the molecular weight of the anodic antigen agree exactly with those of Nash et al. (1974) but not with those of Gold et al. ( 1969), who reported a molecular weight lower than 10,000. Gold et al. also observed their anodic an,tigen in the urine of infected hamsters, whereas in the present study it was the cathodic antigen which was found in hamster urine. As Gold et al. used only immunodiffusion and not immunoelectrophoresis, it might be possible that [their urine antigen was cathodic. The urine they used was unconcentrated and undesalted, which gave unsatisfactory results with immunodiffusion in our experiments. The ultimate goal of our studies, the development of a sensitive method for the demonstration of circulating antigen in hu-
man serum, has so far been hampered by the difficulty of eliciting specific immune sera in rabbits. Our experiments to isolate the specific antibody fraction from immune mouse serum have so far proved unsuccessful, since it has not yet been possible to bind the antigen to a suitable support. Preparation of a specific antiserum against the cathodic antigen might enable the developmen’t of a sensitive test for the demonstration of schistosome antigen in urine. ACKNOWLEDGMENT We wish for carefully
to thank reading
Professor Dr. the manuscript.
A.
W.
Senft
REFERENCES M. P., ANU WELLER, T. H. 1974. Schistosoma mansoni circulating antigen: Detection by complement fixation in sera from infected hamsters and mice. The American Journal of Tropical Medicine and Hygiene 23, 1077-1084. BERGGREN, W. L., AND WELLEH, T. H. 1967. Immunoelectrophoretic demonstration of specific circulating antigen in animals infected with Schistosoma mansoni. The American Journal of Tropical Medicine and Hygiene 16, 606-612. BROWNE, H. G., AND THOMAS, J. I. 1963. A method for isolating pure, viable Schistosoma eggs from host tissues. Journal of Parasitology 49, 371374. DEELDER, A. M. 1973. Immunology of experimental infections with Schistosoma mansoni in the Swiss mouse and with Fasciola hepatica in the rabbit. Acta Leidensia 39, I-107. GOLD, R., ROSEN, F. S., AND WELLER, T. H. 1969. A specific circulating antigen in hamsters infected with Schistosoma mansoni. Detection of antigen in serum and urine, and correlation between antigenic concentration and worm burden. The American Journal of Tropical Medicine and Hygiene 18, 545551. LICHTENBEHG, F. v., BAWDEN, M. P., AND SHEALY, S. H. 1974. Origin of circulating antigen from the schistosome gut. An immunofluorescent study. The American Journal of Tropical Medicine and Hygiene 23, 1088-1091. MARGOLIS, J., A~TD KENRICK, K. G. 1968. Polyacrylamide gel electrophoresis in a continuous molecular sieve gradient. Analytical Biochemistry 25, 347-362. NASH, T. E. 1974. Localization of the circul’ating antigen within the gut of Schistosoma mansoni. The American Journal of Tropical Medicine and Hygiene 23, 1085-1087.
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SCHISTOSOME
ANTIGENS
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IN
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Urine precipitin schistosomiasis japonica. The Kurume Medical Journal 8, 24-37. THE, T. H., AND FELTKAMP, T. E. W. 1970. Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation; II. A reproducible method. lmmunology 18, 865-873, 875-881. reaction
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