EXPERIMENTAL PARASITOLOGY 73, 172-183 (1991)

Trichinella spiralis: Light Microscope Monoclonal Antibody Localization and lmmunochemical Characterization of Phosphorylcholine and Other Antigens in the Muscle Larva WAI FUN CHOY, MUN HON NG, AND PAK LEONG LIM’ Department

of Microbiology,

University

of Hong Kong, Pokfulam

Road, Hong Kong

CHOY, W. F., NC, M. H., AND LIM, P. L. 1990. Trichinellu spiralis: Light microscope monoclonal antibody localization and immunochemical characterization of phosphorylcholine and other antigens in the muscle larva. Experimental Parasitology 73, 172-183. A panel of monoclonal antibodies was used to examine the structure of the muscle larva of Trichinella spiral& under the light microscope. Immunofluorescence and, in some cases, immunoperoxidase staining were used. All four antibodies reacted with the cuticle of the organism, although differences in the staining pattern were observed for some of these. Interestingly, all the antibodies also reacted with the stichosome. One of the antibodies (Ts2Ab) is specific for the hapten, phosphorylcholine. In a binding assay, this antibody also reacted with extracts of Trichuris suis, Ascaris sum, and Fasciolopsis buski, but not with extracts derived from Cysticercus cellulosae, Candida albicans, Salmonella typhi, or Escherichia coli. This crossreactivity was confirmed microscopically in which the cuticle, oviduct and eggs of T. suis, the cuticle, muscle cells, and eggs of A. suum, and the cuticle and vitelline glands of F. buski were seen to be clearly stained by the antibody. In addition, Ts2Ab also reacted with the cuticle and stichosome of the adult T. spiralis worm. In Western blot analysis, Ts2Ab recognized a 43-kDa antigen from T. spiralis muscle larvae extracts, while a previously studied antibody (7C,C,Ab) identified four major antigens (48.5,47, 43, and 39 kDa) in this preparation. Similar results were obtained when the 24-hr excretory-secretory (ES) antigens of T. spiralis were immunoblotted with the antibodies, although the reactivity shown by Ts2Ab was relatively weak. With the 72-hr ES material, on the other hand, major antigens of lower mol wt (44, 28, and 25 kDa) were revealed by 7C,CsAb, and no reactivity was seen with Ts2Ab. However, this antigen preparation reacted well with both antibodies in an enzyme-linked immunoassay. Taken together, the findings suggest that the 72-hr ES antigens probably result from extensive degradation of material originally secreted or excreted by the worm. Similar binding studies on the 24-hr ES preparation indicated that this source may be relatively rich in 7C,C,Ab-reactive epitopes and relatively poor in the antigen identified by Ts2Ab. Other studies performed demonstrated that the antigens recognized by these two antibodies were distinct and physically unassociated. o IW Academic Press, 1”~. INDEX DESCRIPTORS AND ABBREVIATIONS: Trichinella spiralis; Nematode, parasitic; phosphorylcholine (PC); antibody (Ab); enzyme-linked immunosorbent assay (ELISA); excretory-secretory (ES); monoclonal antibody (mAb); phosphate-buffered saline (PBS); pneumococcal C antigen (PnC); phosphorylcholine-coupled human serum albumin (PCHSA); sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); Trisbuffered saline (TBS); whole worm extract (WWE). INTRODUCTION

Recent freeze-fracture studies on the muscle stage larva of Trichinella spiralis revealed an outermost layer of the cuticle which is subsequently lost in the life cycle of the nematode (Lee et al. 1984; Wright i To whom correspondence should be addressed.

and Hong 1988). This accessory layer, which is not a cell membrane, is beheved to comprise an outer component of globular proteins associated with an inner component of filaments. The latter contains various phospholipids including phosphatidylethanolamine and phosphatidvlcholine (Castro and Fairbairn 1969). A different approach based on immuno172

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T. spiralis:

LIGHT MICROSCOPIC mAb LOCALIZATION

cytochemical staining was also used to study the structure of the T. spirulis larva. Used with monoclonal antibodies (mAb) specific for the nematode, this approach has recently revealed interesting observations about its antigenic structure. Antibodies were found which reacted with the cuticle (Ortega-Pierres et al. 1984; Silberstein and Despommier, 1984; McLaren et al. 1987), some presumably with the globular proteins of the accessory layer. Interestingly, all these antibodies also stained the stichosome. In another study, Gamble and Graham (1984) observed that this organ was also stained by an antibody made to excretory-secretory (ES) antigens. However, the reactivity of this antibody with the cuticle was not mentioned. The relationship among antigens found in these anatomically different areas of the parasite is unclear, although McLaren et al. (1987) recently speculated that antigens originating in the stichosome could be excreted or secreted and subsequently absorbed onto the surface of the organism. The specificities of the antibodies used by the different groups of workers were unknown, however, nor were these compared with one another. It thus seems possible that some of the antibodies may have similar specificities, and consequently, the observation that the same larval antigens are widely distributed in different tissues of the parasite may be less general. Indeed, each of the antibodies used identified two to three antigens, a common size range being 45-57 kDa. We have extended the immunocytochemical studies of the previous investigators using our own set of T. spiralis-specific mAbs, including an antibody previously studied by Gamble and Graham (1984). Based on specificity, at least three types of antibodies were employed, including one which is specific for phosphorylcholine (PC). In addition, we examined the antigens recognized by two of the antibodies by immunochemical and antibody binding analyses.

MATERIALSAND

173 METHODS

Parasites T. spiralis muscle larvae were isolated from the diaphragms of Sprague-Dawley rats infected with the nematode 1 month previously. The tissue was digested with 1% pepsin-l% HCl at 37°C overnight, followed by filtration over a wire gauze. Adult worms were obtained from the intestine of rats orally administered with 3000 muscle larvae 1 week previously. Other helminths were obtained from naturally infected pigs at our local abattoir. Ascaris suum and Trichuris suis were recovered from the intestine, Cysticercus cellulosae from the skeletal muscle, and Fasciolopsis buski from the lungs of these animals.

Antigens Crude T. spirafis larval antigen (WWE) was obtained by sonication of the muscle larva (Choy ef al. 1988).Purified preparations were obtained from WWE by aflinity chromatography (Lim 1987) using Ts2Ab (Ts2Ag) or 7C,C,Ab (7C,C5Ag) as the reagent antibody (see below). In the purification, the reagent antibody was coupled to CNBR-activated Sepharose 4B (Sigma Chemical Co., St. Louis, MO), while the bound antigen was isolated by elution with 0.1 M glytine-NaOH buffer (pH 11.O)at room temperature. The elution material was dialyzed against phosphatebuffered saline pH 7.4 (PBS), concentrated with aquacide and redialyzed before clarification. A. suum antigen was obtained by homogenizing (Kinematica GmbH, Switzerland) small pieces of the worm in normal saline (kept ice cold), followed by treatment with 0.1% Triton X-100 overnight at 4°C and subsequent clarification at 8000 rpm for 15 min. Antigens of other helminths were similarly prepared as for T. spiralis. ES material was obtained as described by Gamble and Graham (1984). Briefly, 2 x 10’ muscle larvae were incubated in 50 ml Iscove’s modified Dulbecco’s medium (GIBCO, Grand Island, NY) at 37°C in a humid atmosphere of 5% CO, for 24 (ES24) or 72 ES72) hr. The medium was then separated from the larvae by centrifugation (3000 rpm/lS min) and concentrated to about 1 ml in an Amicon filtration unit. Antigen content was based on protein estimation. The preparation was stored in aliquots at - 20°C. Sonication was also used to prepare an extract of Can&da albicans grown on Sabouraud agar for 48 hr. The yeast cells were harvested in normal saline and treated at 65°C for 7 hr prior to the extraction. SalmoneNa typhi and Escherichia co/i lipopolysaccharide were purchased from DIFCO Laboratories (Detroit, Ml). Pneumococcal C-polysaccharide (PnC) was prepared from cell lysates of S. pneumoniae Type 3 (ATCC) according to the method of Sorensen ef a/. (1984).

174

CHOY,

NG,

Monoclonal Antibodies Murine mAbs specific for T. spiralis muscle larva were produced by somatic cell fusion (Lim and Ho 1983) from BALB/c mice which were orally infected with live larvae 6 months previously. One of the antibodies produced (TsZAb), an IgM protein specific for PC which binds to protein A of Staphylococcus aureus, has been described previously (Lim and Choy 1986; Choy et a/. 1988). Other antibodies (all IgM) produced from the same fusion are described here for the first time: Ts3Ab, Ts4Ab, and Tsl8Ab. In addition, an IgM antibody (7C,C,Ab) described previously by Gamble and Graham (1984) was obtained from the American Type Culture Collection (Rockville, MD). All the antibodies used in the cytochemical studies were obtained from spent culture supernatant following fractionation with 50% cold saturated ammonium sulfate. In other studies, Ts2Ab was purified from mouse ascites fluid by protein-A affinity chromatography (Lim and Choy, 1986).For use in immunoblotting, 7C,C,Ab was obtained from spent culture medium following fractionation with 50% cold saturated ammonium sulfate. When used for preparing an immunoabsorbent, it was cryoprecipitated from mouse ascites fluid.

Absorption Experiments (a) Absorption with TsdAb. T. spiralis WWE (23 pg) was incubated with afftnity-purified Ts2Ab (25 pg) or PBS (control) in a total vol of 250 J.LIin a mixer (Couher Electronic, Luton, Bedfordshire, UK) at 37°C for 1 hr. Protein A-sepharose 4B (Sigma Chemical Co.. St. Louis, MO) (150 ~1) was then added to the mixture and the incubation continued for another hour. The mixture was centrifuged (Micro Centaur, MSE, London) for 1 min and the supernatant obtained was used for subsequent examination. (b) Absorption with 7C,CsAb. T. spiralis, WWE (37 kg) was similarly absorbed with 7C,C,Ab-coupled Sepharose 4B (500 pl) or plain Sepharose 4B (Sigma) using the conditions described in (a). The antibody matrix was previously made by coupling a partially purified fraction (obtained by cryoprecipitation) of 7C,C,Ab with CN-Br activated Sepharose 4B (Sigma) (Lim, 1987).

Enzyme-linked immunosorbent assay (ELZSA) (a) Antibody speciJiciry test. The procedure described previously (Lim and Ho, 1983) was followed. Immulon-2 microtiter plates (Dynatech Laboratories, Alexandria, VA) coated with the parasite or microbial antigen (0.3-10 &ml) and blocked with bovine serum albumin (BSA) were incubated with dilutions of the test antibody. The assay was subsequently developed using peroxidase-labeled goat anti-mouse IgG (whole

AND

LIM

molecule) antibody (Tago Inc, Burlingame, CA). Absorbance readings greater than twice that of buffer control for the respective antigen were considered positive. Inhibition assays were performed by incubating the test antibody with the antigen in the presence of lo-’ M PC chloride (Sigma). (b) Antigen detection. Serial dilutions of the test supematant were used to coat microtiter plates. The assay was developed as in (a) using a standard dilution of Ts2Ab or 7C,C,Ab as the primary antibody, and peroxidase-labeled goat anti-mouse IgG (whole molecule) as the secondary antibody.

Zmmunocytochemical Studies (a) ImrnunoJluorescence stain. Frozen sections (6 pm thick) of the worm were prepared on slides, airdried, and fixed in acetone at -20°C for 5 min. The preparation was washed with PBS for 15 min and incubated with normal goat serum for 30 min at room temperature. The test antibody (50 pl of a 1:2 dilution) was then applied to the section and incubation was allowed for 30 min (room temperature). The slide was washed again three times with PBS, after which fluorescein-conjugated goat anti-mouse IgG (whole molecule) (Tago) used at a dilution of 1:50 was applied to the section. Following a 30-min incubation at room temperature, the slide was washed three times with PBS and then mounted in 60% (v/v) glycerin-PBS before being examined under the light microscope (Dialux 20, Leitz Weztlar, Germany). Control preparations were made exactly as described, using PBS instead of the test antibody. (b) Immunoperoxidase staining. Sections of worms prepared as in (a) were treated with freshly prepared 0.5% H,O, in absolute methanol for 30 min at room temperature. The slide was rinsed in distilled water TABLE I Specificity of Reagent Monoclonal Antibodies” Monoclonal antibody Microorganism Trichinella spiralis Trichuris suis Ascaris suum Cysricercus cellulosae Fasciolopsis buski Candida albicans Salmonella typhi Escherichia co/i

Ts2Ab

Ts3Ab

+ + +

+

-

ND ND

-

+ + + + +

-

+ -

&9

+

a Based on ELISA activities to crude antigen extracts of the microorganism: + , reactive; - , not reactive; ND, not done.

T. spiralis: LIGHT MICROSCOPIC mAb LOCALIZATION and washed twice with TBS for 15 min. The preparation was then incubated with normal goat serum 10 mitt and subsequently with the test antibody as in (a). Following washing three times with TBS, the preparation was incubated with peroxidase-labeled goat anti-mouse IgG (whole molecule) (Tago) for 30 min at room temperature. Following similar washing as before, substrate (3,3’-diaminobenzidine tetrahydrochloride (Sigma)) was added and reaction was allowed for 30 min. The slide was subsequently washed with TBS

175

and distilled water and then stained with hematoxylin for 7 min. The slide was washed again, air dried, dehydrated, and then mounted for examination.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) This was performed as previously described (Lim and Choy, 1986), using 10% polyacrylamide and 4% stacking gel. The samples (10 pg per lane) were pre-

FIG. 1. Immunofluorescence staining by antibody Ts2Ab of various helminths: adult T. spiralis (A), 2’. suis (B), A. suwz (C and D), F. buski (E), and C. celiulosae (F). Note that in (D) both fertilized (tilled) and unfertilized (empty) eggs are present, including the weakly stained protein coat which surrounds the eggs. Abbreviations: c, cuticle; o, oviduct; u, uterus; mc, muscle cell; vg, vitelline gland.

176

CHOY,

NG, AND

LIM

301’ FIG. 2. lmmunofluorescence staining of T. spiralis muscle larva by various antibodies: Ts2Ab (A and B), Ts4Ab (C), 7C,C5Ab (D), Ts3Ab (E), Tsl8Ab (F), and no antibody, i.e., control (G). Abbreviations: s, stichosome; e, esophagus; gp, genital primordium; others, as in the legend to Fig. 1.

pared in 0.125 M Tris-HCl buffer (pH 6.8) containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, and 0.002% bromphenol blue, and incubated at 37°C for 30 min before being loaded to the gel. After electrophoresis, the gel in some cases was stained for protein (Oakley ef al. 1980) and, following destaining, for carbohydrate (Tsai and Frasch 1982) as well.

Western Blot Analysis The unstained gel following SDS-PAGE was blotted onto nitrocellulose paper (Bio-Rad Laboratories, Richmond, CA) at 30 V overnight (10°C) using 25 mM Tris 192 mM glycine buffer (pH 8.3) containing 20% methanol. The nitrocellulose was then cut into OS-cm

T. SJkdiS: LIGHT MICROSCOPIC mAb LOCALIZATION

177

FIG. 3. Immunoperoxidase staining of T. spiralis muscle larva by antibody Ts2Ab (A), Ts3Ab (B), Tsl8Ab (C), and no antibody, i.e., control (D). Abbreviations as in the legend to Fig. 2. strips and incubated individually with PBS containing 5% BSA at 37°C for 1 hr. Following this, incubation with the test serum (3 ml per strip) was performed at 4°C overnight. The strips were washed three times with TBS containing 0.05% Nonidet-P40 (TBS-NP) for

10 min. Biotin-labeled goat anti-mouse IgM (Sigma) antibody was then added and overnight incubation at 4°C was allowed. The strips were washed three times again with TBS-NP, before peroxidase-labeled ExtrAvidin (Sigma) was added. Following overnight incuba-

178

CHOY,

NG,

AND

LIM

In general, all five antibodies stained both the cuticle and stichosome of the parasite, although the intensity and nature of staining, as well as the precise location Miscellaneous Analysis within these structures, were different for Protein estimation was performed according to some of the antibodies. Thus, whereas Lowry et al. (1951) and carbohydrate estimation ac7C2C5Ab reacted very strongly with the cording to Williams and Chase (1968). outermost layer of the cuticle, Ts2Ab RESULTS stained most strongly the lining of the stichosome adjacent to the hypodermis. A Specificity of Reagent mAbs very different pattern of cuticular staining All five antibodies bound to T. spirafis (“mottled”) was observed with Ts3Ab in larval extract in an ELISA, although the both fluorescent and enzymatic preparabinding of Ts2Ab and 7C,CSAb (detectable tions. The genital primordium of the organat 5 rig/ml of antibody) was at least lOO-fold ism was not stained by any of these antibetter than that of Ts3Ab, TsilAb, or bodies. Tsl8Ab. The binding of Ts2Ab could be inhibited by 10V3 M PC, but not that of the Zmmunochemical Analysis of Antigens Identified by 7C,Cdb and Ts2Ab others (data not shown). Ts2Ab also bound to the crude extracts The whole worm extract (WWE) of T. obtained from various microorganisms in spiralis (Fig. 4) was immunoblotted with an ELISA, including T. suis, A. suum, and the mAbs. Ts2Ab revealed a major antigen F. buski, but not C. cellulosae, C. albicans, of 43 kDa mol wt, and 7C,C,Ab four major S. typhi, or E. co/i (Table I). Another anti- antigens (48.5,47, 43, and 39 kDa) (Fig. 5). body (Ts3Ab) bound, surprisingly, to all of No reactivity, however, was observed with the antigens tested. In contrast, a control the other antibodies, Ts3Ab, Ts4Ab, and mAb specific for the Salmonella O-9antigen Tsl8Ab, when used even at 1.7- (Tsl8Ab) bound only to the homologous antigen. The or 15 (Ts3Ab and Ts4Ab)-fold higher antireactivity of Ts2Ab to helminths other than body concentrations (based on ELlSA acT. spirulis was confirmed by light micro- tivities) than that of 7C,C,Ab (data not scope immunofluorescence (Fig. 1). Thus, shown). The possibility that the antigens it stained the cuticle, oviduct, and eggs of recognized by these antibodies could be deT. suis and the cuticle, muscle cells (lining natured by the treatment used in SDSof), and eggs of A. suum. It also stained the PAGE was investigated by incubating the vitelline glands of F. buski which produce WWE antigen in the reducing buffer at 37°C the egg yolk and shell of the organism, as for 30 min and, following dialysis, using it in well as the cuticle. No reactivity was ob- an ELBA. However, the treated antigen served with C. cellulosae. In addition, the was bound as efficiently as the native antiantibody also reacted with the adult T. spi- gen by all these antibodies. Attempts at usrulis worm, staining both the cuticle and the ing another technique based on the immustichosome. noprecipitation of radiolabeled antigen also failed to reveal the antigens recognized by Zmmunocytochemical Studies on T. these antibodies (data not shown). spiralis Muscle Lurva Although the major antigen identified by T. spiralis larval sections were stained TBAb in the immunoblot appears to be the with the different mAbs and the results same as the 43-kDa antigen recognized by based on immunofluorescence (Fig. 2) or 7C&Ab, they differ in two respects. First, the reactivity of Ts2Ab in the immunoblot enzymatic reaction (Fig. 3) are shown.

tion at 4°C and subsequent washing (three times) in TBS-NP, the assay was developed with 4-chloro-lnaphthol (Sigma) for 10 min.

T. SpirafiS: LIGHT MICROSCOPIC mAb LOCALIZATION

581, 4 8.5 * 36.5

*

FIG. 4. SDS-PAGE analysis of whole worm extracts of T. spiralis (A). A. sum (B) and S. pneumoniae (C) stained for protein (1) or carbohydrates (2). Mol wt markers in kDa.

was inhibitable by lop3 M PC whereas that of 7C,C,Ab was not (Fig. 5). Second, the ELISA activity of either of the antibodies to the crude larval extract could be independently removed by prior absorption of the antigen with the respective antibody but not the heterologous antibody (Fig. 6). A corollary of this is that the antigen purified by either of these antibodies was bound only by that antibody (in an ELBA) but not the other antibody (see below, Table II). For comparison, the PC antigen present in A. suum and S. pneumoniae was identified by immunoblotting with Ts2Ab. Figure 4 shows the crude antigenic mixtures of the organisms used for the experiment, which appeared to be quite different from each other and from that of T. spiralis. Figure 5 shows that the PC antigens identified in A. suum (165, 66, and 62 kDa) are different

5. Antigens from WWE of T. spiralis (A), A. (B), and S. pneumoniae (C) separated as shown in Fig. 4 were immunoblotted with 7CzC,Ab (a) or Ts2Ab (b) in the presence (+) or absence (-) of 10m3 M PC. 7C,C5Ab (ELISA titer 1:25000) was used at 150 dilution and Ts2Ab at 12 &ml. MoI wt markers in kDa. FIG.

sum

from those found in S. pneumoniae (21 and 19.5 kDa) or T. spiralis (43 kDa). Since 7C,C,Ab was originally identified from its reactivity with ES antigens (Gamble and Graham, 1984), we examined this material for the presence of the antigens to 7C&Ab and TBAb. The material (ES24) obtained at 24 hr following in vitro culture of the muscle larvae showed patterns of reactivity in the Western blot similar to those of WWE developed with the respective antibody. However, the antigen of the highest mol wt (48.5 kDa) identified by 7C&Ab was less distinct in ES24 and the reaction of TQAb was extremely weak. In contrast, a different pattern of reactivity on ES72 (material obtained after 72 hr culture) was observed with 7C&Ab, which revealed ma-

180

CHOY, NG, AND LIM B

FIG. 6. Reciprocal absorption experiments to determine whether antibody Ts2Ab (A, A) and 7C,C,Ab (0, 0) bind to the same antigen in T. spiralis WWE. The crude antigen was absorbed with insolubilized Ts2Ab (A) or 7C,C,Ab (B) and then examined for residual ELISA activity with either antibody (open symbols). Control, unabsorbed antigen was similarly examined (closed symbols). (Experiments A and B were performed on separate occasions.)

jor antigens of 44, 28, and 25 kDa, and numerous minor antigens (e.g., 48.5, 46, 33, and 23.5 kDa). Virtually no reactivity (except for very faint bands at 38 and 36 kDa) was observed with Ts2Ab on this antigen preparation. The relative distribution of 7C,C,Ab- and Ts2Ab-reactive antigens in the ES preparations was also determined by direct binding analysis. As shown in Table II, based on the relative abundance of these antigens, it appears that ES72 is similar to WWE, whereas ES24 is significantly different. The latter seems to be rich in 7C,C,Ab-reactive epitopes and poor in those for Ts2Ab. It is

also apparent from this study that 7C2C,Ab and Ts2Ab recognize mutually distinct antigens. DISCUSSION

The present investigation extends similar studies done previously (Silberstein and Despommier 1984; Gamble and Graham 1984; McLaren et al. 1987) attempting to localize antigens in the T. spiralis muscle larva with the help of mAbs. At the level of resolution permissible with the light microscope, all the antibodies used in our studies stained both the cuticle and stichosome of the worm. However, the cuticular staining

TABLE II Comparison of the Relative ELISA Activities of Ts2Ab and 7C,C,Ab on Various Antigens” mAb TsZAb(a) 7C&Ab(b) a+b

WWE (6~)

‘JWYg (0.4PC)

ES24

ES72

UPI

UPI

Ts2Ag (0.3PC)

PnC (SC)

PC-HSA (3P)

12,000 3,198 3.752