EXPERIMENTALPARASITOLOGY
Trichinella
73, 1%)-160(1~1)
spiralis: A 76-kDa Excretory/Secretory Larval Antigen Identified by a Monoclonal Antibody
I.R. KEHAYOV,* S.D. KYURKCHIEV,* CH. V. TANKOV,? AND S. K.KOMANDAREW *Institute
of Immunology
and Kentral
Laboratory of Helminthology, Sofia, Bulgaria
Bulgarian
Academy
of Sciences,
R., KYURKCHIEV,% D., TANKOV, CH. V., AND KOMANDAREV,!~. K.1991. spiralis: A 76-kDa excretory/secretory larval antigen identified by a monoclonal antibody. Experimental Parasitology 73, 150-160. Spleen cells from BALB/c mice were fused with myeloma cells following infection of the mice with TrichineUa spiralis larvae and KEHAYOV,~.
Trichinella
an ip booster injection with larval homogenate antigen. A monoclonal antibody (Mab), designated as TS 3G6 which did not react with sera or tissue extracts from noninfected mice, rats, and guinea pigs, was selected for further studies because of its high activity and specificity. When tested in ELISA TS 3G6 did not cross-react with Ascaris suum, A. lumbricoides, Toxocara canis, E. granulosus (larvae), Trichiuris suis, or T. ovis. Western blot analysis showed that Mab 3G6 recognized an antigen of 76 kDa located in the stichosome of the larvae as well as on the surface of the larval cuticle. Digestion of a larval extract with different enzymes suggests that the Mab TS 3G6 corresponding epitope is a polypeptide. The TS 3G6 antigen was detected in culture supernatants of Trichinella muscle larvae and in sera of experimentally infected animals using a sensitive ELISA assay. This secretory antigen also seemed to induce a specific immune response in the host since sera from infected animals could block the binding of Mab TS 3G6 to its target antigen when tested in a competitive ELISA. 8 IW Academicpress,IK. INDEX DESCRIPTORS: Monoclonal antibody; Specific antigen; Shedding of antigen; Stichosome.
antigen from muscle larvae has also been described (Silberstein and Despommier Several workers have used monoclonal 1984). The corresponding antigen, after beantibodies (Mab) as tools for the antigenic ing purified by affinity chromatography, analysis of Trichinella spiralis and have can induce a signiticant protective immune used them to identify epitopes specific to response in mice, causing a statistically sigmorphological structures and to specific nificant reduction in larval numbers in chalstages of the nematode as well. Ortega- lenged animals. A similar phenomenon has Pierres et al. (1984a) have described mono- been observed by Gamble (1985), while clonal antibodies which recognize surface studying the immunological and biological antigens of the different stages-newborn activity of the excretory/secretory antigen larvae, muscle larvae, and adult worms. of 45 and 53 kDa isolated by monoclonal They have partially characterized the cor- antibody 7C2C5. Further studies have responding antigens and determined their shown that immunization with the excrelocalization using sensitive immunohistotory/secretory antigen caused expulsion of chemical techniques (McLaren et al. 1987). adult nematodes and a decreased fecundity It has been reported that anti-cuticular an- of females. tibodies are destructive to newborn larvae, In this paper data are presented on the causing a significant inhibition of infectivity production of a monoclonal antibody (Mab of larvae (Ortega-Pierres et al. 1984b). A TS 3G6) against stichocytes of muscle larmonoclonal antibody against a stichosome vae and partial characteristics of the correINTRODUCTION
150 0014-4894/91$3.00 Copyright0 1991by AcademicPress.Inc. All rightsof reproductionin any form reserved.
ANTIGEN
OF
T. S&+YdiSMUSCLE LARVAE
sponding antigen. Our research is focused toward the development of specific immunodiagnostic tests. MATERIALS
ANDMETHODS
Experimental animals. Male and female BALBlc and C3H mice, Wistar rats, and outbred guinea pigs free of T. spiralis infection were supplied from the breeding farm of the Bulgarian Academy of Sciences. Nematodes. T. spiralis was serially passaged in female BALB/c mice. Muscle larvae were recovered by digestion with 1% pepsin-HCl for 120 min at 37”C, followed by filtration through two layers of gauze. The larvae were washed in saline at 37°C and resuspended in serum-free medium RPM1 1640 (Flow Labs., UK). T. nelsoni, T. nativa, and T. pseudospiralis as well as Ascaris suum, A. lumbricoides, Toxocara canis, Echinococcus granulosus (larvae), Trichiuris ovis, and T. suis were used to test the specificity of the monoclonal antibodies. All variants of T. spiralis as well as other nematodes were supplied by the Central Laboratory of Helminthology, Bulgarian Academy of Sciences. Antigens. Crude extracts from muscle larvae were prepared by homogenization of washed larvae in a glass Potter-Elvehjem homogenizer. This suspension was frozen (at -20°C) and thawed several times and homogenized again. After centrifugation at 10,000rpm for 60 min, the supematant was passed through a 0.22urn filter (Millipore Ltd.) and stored at - 20°C. Protein content was determined by the standard Lowry method. Excretory/secretory antigens were obtained by culturing living muscle larvae in RPM1 1640medium supplemented with 10% fetal bovine serum (Flow Labs.) and antibiotics at 37°C and 5% CO,. Samples from the culture medium were collected on Days 1, 3, 5, and 7. After centrifugation at 1000rpm for 30 min the supernatants were filtered and stored at -20°C. Generation of hybridomas. BALB/c mice were infected orally with 200 living T. spiralis larvae, and 20 days later the mice were boosted ip with 200 ug of a crude larval extract. Cell fusion was carried out following the well-established procedure in our laboratory. Briefly, splenocytes from infected mice were fused with P3x63.Ag8.653 myeloma cells with 40% polyethylene glycol 1500 (Sigma Co.) for 8 min. Cells were washed at 1000 rpm for 10 min, resuspended in hypoxanthineaminopterine-thymidine-supplemented RPM1 1640 with 10% fetal calf serum, and distributed in %-well microtiter plates (Costar Ltd.). Supematants from wells with growing hybrids were screened by ELISA against the total extract from muscle larvae. Highly reactive hybridomas were cloned by limiting
151
dilution. Selected clones were grown in mass culture and frozen and stored in liquid nitrogen. Enzyme-linked immunosorbent assay. Microtiter 96 well plates (Costar Ltd.) were coated with 100 &ml total larval extract in carbonate buffer, pH 9.6, at 4°C and then blocked with 10% inactivated calf serum in 0.1 M Tris-HCl, pH 8.5, containing 0.5 M NaCl for 1 hr at room temperature (RT). After washing in phosphate-buffered saline (PBS) (pH 7.4) containing 0.05% Tween 20 (T-PBS), 50 pl of hybridoma supematants was added to each well for 2 hr at RT. Plates were washed in T-PBS and 50 (~1swine anti-mouse IgG serum conjugated with horseradish peroxidase (RIIPD, Sofia, Bulgaria) was added and incubated for 1 hr at RT. After extensive washing in T-PBS bound peroxidase was developed with 50 ul o-phenylendiamine (Sigma Co.) in citrate buffer, pH 5.0 (2 mg/lO ml buffer with 0.03% H,O,). The enzyme reaction was stopped after 5 min with 50 )LI 10% sulfuric acid and the results were read with a Micro-ELISA reader (Dynatech GmBh, Switzerland) at 492 nm. The presence of specific antigens in culture supernatants of in vitro-cultured larvae or in sera of infected animals was tested by an absorption ELISA. Basically, the procedure was as described above but the monoclonal antibody was preabsorbed with an equal volume of larvae supematant or serum for I hr at 37°C and then added to wells coated with the total Trichinella extract. To test sera from infected animals (60-90 days after infection) for the presence of anti-Trichinella antibodies, antigen-coated plates were treated with serial dilutions of sera from infected animals for 2 hr at RT. After washing three times for 5 min each in T-PBS, the monoclonal antibody TS 3G6 was added and the reaction proceeded as described above. Negative controls were unrelated culture medium samples, and sera from parasite-free animals were used as negative controls. The biochemical nature of the epitope recognized by Mab TS 3G6 was determined after treatment with different enzymes. The larval extract (100 kg/ml) coated on PVC microtiter plates was treated with trypsin, proteinase, pronase E, and subtilysin at a concentration of 1 mg/ml for 12 hr at 37°C. The wells were extensively washed with T-PBS and were processed as described above. All enzymes were purchased from Serva Fein B&hem., Germany. Periodic acid at different molar concentrations (from 5 to 100 rm+4)was used to test its effect on the antigenicity of the larval extract. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). SDS-PAGE under reducing con-
ditions was carried out as described by Laemmli (1970) using a 5% stacking gel and a 10% running gel. Electrophoretic conditions were 60 V while the sample ran through the stacking gel and then 100 V for about 120 min in 25 mM Tris, 192 mh4 glycine, pH 8.3, contain-
152
KEHAYOV ETAL.
ing 0.1% SDS. Molecular markers thyroglobulin (330 kDa), ferritin (220 kDa), albumin (67 kDa), catalase (60 kDa), lactate dehydrogenase (36 kDa), and ferritin (18.5 kDa) were applied. At the end of the run, gel slabs were either used for blotting or staining with 0.25% Coomassie blue. Western
blotting
and immunoenzyme
detection.
Electrotransfer of proteins from the SDS gel to nitrocellulose was done using a Transblot cell (BioRad) and Tris-glycine buffer (pH 8.3) containing 20% methanol. For detection of the specific antigens, nitrocellulose strips were blocked overnight at 4°C using 10% inactivated calf serum in 0.1 M Tris-HCI buffer, pH 8.5, containing 0.5 M NaCl, washed in PBS, and incubated overnight at 4°C in the monoclonal antibody. After washing in PBS bound antibody was visualized using biotin-conjugated anti-mouse IgG serum and the avidin-peroxidase conjugate (Sigma Co.). The enzyme reaction was developed by incubation in I-chloro-4naphthol (Sigma Co.). An irrelevant mouse monoclonal antibody of the same isotype was used to control the specificity of the test system. indirect immuno$7uorescence. Paraffin-embedded tissue sections from infected animals were passed through xylene and a series of alcohols and rehydrated briefly in PBS, pH 7.4. (McLaren et al. 1987). Tissue sections were incubated in the monoclonal antibody or in suitable control supernatants for 60 min at RT. After washing in PBS, pH 7.4, containing 0.1% w/v bovine serum albumin (BSA) the sections were covered with FITC-conjugated anti-mouse IgG serum, 0.1 mglml (RIIPD, Sofia, Bulgaria), and incubated for 60 min. After washing in PBS-BSA the sections were mounted in SO%buffered glycerol and examined using a Laborlux K fluorescent microscope (Leitz, Austria). Indirect immunoperoxidase technique. Rehydrated tissue sections were incubated with the monoclonal antibody or control supernatants for 60 min at RT. The sections were thoroughly washed in PBS-BSA and transferred into a l/50 dilution of swine anti-mouse IgG serum conjugated with horseradish peroxidase (RIIPD) for 60 min. After further washing the slides were incubated in substrate solution (5 mg 3,3’-diaminobenzidine in 10 ml Tris-HCI buffer, pH 7.2, with 0.01% H,O,). The sections were then dehydrated and mounted in oil for light microscopy.
cells. Hybrid cells were found in 134 out of 288 wells and specific antibodies against the Trichinella extract were detected in six supernatants. Cells from these wells were cloned at least three times by limiting dilution and a hybridoma (TS 3G6) was selected for further study. The supernatant from the TS 3G6 hybridoma was tested against sera and extracts from striated muscle from rodents, pigs, and humans by ELISA, and in each case the reactions were completely negative. A similar approach was used to test the same supernatant for cross-reactivity against A. suum, A. lumbricoides, E. granulosus, T. canis, T. suis, and T. ovis. The results presented in Table I show that Mab TS 3G6 recognizes an epitope specific to T. spiralis and that it is not shared with the other nematodes tested. However, when tested against different Trichinella species, Mab TS 3G6 gave positive reactions with similar OD values with extracts from all species tested (T. spiralis, T. nelsoni, T. nativa, and T. pseudospiralis) (Table II). Characterization of the antigen recognized by Mab TS 3G6. When the total ex-
tract from muscle larvae or the extract from noninfected muscle was subjected to SDSPAGE under reducing conditions and stained with 0.025% Coomassie blue, a number of protein bands with a different molecular mass (from 300 to 12 kDa) were seen (Figs. lb and lc). In the larval extract, TABLE I Specificity of Monoclonal Antibody 3G6 against T. spiralis Tested in ELISA Nematode species
RESULTS
Generation and characterization of monoclonal antibodies. High titers of antiTrichinella antibodies were detected by
ELISA 4 days after infected mice were given a booster injection of larval antigen, and accordingly, splenocytes were taken for fusion with P3x63.Ag8.653 myeloma
T. T. A. A. E. T. T.
spiralis canis lumbricoides suum granulosum ovis suis
Mab 3G6 1.012” 0.090 0.045 0.080 0.058 0.045 0.03 1
0 OD492 values in ELISA from three independent assays.
ANTIGEN
OF
T. spiralis MUSCLE
TABLE II Cross-Reactions of Mab TS 3G6 against Different Trichinella Species Trichinella T. T. T. T.
species
spiraiis nelsoni nativa pseudospiralis
Mab TS 3G6 0.988” 0.860 0.790 0.810
a OD492 values in ELISA.
two protein bands in the region of 35-40 kDa are seen which are not present in the extract of noninfected muscle. After transfer to nitrocellulose and treatment with Mab TS 3G6, three bands were specifically stained, with the major band stained having a molecular weight of 76 kDa. There are two additional bands which are less intensively but, still, specifically stained with MW of 41 and 36 kDa, respectively (Fig. la). When the immobilized larval extract was digested with different enzymes the reactivity of Mab TS 3G6 with its corresponding epitope was significantly reduced after treatment with proteases (Table III). The
153
LARVAE
TABLE III Effect of Treatment with Different Enzymes on the Reactivity of Mab TS 3G6 Larval extract treated with PBS Trypsin Proteinase Subtilysin Pronase E Periodic acid (5-25 mk4)
Mab TS 3G6 0.854 k 0.146 k 0.050 2 0.032 + 0.031 ? 0.870 5
0.089” 0.031 0.015 0.012 0.016 0.046
a OD492 in ELISA from three independent experiments.
protein nature of the epitope was confirmed by the fact that oxidation with increasing concentrations of periodic acid did not change the antigenicity of the larval extract. Cellular localization of the TS 3G6 antigen was determined by indirect immunoperoxidase and immunofluorescence using serum from an infected mouse as a positive control (Fig. 2a). In the immunoperoxidase reaction the serum stained striated muscle fibers light yellow and muscle larvae much more intensively (yellow to dark brown). The parasite was uniformly stained and a localization of the reaction product could not be specified. In contrast, Mab TS 3G6 stained quite intensively the stichosome only. Specific binding to the stichosomes was clearly seen in both longitudinal and 76 kD cross sections of the larvae (Fig. 2b). Dark deposits of the reaction product in the cytoplasm of nurse cells should be noted. A typical finding is illustrated in Fig. 2b. The 41 kD nuclei of the nurse cells seem to be un36kD stained as do the inflammatory cells (Fig. 2b, lower left corner of nurse cell). The cuticle adjacent to the stichosome was also intensively stained. This specific staining was most clearly seen at a higher magnification (Fig. 2~). There was no staining of FIG. 1. SDS-PAGE electrophoresis and electrothe esophagus, intestine, or genital ruditransfer of Trichinella extract (lane a,b) and extract ment of the larvae. No positive reaction from noninfected muscle (c). Lane a is stained with Mab TS 3G6; lanes b and c are stained with Coomassie was seen on the parasite capsule or the surrounding muscle fibers. blue.
154
KEHAYOV
b
ET AL.
ANTIGEN
OF
T. Spiralis MUSCLE
LARVAE
The immunocytochemical findings were confirmed by the indirect immunofluorescence technique. When Mab TS 3G6 was used as the first layer and FITC anti-mouse IgG serum as the second, intensive bright green fluorescence was seen on the surface of the stichosome and the outer cuticular layer only (Figs. 3a-3c). The irrelevant monoclonal antibody from the same immunoglobulin isotype applied as the first antibody gave a completely negative reaction (Fig. 3d).
155
rectly in ELISA. However, when Mab TS 3G6 was preabsorbed with serum samples and then applied to Trichinella-coated plates in an absorption ELISA, evidence was obtained for the presence of the specific antigen, the percentage of the activity absorbed rising from 13.50% on Day 3 to a maximum of 3uO% on Day 5 (Fig. 5). The fact that the TS 3G6 antigen was localized in the stichosome and was secreted by muscle larvae made it likely that this antigen would induce a humoral immune response against T. spiralis. If that was the case, Mab Immunogenicity of the antigen recog3G6 might be used for development of an nized by Mab XX. To test the possibility that the antigen recognized by Mab TS 3G6 immunoenzyme test for diagnosis of the inwas an excretory/secretory component, its fection. To test this assumption, sera from shedding by in vitro-cultured muscle larvae guinea pigs taken 60 days after the infection was followed. Trichinella larvae isolated, and from rats taken 90 days after infection antibodtransferred into RPM1 1640 medium with were screened for anti-Trichinella 10% FCS, and samples of culture medium ies by a competitive ELISA. All sera tested were collected on Days 1,3, 5, and 7. Using could block the binding of Mab TS 3G6 to competitive ELBA, the TS 3G6 antigen its target antigen. A representative dosewas detected in the culture supematant as dependent curve of the blocking effect is early as the first day of culture. A represen- shown in Fig. 6, where it can be seen that tative curve formed when Mab TS 3G6 was OD values increase with the dilution of the preabsorbed with culture medium is shown sera tested. in Fig. 4, where it was demonstrated that DISCUSSION 120 pg/ml total protein absorbed about 50% Elucidation of the complicated immunoof the activity of Mab TS 3G6 in ELISA. Since the TS 3G6 antigen was secreted logical interrelations between parasitic by in vitro-cultured larvae, we reasoned nematodes and their hosts is not possible that it might be present in the serum of in- without precise data on the morphologic fected animal. This assumption was tested. and cellular localization of the parasite anRats and guinea pigs were infected orally tigens and particularly on their structure with 2000 larvae and blood samples were and expression. T. spiralis provides a good collected on Days 3,4, 5, 6, and 7 after the model for such studies and several investiinfection. No antigen was detected in any gators have described antigens which can of the samples when they were tested di- induce specific humoral or cell-mediated
FIG. 2. (a) Section of paraffin-embedded muscle from a guinea pig infected with T. spiralis. Immunoperoxidase staining was done with serum from a BALB/c mouse immunized against T. spiralis. The reaction product is uniformly layered on the whole larval surface and completely tills the capsular space. Striated muscle tissue is also stained (magnification X205). (b) Indirect immunoperoxidase technique with Mab TS 3G6 and anti-mouse IgG serum conjugated with peroxidase. Intensive specific staining in the stichosome is seen while the parasite capsule and striated muscles are completely negative (magnification x410). (c) Immunoperoxidase technique with Mab TS 3G6. The stichosome is intensively stained and a positive reaction is seen on the cuticle adjacent to the stichosome (magnification X410).
156
KEHAYOV
ET AL.
FIG. 3. (a,b) Immunofluorescent staining with Mab TS 3G6 on paraffin-embedded sections of muscle from guinea pig infected with T. spiralis. Bright green fluorescence is seen in the cells of the stichosome (magnification x 125; x2.50). (c) Transversal section of T. spiralis larvae. There is bright staining of the stichosome and of the cuticular surface layer (magnification x500). (d) A negative control section treated with an irrelevant monoclonal antibody from the same immunoglobulin class as Mab TS 3G6 (magnification x500).
imnnune responses (Komandarev 1978; DUI.ham el al. 1984) or which can stimulate pro1tective immunity. In this respect attention I has been focused on antigens located on 1the cuticle (Grencis et al. 1986; OrtegaPie1-res 1984a) or present in excretoryke-
cretory products of muscle larva (Silt stein and Despommier 1985). The studies presented in this paper w aimed at identification of antigens relea sed by T. spiralis which might be valuable for immunodiagnosis or for experimental stud-
ANTIGEN
OF
T. SpiraliS MUSCLE
LARVAE
1.57
FIG. 3-Continued.
ies. A hybridoma was constructed (TS 3G6) secreting a monoclonal antibody that reacted against all four Trichinella species (T. spiralis, T. nelsoni, T. nativa, and T. pseudospiralis) but did not cross-react with
other helminths. It was established by immunoenzyme and immunofluorescent techniques that the major deposition of the reaction products with this monoclonal antibody was in the stichosome, with the
158
KEHAYOV
ET AL.
4
A 1.100. 1.000
0.600
0.800
0.700
0.600-
: d 0
N
0.600
0.700
nz
0.500
0.600~
0
0.400
0.500
4
c\
0
0
0.300 0.200 0
0.100
7/* 0
undiluted
I:10
1:50
sera
total
protein
(gg/mO
FIG. 4. A representative curve of ELBA with Mab TS 3G6 preabsorbed with serial dilutions of T. spirulis larvae supematant after 18 hr in culture (0) or just culture medium (A). Plates have been coated with T. spiralis extract.
stichocytes being intensively stained. The presence of positive staining in the cytoplasm of nurse cells (Figs. 2b and 2c) probably shows that at least one of the excretory/secretory antigens (76,41, and 36 kDa) is related to some tissue component of the host. Still, another possibility is that some of the secreted antigen has been digested by the nurse cell. The intensity of the reaction did not allow discrimination between (Y-or P-stichocytes. The cellular distribution of 1.2% 3
40% 26.
6%
/
i 4
I! 5
Days
after
6
infection
FIG. 5. Presence of TS 3G6 antigen in sera of T. spiral&-infected animals as detected by an absorption ELBA with Mab TS 3G6. Hatched bars show the percentage of Mab TS 3G6 reactivity absorbed by sera of infected animals at different days after infection.
1:100
1:200
1:400
dilution
FIG. 6. A representative curve of blocking experiments. Plates coated with T. spiralis were treated with serial dilutions of sera from animals (0) or from healthy donors (A) and acted with Mab TS 3G6.
ELBA extract infected then re-
the antigen identified in our experiments is quite similar to that described by Silberstein and Despommier (1984) and by Gamble (1985). Recently, McLaren et al. (1987) have determined the location of the antigens recognized by the NIM-Ml and NIMM2 antibodies which stained the epicuticle, stichocytes, and the surface layer of the intestine of muscle larvae. It was also shown in these investigations that the antigens located in the stichosome of the larvae were released in excretory/secretory products. Quite similar results were obtained in our laboratory with Mab TS 3G6. When Mab TS 3G6 was absorbed with increasing concentrations of larvae culture medium and tested in ELISA, it was established that the corresponding antigen was secreted in the medium (Fig. 4). Based on this finding it was assumed that this excretory/secretory antigen might be shed in the circulation of naturally infected animals. Confirmation of this assumption was provided by detection of circulating antigen in guinea pigs and rats heavily infected with Trichinella larvae. Although the OD values were not high, sera from infected animals absorbed the activity of Mab TS 3G6 against the crude extract from muscle larvae (Fig. 5). The experimental design used for detection of circulating Trichinelfa antigens in our laboratory
ANTIGEN
OF
T. Spirdis MUSCLE
was different from that of Gamble and Graham (1984) who did not find secretory antigens in the sera of 2-month-old piglets infected with 500 larvae of T. spiralis. The infective dose used in our experiments was significantly higher (2000 larvae per animal) and the circulating antigen was detected from Day 3 after infection. These early stages of infection (Days 3-7) were chosen to avoid elimination of any circulating antigens as a result of an immune response. Mab TS 3G6 is similar to 7C2CS (Gamble 1985), 7B2.3 (Silberstein and Despommier 1984), and NIM-Ml (McLaren et al. 1987) as far as its immunohistochemical reactions and the excretory/secretory nature of the corresponding antigen are concerned. However, the basic difference between Mab TS 3G6 and the other monoclonal antibodies described so far is that Mab TS 3G6 reacts with a 76-kDa protein as shown by immunoblotting (Fig. 1). The epitope recognized specifically by Mab TS 3G6 seems to be of a protein nature since it is sensitive to protease digestion and is resistant to oxidation with periodic acid (Table III). This antibody stains two additional bands of approximately 41 and 36 kDa, respectively. There are at least two alternative explanations of this finding. One possibility is that Mab TS 3G6 reacts with a protein epitope that is detected on three different polypeptides with IV, 76, 41, and 36 kDa. Another possibility is that Mab TS 3G6 recognizes a 76-kDa antigen and that the other two bands represent degradation products or post-translational moditications of the 76-kDa molecule. Detailed immunochemical studies are in progress to distinguish between these alternatives. It should be stressed, however, that the antigen recognized by Mab TS 3G6 is quite different in its molecular weight from the antigens deother Trichinella-specific scribed so far: 7C2C5 (45, 49, 53 kDa), 7B2.3.1. (O/55 kDa) NIM-Ml (105, 90, 57, 47 kDa), or NIM-M2 (90, 47 kDa). The antigen recognized by Mab TS 3G6
LARVAE
159
is located in the stichosome and in the outer layer of the cuticle, particularly on the portion adjacent to the stichosome (Figs. 2 and 3). This specific localization is difficult to reconcile with the suggestion of McLaren et al. (1987) that the sources of the excretory/secretory antigens are the stichocytes and that the antigens are incorporated in the cuticle after their passage through the esophagus and intestines and their release through the mouth and anus to the perilarval space of the capsule. It seems more likely, as also suggested by these authors, that the antigens are actively synthesized by the cells of the hypodermis or bacillary band cells, which show antigenic determinants common to the antigens of the stichocytes. It is of course quite possible that the antigen is directly transferred from the stichocytes toward the cuticle by a different but presently unknown mechanism. The data reported here suggest that Mab TS 3G6 could be used in diagnostic tests for a semiquantative determination of antiTrichinella antibodies in the sera of infected pigs or patients. Gamble et al. (1983) have already developed a competitive ELISA with monoclonal antibody 7C2C5 against excretory/secretory antigens (45, 49, 53 kDa) for the immunodiagnosis of T. spiralis in experimentally infected animals. This method has demonstrated a high efficiency in the detection of specific anti-Trichinella antibodies and there has not been a single false positive result with sera from pigs infected with Trichuris suis. In our experiments using a competitive ELISA and a crude Trichinella extract or a purified Trichinella antigen (Difco Lab.), all sera from infected animals blocked the reaction of Mab TS 3G6 with a solid-phase bound Trichinella antigen (Fig. 6). At a dilution of l/100 the blocking effect was about 50%. Tests of sera from patients infected after consumption of infected meat are now in progress and some preliminary data show that this test has considerable potential for immunodiagnosis of human trichinellosis.
KEHAYOVETAL.
160 REFERENCES
ALMOND, N. M., MCLAREN, D. I., AND PARKHOUSE, R. M. E. 1986. A comparison of the surface and secretions of Trichinella pseudospiralis and T. spiralis. Immunology 93, 163-116. BOEV, S. N., BRITOV, V. A., AND ORLOV, I. V. 1979. Species composition of Trichinellae. Wiadomosci Parazitologiczne 25, 495-503. DURHAM, C. P., MURREL, K. D., AND LEE, C. M. 1984. Trichinella spiralis: Immunization of rats with an antigen fraction enciched for allergenicity. Experimental Parasitology 57, 297-306. GAMBLE, H. R. 1985. Trichinella spiralis: Immunization of mice using monoclonal antibody affmityisolated antigens. Experimental Parasitology 59, 398-404. GAMBLE, H. R., ANDERSON, W. R., GRAHAM, C. E., AND MURRELL, K. D. 1983. Diagnosis of swine trichinosis by enzyme linked immunosorbent assay (ELISA) using an excretory/secretory antigen. Veferinary Parasitology 13, 349-361. GAMBLE, H. R., AND GRAHAM, C. E. 1984. Comparison of monoclonal antibody-based competitive and indirect enzymelinked immunosorbent assays for the diagnosis of swine trichinosis. Veterinary Immunology and Immunopathology 6, 379-389. GRENSIS, R. K., CRAUFORD, C., PRITCHARD, D. I., BEHNKE, I. M., AND WAKELIN, D. 1986. Immunization of mice with surface antigens from the muscle larvae of Trichinella spiralis. Parasite Immunology 8, 587-596. KOMANDAREV, S. 1978. “Immunity in Trichinellosis.” Sofia, BAS. KOMANDAREV, S., MIHOV, L., AND HOVORKA, I. 1986a. Immunological studies on different Trichinella species. I. Antigenic characteristics by means of linear immunoelectrophoresis. Helminthology 21, 13-19. KOMANDAREV, S., MIHOV, L., AND HOVORKA, I. 1986b. Immunological studies on different Trichinella species. II. Antigenic characteristics by means of
two dimensional immunoelectrophoresis. Helminrhology 21, 20-26. KRAMAR, M., STEWARD, G. L., AND CHARNIGA, L. 1981. A comparative study of Trichinella spiralis (Owen, 1835) and Trichinella pseudospiralis (Garkavi, 1972). Journal of Parasitology 67, 91 l916. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the cesserubly of the head of bacteriophage T4. Nature 227, 680-685. LICHTENFELS, J. R., MURRELL, K. D., AND PILITT, P. A. 1983. Comparison of three subspecies of Trichinella spiralis by scanning electron microscopy. Parasitology 69, 1131-l 140. MARTINEZ-FERNANDEZ, A. R., AND SANMARTINDURAN, K. L. 1981. Some difference in the biological behaviour of various sibling species of Trichinella. In “Proc. of the V Intern. Conference of Trichinella” (C. W. Kim et al., Eds.), pp. 35-39. MCLAREN, D. I., ORTEGA-PIERRES, G., AND PARKHOUSE, R. M. E. 1987. Trichinella spiralis: Immunocytochemical localization of surface and intracellular antigens using monoclonal antibody probes. Parasifology 94, 101-l 14. ORTEGA-PIERRES, G., CHAYEN, A., CLARK, N. T., AND PARKHOUSE, R. M. E. 1984a. The occurrence of antibodies to hidden and exposed determinants of surface antigens of Trichinella spiralis. Parasitology 83, 359-369. ORTEGA-PIERRES, G., MACKENZIE, C. D., AND PARKHOUSE, R. M. E. 1984b. Protection against Trichinella spiralis induced by a monoclonal antibody that promotes killing on newborn larvae by granulocytes. Parasife Immunology 6, 275-284. SILBERSTEIN, D. S., AND DESPOMMIER, D. D. 1984. Antigens from Trichinella spiralis that induce a protective response in the mouse. Journal oflmmunology 132(2), 898-904. SILBERSTEIN, D. S., AND DESPOMMIER, D. D. 1985. Effect of Trichinella spiralis of host responses to purified antigens. Science 221, 948-950. Received 2 July 1990; accepted with revision 1991
19 March
Trichinella
73, 1%)-160(1~1)
spiralis: A 76-kDa Excretory/Secretory Larval Antigen Identified by a Monoclonal Antibody
I.R. KEHAYOV,* S.D. KYURKCHIEV,* CH. V. TANKOV,? AND S. K.KOMANDAREW *Institute
of Immunology
and Kentral
Laboratory of Helminthology, Sofia, Bulgaria
Bulgarian
Academy
of Sciences,
R., KYURKCHIEV,% D., TANKOV, CH. V., AND KOMANDAREV,!~. K.1991. spiralis: A 76-kDa excretory/secretory larval antigen identified by a monoclonal antibody. Experimental Parasitology 73, 150-160. Spleen cells from BALB/c mice were fused with myeloma cells following infection of the mice with TrichineUa spiralis larvae and KEHAYOV,~.
Trichinella
an ip booster injection with larval homogenate antigen. A monoclonal antibody (Mab), designated as TS 3G6 which did not react with sera or tissue extracts from noninfected mice, rats, and guinea pigs, was selected for further studies because of its high activity and specificity. When tested in ELISA TS 3G6 did not cross-react with Ascaris suum, A. lumbricoides, Toxocara canis, E. granulosus (larvae), Trichiuris suis, or T. ovis. Western blot analysis showed that Mab 3G6 recognized an antigen of 76 kDa located in the stichosome of the larvae as well as on the surface of the larval cuticle. Digestion of a larval extract with different enzymes suggests that the Mab TS 3G6 corresponding epitope is a polypeptide. The TS 3G6 antigen was detected in culture supernatants of Trichinella muscle larvae and in sera of experimentally infected animals using a sensitive ELISA assay. This secretory antigen also seemed to induce a specific immune response in the host since sera from infected animals could block the binding of Mab TS 3G6 to its target antigen when tested in a competitive ELISA. 8 IW Academicpress,IK. INDEX DESCRIPTORS: Monoclonal antibody; Specific antigen; Shedding of antigen; Stichosome.
antigen from muscle larvae has also been described (Silberstein and Despommier Several workers have used monoclonal 1984). The corresponding antigen, after beantibodies (Mab) as tools for the antigenic ing purified by affinity chromatography, analysis of Trichinella spiralis and have can induce a signiticant protective immune used them to identify epitopes specific to response in mice, causing a statistically sigmorphological structures and to specific nificant reduction in larval numbers in chalstages of the nematode as well. Ortega- lenged animals. A similar phenomenon has Pierres et al. (1984a) have described mono- been observed by Gamble (1985), while clonal antibodies which recognize surface studying the immunological and biological antigens of the different stages-newborn activity of the excretory/secretory antigen larvae, muscle larvae, and adult worms. of 45 and 53 kDa isolated by monoclonal They have partially characterized the cor- antibody 7C2C5. Further studies have responding antigens and determined their shown that immunization with the excrelocalization using sensitive immunohistotory/secretory antigen caused expulsion of chemical techniques (McLaren et al. 1987). adult nematodes and a decreased fecundity It has been reported that anti-cuticular an- of females. tibodies are destructive to newborn larvae, In this paper data are presented on the causing a significant inhibition of infectivity production of a monoclonal antibody (Mab of larvae (Ortega-Pierres et al. 1984b). A TS 3G6) against stichocytes of muscle larmonoclonal antibody against a stichosome vae and partial characteristics of the correINTRODUCTION
150 0014-4894/91$3.00 Copyright0 1991by AcademicPress.Inc. All rightsof reproductionin any form reserved.
ANTIGEN
OF
T. S&+YdiSMUSCLE LARVAE
sponding antigen. Our research is focused toward the development of specific immunodiagnostic tests. MATERIALS
ANDMETHODS
Experimental animals. Male and female BALBlc and C3H mice, Wistar rats, and outbred guinea pigs free of T. spiralis infection were supplied from the breeding farm of the Bulgarian Academy of Sciences. Nematodes. T. spiralis was serially passaged in female BALB/c mice. Muscle larvae were recovered by digestion with 1% pepsin-HCl for 120 min at 37”C, followed by filtration through two layers of gauze. The larvae were washed in saline at 37°C and resuspended in serum-free medium RPM1 1640 (Flow Labs., UK). T. nelsoni, T. nativa, and T. pseudospiralis as well as Ascaris suum, A. lumbricoides, Toxocara canis, Echinococcus granulosus (larvae), Trichiuris ovis, and T. suis were used to test the specificity of the monoclonal antibodies. All variants of T. spiralis as well as other nematodes were supplied by the Central Laboratory of Helminthology, Bulgarian Academy of Sciences. Antigens. Crude extracts from muscle larvae were prepared by homogenization of washed larvae in a glass Potter-Elvehjem homogenizer. This suspension was frozen (at -20°C) and thawed several times and homogenized again. After centrifugation at 10,000rpm for 60 min, the supematant was passed through a 0.22urn filter (Millipore Ltd.) and stored at - 20°C. Protein content was determined by the standard Lowry method. Excretory/secretory antigens were obtained by culturing living muscle larvae in RPM1 1640medium supplemented with 10% fetal bovine serum (Flow Labs.) and antibiotics at 37°C and 5% CO,. Samples from the culture medium were collected on Days 1, 3, 5, and 7. After centrifugation at 1000rpm for 30 min the supernatants were filtered and stored at -20°C. Generation of hybridomas. BALB/c mice were infected orally with 200 living T. spiralis larvae, and 20 days later the mice were boosted ip with 200 ug of a crude larval extract. Cell fusion was carried out following the well-established procedure in our laboratory. Briefly, splenocytes from infected mice were fused with P3x63.Ag8.653 myeloma cells with 40% polyethylene glycol 1500 (Sigma Co.) for 8 min. Cells were washed at 1000 rpm for 10 min, resuspended in hypoxanthineaminopterine-thymidine-supplemented RPM1 1640 with 10% fetal calf serum, and distributed in %-well microtiter plates (Costar Ltd.). Supematants from wells with growing hybrids were screened by ELISA against the total extract from muscle larvae. Highly reactive hybridomas were cloned by limiting
151
dilution. Selected clones were grown in mass culture and frozen and stored in liquid nitrogen. Enzyme-linked immunosorbent assay. Microtiter 96 well plates (Costar Ltd.) were coated with 100 &ml total larval extract in carbonate buffer, pH 9.6, at 4°C and then blocked with 10% inactivated calf serum in 0.1 M Tris-HCl, pH 8.5, containing 0.5 M NaCl for 1 hr at room temperature (RT). After washing in phosphate-buffered saline (PBS) (pH 7.4) containing 0.05% Tween 20 (T-PBS), 50 pl of hybridoma supematants was added to each well for 2 hr at RT. Plates were washed in T-PBS and 50 (~1swine anti-mouse IgG serum conjugated with horseradish peroxidase (RIIPD, Sofia, Bulgaria) was added and incubated for 1 hr at RT. After extensive washing in T-PBS bound peroxidase was developed with 50 ul o-phenylendiamine (Sigma Co.) in citrate buffer, pH 5.0 (2 mg/lO ml buffer with 0.03% H,O,). The enzyme reaction was stopped after 5 min with 50 )LI 10% sulfuric acid and the results were read with a Micro-ELISA reader (Dynatech GmBh, Switzerland) at 492 nm. The presence of specific antigens in culture supernatants of in vitro-cultured larvae or in sera of infected animals was tested by an absorption ELISA. Basically, the procedure was as described above but the monoclonal antibody was preabsorbed with an equal volume of larvae supematant or serum for I hr at 37°C and then added to wells coated with the total Trichinella extract. To test sera from infected animals (60-90 days after infection) for the presence of anti-Trichinella antibodies, antigen-coated plates were treated with serial dilutions of sera from infected animals for 2 hr at RT. After washing three times for 5 min each in T-PBS, the monoclonal antibody TS 3G6 was added and the reaction proceeded as described above. Negative controls were unrelated culture medium samples, and sera from parasite-free animals were used as negative controls. The biochemical nature of the epitope recognized by Mab TS 3G6 was determined after treatment with different enzymes. The larval extract (100 kg/ml) coated on PVC microtiter plates was treated with trypsin, proteinase, pronase E, and subtilysin at a concentration of 1 mg/ml for 12 hr at 37°C. The wells were extensively washed with T-PBS and were processed as described above. All enzymes were purchased from Serva Fein B&hem., Germany. Periodic acid at different molar concentrations (from 5 to 100 rm+4)was used to test its effect on the antigenicity of the larval extract. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). SDS-PAGE under reducing con-
ditions was carried out as described by Laemmli (1970) using a 5% stacking gel and a 10% running gel. Electrophoretic conditions were 60 V while the sample ran through the stacking gel and then 100 V for about 120 min in 25 mM Tris, 192 mh4 glycine, pH 8.3, contain-
152
KEHAYOV ETAL.
ing 0.1% SDS. Molecular markers thyroglobulin (330 kDa), ferritin (220 kDa), albumin (67 kDa), catalase (60 kDa), lactate dehydrogenase (36 kDa), and ferritin (18.5 kDa) were applied. At the end of the run, gel slabs were either used for blotting or staining with 0.25% Coomassie blue. Western
blotting
and immunoenzyme
detection.
Electrotransfer of proteins from the SDS gel to nitrocellulose was done using a Transblot cell (BioRad) and Tris-glycine buffer (pH 8.3) containing 20% methanol. For detection of the specific antigens, nitrocellulose strips were blocked overnight at 4°C using 10% inactivated calf serum in 0.1 M Tris-HCI buffer, pH 8.5, containing 0.5 M NaCl, washed in PBS, and incubated overnight at 4°C in the monoclonal antibody. After washing in PBS bound antibody was visualized using biotin-conjugated anti-mouse IgG serum and the avidin-peroxidase conjugate (Sigma Co.). The enzyme reaction was developed by incubation in I-chloro-4naphthol (Sigma Co.). An irrelevant mouse monoclonal antibody of the same isotype was used to control the specificity of the test system. indirect immuno$7uorescence. Paraffin-embedded tissue sections from infected animals were passed through xylene and a series of alcohols and rehydrated briefly in PBS, pH 7.4. (McLaren et al. 1987). Tissue sections were incubated in the monoclonal antibody or in suitable control supernatants for 60 min at RT. After washing in PBS, pH 7.4, containing 0.1% w/v bovine serum albumin (BSA) the sections were covered with FITC-conjugated anti-mouse IgG serum, 0.1 mglml (RIIPD, Sofia, Bulgaria), and incubated for 60 min. After washing in PBS-BSA the sections were mounted in SO%buffered glycerol and examined using a Laborlux K fluorescent microscope (Leitz, Austria). Indirect immunoperoxidase technique. Rehydrated tissue sections were incubated with the monoclonal antibody or control supernatants for 60 min at RT. The sections were thoroughly washed in PBS-BSA and transferred into a l/50 dilution of swine anti-mouse IgG serum conjugated with horseradish peroxidase (RIIPD) for 60 min. After further washing the slides were incubated in substrate solution (5 mg 3,3’-diaminobenzidine in 10 ml Tris-HCI buffer, pH 7.2, with 0.01% H,O,). The sections were then dehydrated and mounted in oil for light microscopy.
cells. Hybrid cells were found in 134 out of 288 wells and specific antibodies against the Trichinella extract were detected in six supernatants. Cells from these wells were cloned at least three times by limiting dilution and a hybridoma (TS 3G6) was selected for further study. The supernatant from the TS 3G6 hybridoma was tested against sera and extracts from striated muscle from rodents, pigs, and humans by ELISA, and in each case the reactions were completely negative. A similar approach was used to test the same supernatant for cross-reactivity against A. suum, A. lumbricoides, E. granulosus, T. canis, T. suis, and T. ovis. The results presented in Table I show that Mab TS 3G6 recognizes an epitope specific to T. spiralis and that it is not shared with the other nematodes tested. However, when tested against different Trichinella species, Mab TS 3G6 gave positive reactions with similar OD values with extracts from all species tested (T. spiralis, T. nelsoni, T. nativa, and T. pseudospiralis) (Table II). Characterization of the antigen recognized by Mab TS 3G6. When the total ex-
tract from muscle larvae or the extract from noninfected muscle was subjected to SDSPAGE under reducing conditions and stained with 0.025% Coomassie blue, a number of protein bands with a different molecular mass (from 300 to 12 kDa) were seen (Figs. lb and lc). In the larval extract, TABLE I Specificity of Monoclonal Antibody 3G6 against T. spiralis Tested in ELISA Nematode species
RESULTS
Generation and characterization of monoclonal antibodies. High titers of antiTrichinella antibodies were detected by
ELISA 4 days after infected mice were given a booster injection of larval antigen, and accordingly, splenocytes were taken for fusion with P3x63.Ag8.653 myeloma
T. T. A. A. E. T. T.
spiralis canis lumbricoides suum granulosum ovis suis
Mab 3G6 1.012” 0.090 0.045 0.080 0.058 0.045 0.03 1
0 OD492 values in ELISA from three independent assays.
ANTIGEN
OF
T. spiralis MUSCLE
TABLE II Cross-Reactions of Mab TS 3G6 against Different Trichinella Species Trichinella T. T. T. T.
species
spiraiis nelsoni nativa pseudospiralis
Mab TS 3G6 0.988” 0.860 0.790 0.810
a OD492 values in ELISA.
two protein bands in the region of 35-40 kDa are seen which are not present in the extract of noninfected muscle. After transfer to nitrocellulose and treatment with Mab TS 3G6, three bands were specifically stained, with the major band stained having a molecular weight of 76 kDa. There are two additional bands which are less intensively but, still, specifically stained with MW of 41 and 36 kDa, respectively (Fig. la). When the immobilized larval extract was digested with different enzymes the reactivity of Mab TS 3G6 with its corresponding epitope was significantly reduced after treatment with proteases (Table III). The
153
LARVAE
TABLE III Effect of Treatment with Different Enzymes on the Reactivity of Mab TS 3G6 Larval extract treated with PBS Trypsin Proteinase Subtilysin Pronase E Periodic acid (5-25 mk4)
Mab TS 3G6 0.854 k 0.146 k 0.050 2 0.032 + 0.031 ? 0.870 5
0.089” 0.031 0.015 0.012 0.016 0.046
a OD492 in ELISA from three independent experiments.
protein nature of the epitope was confirmed by the fact that oxidation with increasing concentrations of periodic acid did not change the antigenicity of the larval extract. Cellular localization of the TS 3G6 antigen was determined by indirect immunoperoxidase and immunofluorescence using serum from an infected mouse as a positive control (Fig. 2a). In the immunoperoxidase reaction the serum stained striated muscle fibers light yellow and muscle larvae much more intensively (yellow to dark brown). The parasite was uniformly stained and a localization of the reaction product could not be specified. In contrast, Mab TS 3G6 stained quite intensively the stichosome only. Specific binding to the stichosomes was clearly seen in both longitudinal and 76 kD cross sections of the larvae (Fig. 2b). Dark deposits of the reaction product in the cytoplasm of nurse cells should be noted. A typical finding is illustrated in Fig. 2b. The 41 kD nuclei of the nurse cells seem to be un36kD stained as do the inflammatory cells (Fig. 2b, lower left corner of nurse cell). The cuticle adjacent to the stichosome was also intensively stained. This specific staining was most clearly seen at a higher magnification (Fig. 2~). There was no staining of FIG. 1. SDS-PAGE electrophoresis and electrothe esophagus, intestine, or genital ruditransfer of Trichinella extract (lane a,b) and extract ment of the larvae. No positive reaction from noninfected muscle (c). Lane a is stained with Mab TS 3G6; lanes b and c are stained with Coomassie was seen on the parasite capsule or the surrounding muscle fibers. blue.
154
KEHAYOV
b
ET AL.
ANTIGEN
OF
T. Spiralis MUSCLE
LARVAE
The immunocytochemical findings were confirmed by the indirect immunofluorescence technique. When Mab TS 3G6 was used as the first layer and FITC anti-mouse IgG serum as the second, intensive bright green fluorescence was seen on the surface of the stichosome and the outer cuticular layer only (Figs. 3a-3c). The irrelevant monoclonal antibody from the same immunoglobulin isotype applied as the first antibody gave a completely negative reaction (Fig. 3d).
155
rectly in ELISA. However, when Mab TS 3G6 was preabsorbed with serum samples and then applied to Trichinella-coated plates in an absorption ELISA, evidence was obtained for the presence of the specific antigen, the percentage of the activity absorbed rising from 13.50% on Day 3 to a maximum of 3uO% on Day 5 (Fig. 5). The fact that the TS 3G6 antigen was localized in the stichosome and was secreted by muscle larvae made it likely that this antigen would induce a humoral immune response against T. spiralis. If that was the case, Mab Immunogenicity of the antigen recog3G6 might be used for development of an nized by Mab XX. To test the possibility that the antigen recognized by Mab TS 3G6 immunoenzyme test for diagnosis of the inwas an excretory/secretory component, its fection. To test this assumption, sera from shedding by in vitro-cultured muscle larvae guinea pigs taken 60 days after the infection was followed. Trichinella larvae isolated, and from rats taken 90 days after infection antibodtransferred into RPM1 1640 medium with were screened for anti-Trichinella 10% FCS, and samples of culture medium ies by a competitive ELISA. All sera tested were collected on Days 1,3, 5, and 7. Using could block the binding of Mab TS 3G6 to competitive ELBA, the TS 3G6 antigen its target antigen. A representative dosewas detected in the culture supematant as dependent curve of the blocking effect is early as the first day of culture. A represen- shown in Fig. 6, where it can be seen that tative curve formed when Mab TS 3G6 was OD values increase with the dilution of the preabsorbed with culture medium is shown sera tested. in Fig. 4, where it was demonstrated that DISCUSSION 120 pg/ml total protein absorbed about 50% Elucidation of the complicated immunoof the activity of Mab TS 3G6 in ELISA. Since the TS 3G6 antigen was secreted logical interrelations between parasitic by in vitro-cultured larvae, we reasoned nematodes and their hosts is not possible that it might be present in the serum of in- without precise data on the morphologic fected animal. This assumption was tested. and cellular localization of the parasite anRats and guinea pigs were infected orally tigens and particularly on their structure with 2000 larvae and blood samples were and expression. T. spiralis provides a good collected on Days 3,4, 5, 6, and 7 after the model for such studies and several investiinfection. No antigen was detected in any gators have described antigens which can of the samples when they were tested di- induce specific humoral or cell-mediated
FIG. 2. (a) Section of paraffin-embedded muscle from a guinea pig infected with T. spiralis. Immunoperoxidase staining was done with serum from a BALB/c mouse immunized against T. spiralis. The reaction product is uniformly layered on the whole larval surface and completely tills the capsular space. Striated muscle tissue is also stained (magnification X205). (b) Indirect immunoperoxidase technique with Mab TS 3G6 and anti-mouse IgG serum conjugated with peroxidase. Intensive specific staining in the stichosome is seen while the parasite capsule and striated muscles are completely negative (magnification x410). (c) Immunoperoxidase technique with Mab TS 3G6. The stichosome is intensively stained and a positive reaction is seen on the cuticle adjacent to the stichosome (magnification X410).
156
KEHAYOV
ET AL.
FIG. 3. (a,b) Immunofluorescent staining with Mab TS 3G6 on paraffin-embedded sections of muscle from guinea pig infected with T. spiralis. Bright green fluorescence is seen in the cells of the stichosome (magnification x 125; x2.50). (c) Transversal section of T. spiralis larvae. There is bright staining of the stichosome and of the cuticular surface layer (magnification x500). (d) A negative control section treated with an irrelevant monoclonal antibody from the same immunoglobulin class as Mab TS 3G6 (magnification x500).
imnnune responses (Komandarev 1978; DUI.ham el al. 1984) or which can stimulate pro1tective immunity. In this respect attention I has been focused on antigens located on 1the cuticle (Grencis et al. 1986; OrtegaPie1-res 1984a) or present in excretoryke-
cretory products of muscle larva (Silt stein and Despommier 1985). The studies presented in this paper w aimed at identification of antigens relea sed by T. spiralis which might be valuable for immunodiagnosis or for experimental stud-
ANTIGEN
OF
T. SpiraliS MUSCLE
LARVAE
1.57
FIG. 3-Continued.
ies. A hybridoma was constructed (TS 3G6) secreting a monoclonal antibody that reacted against all four Trichinella species (T. spiralis, T. nelsoni, T. nativa, and T. pseudospiralis) but did not cross-react with
other helminths. It was established by immunoenzyme and immunofluorescent techniques that the major deposition of the reaction products with this monoclonal antibody was in the stichosome, with the
158
KEHAYOV
ET AL.
4
A 1.100. 1.000
0.600
0.800
0.700
0.600-
: d 0
N
0.600
0.700
nz
0.500
0.600~
0
0.400
0.500
4
c\
0
0
0.300 0.200 0
0.100
7/* 0
undiluted
I:10
1:50
sera
total
protein
(gg/mO
FIG. 4. A representative curve of ELBA with Mab TS 3G6 preabsorbed with serial dilutions of T. spirulis larvae supematant after 18 hr in culture (0) or just culture medium (A). Plates have been coated with T. spiralis extract.
stichocytes being intensively stained. The presence of positive staining in the cytoplasm of nurse cells (Figs. 2b and 2c) probably shows that at least one of the excretory/secretory antigens (76,41, and 36 kDa) is related to some tissue component of the host. Still, another possibility is that some of the secreted antigen has been digested by the nurse cell. The intensity of the reaction did not allow discrimination between (Y-or P-stichocytes. The cellular distribution of 1.2% 3
40% 26.
6%
/
i 4
I! 5
Days
after
6
infection
FIG. 5. Presence of TS 3G6 antigen in sera of T. spiral&-infected animals as detected by an absorption ELBA with Mab TS 3G6. Hatched bars show the percentage of Mab TS 3G6 reactivity absorbed by sera of infected animals at different days after infection.
1:100
1:200
1:400
dilution
FIG. 6. A representative curve of blocking experiments. Plates coated with T. spiralis were treated with serial dilutions of sera from animals (0) or from healthy donors (A) and acted with Mab TS 3G6.
ELBA extract infected then re-
the antigen identified in our experiments is quite similar to that described by Silberstein and Despommier (1984) and by Gamble (1985). Recently, McLaren et al. (1987) have determined the location of the antigens recognized by the NIM-Ml and NIMM2 antibodies which stained the epicuticle, stichocytes, and the surface layer of the intestine of muscle larvae. It was also shown in these investigations that the antigens located in the stichosome of the larvae were released in excretory/secretory products. Quite similar results were obtained in our laboratory with Mab TS 3G6. When Mab TS 3G6 was absorbed with increasing concentrations of larvae culture medium and tested in ELISA, it was established that the corresponding antigen was secreted in the medium (Fig. 4). Based on this finding it was assumed that this excretory/secretory antigen might be shed in the circulation of naturally infected animals. Confirmation of this assumption was provided by detection of circulating antigen in guinea pigs and rats heavily infected with Trichinella larvae. Although the OD values were not high, sera from infected animals absorbed the activity of Mab TS 3G6 against the crude extract from muscle larvae (Fig. 5). The experimental design used for detection of circulating Trichinelfa antigens in our laboratory
ANTIGEN
OF
T. Spirdis MUSCLE
was different from that of Gamble and Graham (1984) who did not find secretory antigens in the sera of 2-month-old piglets infected with 500 larvae of T. spiralis. The infective dose used in our experiments was significantly higher (2000 larvae per animal) and the circulating antigen was detected from Day 3 after infection. These early stages of infection (Days 3-7) were chosen to avoid elimination of any circulating antigens as a result of an immune response. Mab TS 3G6 is similar to 7C2CS (Gamble 1985), 7B2.3 (Silberstein and Despommier 1984), and NIM-Ml (McLaren et al. 1987) as far as its immunohistochemical reactions and the excretory/secretory nature of the corresponding antigen are concerned. However, the basic difference between Mab TS 3G6 and the other monoclonal antibodies described so far is that Mab TS 3G6 reacts with a 76-kDa protein as shown by immunoblotting (Fig. 1). The epitope recognized specifically by Mab TS 3G6 seems to be of a protein nature since it is sensitive to protease digestion and is resistant to oxidation with periodic acid (Table III). This antibody stains two additional bands of approximately 41 and 36 kDa, respectively. There are at least two alternative explanations of this finding. One possibility is that Mab TS 3G6 reacts with a protein epitope that is detected on three different polypeptides with IV, 76, 41, and 36 kDa. Another possibility is that Mab TS 3G6 recognizes a 76-kDa antigen and that the other two bands represent degradation products or post-translational moditications of the 76-kDa molecule. Detailed immunochemical studies are in progress to distinguish between these alternatives. It should be stressed, however, that the antigen recognized by Mab TS 3G6 is quite different in its molecular weight from the antigens deother Trichinella-specific scribed so far: 7C2C5 (45, 49, 53 kDa), 7B2.3.1. (O/55 kDa) NIM-Ml (105, 90, 57, 47 kDa), or NIM-M2 (90, 47 kDa). The antigen recognized by Mab TS 3G6
LARVAE
159
is located in the stichosome and in the outer layer of the cuticle, particularly on the portion adjacent to the stichosome (Figs. 2 and 3). This specific localization is difficult to reconcile with the suggestion of McLaren et al. (1987) that the sources of the excretory/secretory antigens are the stichocytes and that the antigens are incorporated in the cuticle after their passage through the esophagus and intestines and their release through the mouth and anus to the perilarval space of the capsule. It seems more likely, as also suggested by these authors, that the antigens are actively synthesized by the cells of the hypodermis or bacillary band cells, which show antigenic determinants common to the antigens of the stichocytes. It is of course quite possible that the antigen is directly transferred from the stichocytes toward the cuticle by a different but presently unknown mechanism. The data reported here suggest that Mab TS 3G6 could be used in diagnostic tests for a semiquantative determination of antiTrichinella antibodies in the sera of infected pigs or patients. Gamble et al. (1983) have already developed a competitive ELISA with monoclonal antibody 7C2C5 against excretory/secretory antigens (45, 49, 53 kDa) for the immunodiagnosis of T. spiralis in experimentally infected animals. This method has demonstrated a high efficiency in the detection of specific anti-Trichinella antibodies and there has not been a single false positive result with sera from pigs infected with Trichuris suis. In our experiments using a competitive ELISA and a crude Trichinella extract or a purified Trichinella antigen (Difco Lab.), all sera from infected animals blocked the reaction of Mab TS 3G6 with a solid-phase bound Trichinella antigen (Fig. 6). At a dilution of l/100 the blocking effect was about 50%. Tests of sera from patients infected after consumption of infected meat are now in progress and some preliminary data show that this test has considerable potential for immunodiagnosis of human trichinellosis.
KEHAYOVETAL.
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