EXPERIMENTAL
PARASITOLOGY
Schistosoma
(1990)
mansoni:
Schistosomiasis I.S. Departments
71, 107-113
Detection of Circulating Antigens in Murine by Antigen-Capture Sandwich ELISA Using a Monoclonal Antibody
BARSOUM, D.G.
COLLEY, AND K. A. KAMAL*
of Microbiology and Immunology, and Medicine, Vanderbilt University School of Medicine Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212, U.S.A., and *U.S. Naval Medical Unit No. 3, Cairo, Egypt
and
BARSOUM, I. S., COLLEY, D. G., AND KAMAL, K. A. 1990. Schistosoma mansoni: Detection of circulating antigens in murine schistosomiasis by antigen-capture sandwich ELISA using a monoclonal antibody. Experimental Parasitology 71, 107-l 13. A monoclonal antibody (MAb) SH 1 l/B 1 that reacts with a repeating epitope on an excretory-secretory (E + S) antigen of adult worms of Schistosoma mansoni was used in the detection of circulating antigen (CA) in sera from S. mansoni-infected mice using an antigen-capture sandwich ELISA. Trichloroacetic acid (TCA) pretreatment of sera from mice infected for 8 or 16 weeks precipitated immune complexes and/or dissociated CA and allowed its detection. Sera obtained 8 weeks after infection contained high levels of CA. Upon treatment with praziquantel (100 mgikg body wt), this level was significantly less within 1 week. A strong correlation was found between the worm count determined by perfusion and the level of antigenemia detected by the 5Hl l/B1 assay in light and heavy infection (r = 0.80). Based on the results of both TCA pretreatment and sodium periodate treatment, the SHll/Bl sandwich ELISA assay detects a repeating carbohydrate epitope on an E + S antigen. This system appears to be a sensitive assay for the detection of schistosomal antigenemia in murine schistosomiasis. Studies on the detection of antigenemia in human schistosomiasis Using this assay are in progress. 8 1!390 Academic press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Schistosoma mansoni; Trematode; Excretory-secretory antigen (E + S); Circulating antigen (CA); Antigenemia; Monoclonal antibody; Sandwich ELISA; Serum; Mouse: Human; Treatment; Soluble worm antigen preparation (SWAP); Soluble egg antigen (SEA); Antibody (Ab); Monoclonal antibody (MAb); Enzyme-linked immunosorbant assay (ELBA); Indirect hemagglutination assay (IHA); Trichloroacetic acid (TCA); Phosphate-buffered saline (PBS); Room temperature (RT).
their intensity (i.e., worm burden), and that could evaluate the efficacy of chemotherapy would be highly desirable. Such a tool may also be necessary for practical human evaluation of the effectiveness of future candidate vaccines. The detection of antigenemia has been used successfully in the diagnosis of many parasitic diseases (Whither 1987). MAbs have been successfully used in the detection of circulating antigens (CA) in sera and urine of filariasis patients (Huijun et al. 1987; Lunde er al. 1988; Weil et al. 1988). Similarly, a MAb-based enzyme immunoassay was used successfully in the detection of circulating DirojZuriu immifis antigen in canine infection (Weil et al. 1986). In
INTRODUCTION
Schistosomiasis is a chronic helminthic infection of more than 200 million people throughout the world (Warren 1982). Estimation of the intensity of schistosomal infection is currently based on quantitative egg counts which can be highly variable (Deelder et al. 1989) and may depend on the immune status of the host (Doenhoff et al. 1978; Damian 1987). Standard serologic detection of anti-schistosomal antibody levels does not discriminate between previous exposure and a current infection (Simpson and Smithers 1985). A test that could measure antigen secreted by live parasites, that would discriminate infections based on 107
0014-4894/90
$3.00
Copyright 0 1990 by Academic F’ress, Inc. AU rights of reproduction in any form reserved.
108
BARSOUM,COLLEY,
schistosomiasis, attempts to diagnose and quantify infection by detection of CA have been reviewed by Simpson and Smithers (1985). CA were first reported in the serum
and urine of mice and hamsters infected with Schistosoma mansoni in 1967 (Berggren and Weller 1967). The detection of CA in human schistosomiasis using MAb and a sensitive antigen-capture sandwich ELISA has been recently reported (Deelder et al. 1989). A major CA was shown to be a negatively charged polysaccharide and was designated circulating anodic antigen (Deelder et al. 1976). A second CA has the characteristics of a polysaccharide with a cathodic migration and was designated circulating cathodic antigen (Deelder et al. 1976). Both CA are extracted from adult worms in the TCA-soluble fraction and are also present in the E + S antigen obtained from schistosome adult worms incubated in culture medium (Deelder et al. 1980). They are heat stable and resistant to proteolytic enzymes, DNase, and RNase, but are destroyed by periodate treatment (Gold et al. 1969; Nash 1974; Carlier et al. 1978; Nash and Deelder 1985). In the present study we have developed a sensitive antigen-capture sandwich ELISA using an IgM MAb that detects antigenemia in the serum during murine schistosomiasis mansoni. MATERIALS
AND METHODS
Animals and sera. Female CBA/J mice and BALBlc mice were obtained under the auspices of the Veterans Administration through a National Cancer Institute supply conttact and CFl mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). All animals were housed in facilities approved by the American Association for Accreditation of Laboratory Animal Care. Mice were infected subcutaneously with S. mansoni cercariae (Puerto Rican strain). At various times after infection, mice were bled from the retroorbital chamber while under ether anesthesia and then further euthanized by intraperitoneal injection of sodium pentobarbital and perfused for worm counts. Blood was allowed to clot. Sera obtained by centrifugation were stored at -20°C until used.
AND KAMAL Preparation of E + S antigen. E + S was prepared as described by Rotmans et al. (1981). Briefly, adult worms obtained from mice infected for 8 weeks with S. mansoni were incubated under sterile conditions in petri dishes with RPM1 1640 medium (GIBCO) without serum and with 3% penicillin-streptomycin (GIBCO) for 2U8 hr in 5% CO, at 37°C. Supematant fluids from the cultures were centrifuged at 12OOgfor 10 min, dialysed in Dulbecco’s PBS with two changes of buffer, and concentrated in an Amicon concentrating chamber (Amicon, Danvers, MA) with 10,000 MW cut-off membrane (Pharmacia, Piscataway, NJ). The E + S antigen was filter sterilized, ahquoted, and kept frozen at -20°C. Protein content was determined by the Lowry method (Lowry et al. 1951) and carbohydrate content was determined by the phenolsulfuric acid method (Dubois et al. 1956). Preparation of monoclonal antibody. BALBlc mice were immunized with adult schistosome worm antigen preparation (SWAP) (Colley et al. 1977) in complete Freund’s adjuvant with subsequent injections in incomplete Freund’s adjuvant. Sensitized spleen cells were fused with murine myeloma cells 8653 using polyethylene glycol as described previously (Barsoum et al. 1989). After HAT selection, hybridomas were screened for antibodies against E + S, soluble egg (SEA), and SWAP by ELISA. Hybrids that were highly reactive to E + S, with or without reactivity to SWAP and with minimal reactivity to SEA, were cloned by limiting dilution, and their isotypes were determined using an anti-mouse immunoglobulin subclass kit (Zymed laboratories, San Francisco, CA). Antibody production by the clones was retested by ELISA and hybridoma cells were injected intraperitoneally into BALB/c mice for ascites production. MAb SHll/Bl (an IgM) was purified from ascitic fluids by a simple procedure of euglobulin precipitation in distilled water (Garcia-Gonzalez et al. 1988). Antigen-capture sandwich ELISA. Wells of microtiter plates (Immulon-2) (Dynatech Laboratories, Chantilly, VA) were coated with 50 pl of MAb (10 kg/ml) in carbonate buffer, pH 9.6, and incubated for 2 hr at room temperature (RT) and overnight at 4°C. Plates were washed with PBS-Tween 20 (O.OS%), incubated for 45 min with PBS-milk (5%), and washed. Mouse serum was tested untreated or treated with 2% TCA to precipitate protein and/or dissociate CA from immune complexes as described by De Jonge et al. (1987). Briefly, equal volumes of serum and 4% TCA were mixed at RT for 15 min and centrifuged at 10,OOOgfor 5 min. The supematant was collected, neutralized with carbonate buffer (pH 9.6), added to the wells, and incubated for 3040 min at RT. Plates were washed and an optimum dilution of the same MAb conjugated to biotin (Pierce, Rockford, IL) was added, incubated, and washed as before. Avidin-peroxidase (Sigma Chemical Co., St. Louis, MO) was added to
DETECTION
OF
CIRCULATING
the wells, and plates were incubated and washed as above. ABTS peroxidase substrate (Rirkegaard and Perry Laboratories, Gaithersburg, MD) was added and the blue-green color was read at the 410-nm wavelength using an MR 600 ELISA reader (Dynatech). Readings that were above mean -1- 3 SD of control wells were considered positive. Pools of normal mouse serum and serum from mice infected for 8 weeks were included as controls in each assay. Biotinylation of MAb 5HllIBl. The procedure was done as recommended by the manufacturer (Pierce). Briefly, MAb 5Hll/Bl was dialyzed in bicarbonate buffer, pH 8.5, and mixed with NHS-LC-biotin for 2 hr in ice. The mixture was centrifuged at 1OOOgin Amicon centrifuge tubes (CentriconMicroconcentrators, Amicon), resuspended in bicarbonate buffer and mixed again with NHS-LC-biotin, incubated, and centrifuged as before. Biotinylated 5Hl l/B 1 was washed 2x in PBS and the protein content was determined by absorption at the 280~nm wavelength and adjusted to 2 mg protein/ml. RESULTS
Selection
of h4Ab 5HllIBI
From a panel of nine positive hybridomas, three were selected that strongly reacted with E + S with some crossreactivity with SWAP and minimal reactivity with SEA. These hybrids were cloned and injected into BALB/c mice for ascites production. Two of these MAbs were IgM (4F7/FlO and 5Hl l/Bl) and one was IgG2a (2G2/D9). The reactivity of these MAbs with SEA, SWAP, and E + S is shown in Fig. 1. MAb 5H1 l/B1 showed the strongest reaction with E + S and was selected as both the capture Ab and the second Ab conjugated with biotin. MAb 5HlllBl was tested against E + S untreated or treated with sodium periodate as described by Stewart et al. (1977). Briefly, 0.02 M sodium periodate was dissolved in 0.1 M sodium acetate and the pH was adjusted to 4.5 with glacial acetic acid. Equal volumes of E + S and sodium periodate buffer were mixed and incubated in ice for 2.5 hr and dialyzed in PBS. Both untreated and periodate-treated antigens were diluted in sodium carbonate buffer (pH 9.6) to 10 pg protein/ml and added to the wells. The
ANTIGENS
IN
109
SCHISTOSOMIASIS
@ I 0
4F7/FIO
5Hll/Bl
SEA E+S SWAP
2G2/D9
FIG. 1. Reactivity of MAbs 4F7/FlO (IgM), 5Hl l/B1 (IgM), and 2G2/D9 (IgG,a) (1: 160 dilution) against soluble egg antigen (SEA), excretorysecretory (E + S) antigen, and soluble worm antigen preparation (SWAP) measured by ELISA.
treatment of E + S with sodium periodate completely abrogated the reactivity of 5Hll/Bl with E + S as shown in Fig. 2. The Effect of Immune Complexes on the Detection of E + S in Mouse Serum E + S (40 &ml) was added to normal, 8-week and ldweek-infected mouse sera and each tested either as such (untreated) or after pretreatment with 2% TCA. In untreated samples, normal mouse serum showed the highest E + S level followed by 16-week-infected serum while &weekinfected serum showed the lowest level of detectable E + S (Fig. 3). When the sera were pretreated with TCA, all sera showed parallel levels of E + S which were com-
060: OSa 04z 030.2Ol-
FIG. 2. Reactivity of MAb 5HlllBl against E + S antigen untreated (0) or treated (0) with 0.02 M sodium periodate as measured by ELISA.
110
BARSOUM,
COLLEY,
0.1 -
8
16
32 RECIPROCAL
64
128
256
512
OF DILUTIONS
FIG. 3. Detection of E + S (40 pg protein/ml) in normal mouse serum (ff; NMS), 8-week-infected (0; I-week MS), and lbweek-infected mouse serum (0; ldweek MS) by sandwich ELISA using 5HlllBl without treatment with 2% trichloroacetic acid (TCA).
parable to the added levels seen in untreated normal serum as shown in Fig. 4. When 8- and 16-week-infected sera were tested for anti-E + S Abs (on E + S-coated plates), 8-week-infected serum showed a higher response than 16-week-infected serum (OD readings at I:1000 serum dilution of 0.54 vs 0.18 for 8-week and 16weekinfected sera, respectively). E + S in Sera of CBAIJ Mice before and after Praziquantel Treatment CBA/J mice infected 8 weeks earlier with 45 cercariae of S. mansoni were treated with praziquantel. The drug was given orally in three doses of 100 mg/kg body wt
AND
KAMAL
at 4-hr intervals (Tawfik et al. 1986). A control group was left untreated. At weekly intervals mice were bled, perfused, and the number of worms counted. Mesenteric venules were examined and livers were examined after pressing between glass plates to assure complete perfusions. Each week, sera were pooled from a group of three untreated mice and from a group of three treated mice and tested for CA after TCA exposure. CA was detected in both groups before treatment. A significant drop in CA was observed on the first week after treatment (Fig. 5). This decreased level of CA in the sera of treated mice continued through the end of the experiment 4 weeks after treatment. The level of CA in the sera of untreated mice did not alter over this time period (Fig. 5). The numbers of worms perfused from mice the first week after treatment were 3.3 * 0.6 (mean + SEM) and 0.6 + 0.6, in the control and treated groups, respectively. The mean worm counts of the mice perfused the fourth week after treatment were 5.6 2 2.0 in the control group and 0 in the treated group. Similar results were obtained in a second experiment. Decreases in the levels of CA were observed by the first day after treatment and re0.61 0.5
0.6 -
0.4
NMS
5
-16WkMS
0.5 -
o---o
;
8 Wk MS
0.3
\.
0.4 -
2
E
0.2
$j 0.3 -
01 z
02-
0 01 o-
0
O-Control
GD
.-Treated
Gp
bc-5 Before
Treatment
FIG.
b
1’6
I28 256 i, 6’4 RECIPROCAL OF DILUTIONS
512
FIG. 4. Detection of E + S (40 pg protein/ml) in NMS, 8-week MS, and 16-week MS by sandwich ELISA using 5HlllBl after pretreatment with 2% TCA.
5. Detection
1 WEEKS
2
AFTER
3
4
TREATMENT
of E + S in sera of mice infected
with 45 S. mansoni cercariae 8 weeks before treatment and either treated orally with praziquantel(lO0 m&g body wt, three doses at 4-hr intervals) (0; treated gp) or untreated (0; control gp). Serum was pooled from a group of three mice at each time interval and pretreated with 2% TCA.
DETECTION
OF
CIRCULATING
ANTIGENS
mained low through the next 4 weeks (data not shown). Correlation between Worm Burden and the Level of Antigenemia in Mice CFI mice were divided into four groups of 12 mice each and infected with 200, 100, 50, or 25 S. mansoni cercariae. Eight weeks after infection, mice were bled and sera collected for detection of CA after TCA treatment of serum. Mice were then euthanized using sodium pentobarbital and perfused for worm counts. Figure 6 shows a scatter plot of the number of worms from each mouse, relative to the level of antigenemia detected in the TCA-treated serum of that mouse by sandwich ELISA using MAb 5Hl l/Bl. A strong coefficient of correlation (r = 0.80) was found between the two parameters (n = 42). DISCUSSION
We have developed an antigen-capture sandwich ELISA using a new MAb to an epitope on an E + S antigen of adult schistosome worms. A significant correlation was found between the number of worms perfused from mice and their circulating levels of CA. Recent reports have shown strong correlation between fecal egg counts and detection of a CA (Deelder et al. 1989) 1.0,
I .
0.8 .
WORM
.
.
COUNT
FIG. 6. Correlation between the worm count and the detection of CA in individual mice. Mice were infected with 200, 100, 50, or 25 S. mansoni cercariae 8 weeks earlier. Individual mice were bled and perfused for worm count. Serum was used to detect CA after 2% TCA pretreatment (n = 42).
IN
SCHISTOSOMIASIS
111
and a reduction in the level of CA after treatment with praziquantel in human schistosomiasis (De Jonge et al. 1989). These authors have used an IgG, MAb as the capture Ab and the same MAb conjugated with alkaline phosphatase as the second Ab in a sandwich ELISA or an indirect hemagglutination (IHA) assays. In their studies using the IHA, CA was detectable only in patients excreting >500 eggs/g of feces, while they could detect CA corresponding to a level of 10 eggs/g of feces using the sandwich ELISA. However, Deelder et al. (1989) stated that anti-CAA IgM MAbs did not perform well in the ELISA assay. We have successfully used an IgM MAb (5Hll/Bl) as the capture Ab and the same biotin-conjugated MAb as the second Ab. In a previous study, a direct relation was found between the number of worms in hamsters and the amount of antigen in the serum (Gold et al. 1969). Because the same MAb is used to capture and detect CA, it is likely that a repeating epitope on an E + S is being detected by 5Hl l/Bl. This epitope is almost certainly a carbohydrate because it withstands 2% TCA treatment and is sensitive to sodium periodate treatment (Fig. 2). It is evident from our results (Figs. 3 and 4), as well as those of others (De Jonge et al. 1988; Deelder et al. 1989), that TCA treatment of sera from infected animals or patients dissociates these CA from immune complexes that are formed between antibody and antigen during the infection and allows their detection. In our study, the level of detection of added E + S in 8week-infected mice is presumed to be low before TCA treatment because of the existence of antibodies that form immune complexes (Fig. 3). These levels approximated the same level as in normal serum after TCA treatment of the serum (Fig. 4). Using our MAb in this system, we demonstrated a significant decrease in the level of CA detected after treatment of infected mice with praziquantel (Fig. 5). Previous
112
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COLLEY,
studies in murine schistosomiasis have also demonstrated a rapid decrease in the level of antigenemia (72-96 hr) following treatment with praziquantel (Weltman 1982; Abdel-Hafez et al. 1983). Similar results were obtained in human schistosomiasis mansoni by De Jonge et al. (1989), using an IgG, MAb alone and conjugated with alkaline phosphatase in a sandwich ELISA. Based on these results, the sandwich ELISA using MAb 5Hl l/B 1 appears to be a sensitive assay for the detection of CA in murine schistosomiasis. This system is currently being evaluated with sera from patients with schistosomiasis and other infections. In preliminary studies it appears to have promise in the immunodiagnosis of human schistosomiasis and in the monitoring of chemotherapy and possible future vaccines. ACKNOWLEDGMENT
The authors gratefully acknowledge the secretarial and administrative assistance of Judith O’Connell. This work was supported by a grant from the Department of Veterans Affairs and the Department of Defense. REFERENCES
ABDEL-HAFEZ, S. K., PHILIPS, S. M., AND ZODDA, D. M. 1983. Schisrosomu mansoni: Detection and characterisation of antigens and antigenemia by inhibition enzyme-linked immunosorbent assay. Experimental Parasitology 55, 219-232. BARSOUM, I. S., GOODMAN, T., HARDIN, L. K., AND COLLEY, D. G. 1989. Monoclonal antibody neutralization of unmanipulated Chlamydia trachomatis serovar A infection of human epithelioid cells (A-43 1). Medical Microbiology and Immunology 178, 113120. BERGGREN, W. L., AND WELLER, T. H. 1%7. Immunoelectrophoretic demonstration of specific circulating antigen in animals infected with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 16, 606-612. CARLIER, Y., BOUT, D., AND CAPRON, A. 1978. Further studies on the circulating M antigen in human and experimental Schistosoma mansoni infections. Annales Immunology (Paris) 129, 81 l-818. COLLEY, D. G., COOK, J. A., FREEMAN, G. L., BARTHOLOMEW, R. K., AND JORDAN, P. 1977. Immune responses during human schistosomiasis mansoni. I.
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In vitro lymphocyte blastogenic responses to heterogenous antigenic preparations from schistosome eggs, worms and cercariae. International Archives of Allergy and Applied Immunology 53,42U33. DAMIAN, R. T. 1987. Presidential address. Journal of Parasitology 73, 3-13. DEELDER, A. M., KLAPPE, H. T. M., VAN DEN AARDWEG, G. J. M. J., AND VAN MEERBEKE, E. H. E. M. 1976. Schistosoma mansoni: Demonstration of two circulating antigens in infected hamsters. Experimental Parasitology 40, 189-197. DEELDER, A. M., KORNELIS, D., VAN MARK, E. A. E., EVELEIGH, P. C., AND VAN EGMOND, J. G. 1980. Schistosoma mansoni: Characterisation of two circulating polysaccharide antigens and the immunological response to these antigens in mouse, hamster, and human infections. Experimental Parasitology 50, 16-32. DEELDER, A. M., DE JONGE, N., FILLIE, Y. E., KORNELIS, D., HELAHA, D., QIAN, Z. L., DE CALUWE, P., AND POLDERMAN, A. M. 1989. Quantitative determination of circulating antigens in human schistosomiasis mansoni using an indirect hemagglutination assay. American Journal of Tropical Medicine and Hygiene 40, 5654. DE JONGE, N., FILLIE, Y. E., AND DEELDER, A. M. 1987. A simple and rapid treatment (trichloroacetic acid precipitation) of serum samples to prevent nonspecific reactions in the immunoassay of a proteoglycan. Journal of Immunological Methods 99, 195197. DE JONGE, N., GRYSEELS, B., HILBERATH, G. W., POLDERMAN, A. M., AND DEELDER, A. M. 1988. Detection of circulating anodic antigen by ELISA for seroepidemiology of schistosomiasis mansoni. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 591-594. DE JONGE, N., DE CALUWE, P., HILBERATH, G. W., KRUGER, F. W., POLDERMAN, A. M., AND DEELDER, A. M. 1989. Circulating anodic antigen levels in serum before and after chemotherapy with praziquantel in schistosomiasis mansoni. Trunsactions of the Royal Society of Tropical Medicine and Hygiene 83, 368-372. DOENHOFF, M., MUSALLAM, R., BAIN, J., AND McGREGOR, A. 1978. Studies on the host-parasite relationship in Schistosoma mansoni-infected mice: The immunological dependence of parasite egg excretion. Immunology 35, 771-778. DUBOIS, M., GILLS, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. 1956. Calorimetric method for determination of sugars. Annales of Chemistry 28, 350-356. GARCIA-GONZALEZ, M., BETTINGER, S., OTT, S., OLIVIER, P., KADOUCHE, J., AND POURLETTY, P. 1988. Purification of murine IgGs and IgM monoclo-
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OF
CIRCULATING
naJ antibodies by euglobulin precipitation. of Immunological GOLD, R., ROSEN,
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Methods 111, 17-23. F. S., AND WELLER, T.
H. 1%9. A specific circulating antigen in hamsters infected with
Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 18, 545-552. HUUUN, Z., ZHENGHON, T., REDDY, M. V. R., HARINATH, B. C., AND PIESSENS, W. F. 1987. Parasite
antigen in sera and urine of patients with bancroftian and brugian tilariasis detected by sandwich ELISA with monoclonal antibodies. American Journal of Tropical Medicine and Hygiene 36, 554560. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. LIJNDE, M. N., PARANJAPE, R., LAWLEY, T. J., AND OTTESEN, E. A. 1988. Filarial antigen in circulating immune complexes from patients with Wuchereria bancrofti filariasis. American Journal of Tropical Medicine and Hygiene 38, 366-371. NASH, T. E. 1974. Localization of the circulating antigen within the gut of Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 23,
1085-1087. NASH, T. E., AND DEELDER, A. M. 1985. Comparison of four schistosome excretory-secretory antigens: Phenol sulfuric test active peak, cathodic circulating antigen, gut-associated proteoglycan, and circulating anodic antigen. American Journal of Tropical Medicine and Hygiene 34, 326-341. ROTMANS, MOOLJ, tosoma
J. P., VAN DER VOORT, M. J., LOOSE, M., G. W., AND DEELDER, A. M. 1981. Schismnnsoni: Use of antigens from excretions and secretions in immunodiagnosis. Experimenral Parasitology 52, 319-330. SIMPSON, A. J. G., AND SMITHERS, S. R. 1985. Schis-
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tosomes: Surface, egg and circulating antigens. Current Topics 205-239. STEWART, STEWART,
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W. E., LIN, L. S., WIRANOWSKAM., AND CANTELL, K. 1977. Elimination of size and charge heterogeneities of human leukocyte interferons by chemical cleavage. Proceedings
of the National Academy of Sciences USA 74,42@4L 4204. TAWFIK, A. F., CARTER, C. E., AND COLLEY, D. G.
1986. Effects of anti-schistosomal chemotherapy on immune responses, protection and humoral responses. American Journal of Tropical Medicine and Hygiene WARREN, K.
35, 100-109.
S. 1982. Selective primary health care: Strategies for control of disease in the developing world. I. Schistosomiasis. Reviews of Infectious
Diseases 4, 715-726. WEIL, G. J., BLAIR, MALATESTA, P. F.
L. S.,
EWANCIW,
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1986. Use of parasite antigen detection to monitor the success of drug therapy in Dirofilaria immitis infected dogs. Journal of Para-
sitology 72, 737-740. WEIL, G. J., SETHUMADHAVAN, K. NAM, S., JAIN, D. C., AND GOSH,
V. P., SANTHAT. K. 1988. Persistence of parasite antigenemia following diethylcarbamazine therapy of bancroftian fdariasis. Amer-
ican Journal of Tropical Medicine and Hygiene 38, 589-595. WELTMAN, J. F. 1982. Effect of praziquantel on antigenemia in murine schistosomiasis. American Journal of Tropical Medicine and Hygiene 31, 1294-
12%. K. (Ed.). 1987. “Microbial Antigenodiagnosis,” Vol. l-2. CRC Press, Boca Raton, FL. Received 4 December 1989; accepted with revision 8 February 1990 WHICHER,
PARASITOLOGY
Schistosoma
(1990)
mansoni:
Schistosomiasis I.S. Departments
71, 107-113
Detection of Circulating Antigens in Murine by Antigen-Capture Sandwich ELISA Using a Monoclonal Antibody
BARSOUM, D.G.
COLLEY, AND K. A. KAMAL*
of Microbiology and Immunology, and Medicine, Vanderbilt University School of Medicine Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212, U.S.A., and *U.S. Naval Medical Unit No. 3, Cairo, Egypt
and
BARSOUM, I. S., COLLEY, D. G., AND KAMAL, K. A. 1990. Schistosoma mansoni: Detection of circulating antigens in murine schistosomiasis by antigen-capture sandwich ELISA using a monoclonal antibody. Experimental Parasitology 71, 107-l 13. A monoclonal antibody (MAb) SH 1 l/B 1 that reacts with a repeating epitope on an excretory-secretory (E + S) antigen of adult worms of Schistosoma mansoni was used in the detection of circulating antigen (CA) in sera from S. mansoni-infected mice using an antigen-capture sandwich ELISA. Trichloroacetic acid (TCA) pretreatment of sera from mice infected for 8 or 16 weeks precipitated immune complexes and/or dissociated CA and allowed its detection. Sera obtained 8 weeks after infection contained high levels of CA. Upon treatment with praziquantel (100 mgikg body wt), this level was significantly less within 1 week. A strong correlation was found between the worm count determined by perfusion and the level of antigenemia detected by the 5Hl l/B1 assay in light and heavy infection (r = 0.80). Based on the results of both TCA pretreatment and sodium periodate treatment, the SHll/Bl sandwich ELISA assay detects a repeating carbohydrate epitope on an E + S antigen. This system appears to be a sensitive assay for the detection of schistosomal antigenemia in murine schistosomiasis. Studies on the detection of antigenemia in human schistosomiasis Using this assay are in progress. 8 1!390 Academic press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Schistosoma mansoni; Trematode; Excretory-secretory antigen (E + S); Circulating antigen (CA); Antigenemia; Monoclonal antibody; Sandwich ELISA; Serum; Mouse: Human; Treatment; Soluble worm antigen preparation (SWAP); Soluble egg antigen (SEA); Antibody (Ab); Monoclonal antibody (MAb); Enzyme-linked immunosorbant assay (ELBA); Indirect hemagglutination assay (IHA); Trichloroacetic acid (TCA); Phosphate-buffered saline (PBS); Room temperature (RT).
their intensity (i.e., worm burden), and that could evaluate the efficacy of chemotherapy would be highly desirable. Such a tool may also be necessary for practical human evaluation of the effectiveness of future candidate vaccines. The detection of antigenemia has been used successfully in the diagnosis of many parasitic diseases (Whither 1987). MAbs have been successfully used in the detection of circulating antigens (CA) in sera and urine of filariasis patients (Huijun et al. 1987; Lunde er al. 1988; Weil et al. 1988). Similarly, a MAb-based enzyme immunoassay was used successfully in the detection of circulating DirojZuriu immifis antigen in canine infection (Weil et al. 1986). In
INTRODUCTION
Schistosomiasis is a chronic helminthic infection of more than 200 million people throughout the world (Warren 1982). Estimation of the intensity of schistosomal infection is currently based on quantitative egg counts which can be highly variable (Deelder et al. 1989) and may depend on the immune status of the host (Doenhoff et al. 1978; Damian 1987). Standard serologic detection of anti-schistosomal antibody levels does not discriminate between previous exposure and a current infection (Simpson and Smithers 1985). A test that could measure antigen secreted by live parasites, that would discriminate infections based on 107
0014-4894/90
$3.00
Copyright 0 1990 by Academic F’ress, Inc. AU rights of reproduction in any form reserved.
108
BARSOUM,COLLEY,
schistosomiasis, attempts to diagnose and quantify infection by detection of CA have been reviewed by Simpson and Smithers (1985). CA were first reported in the serum
and urine of mice and hamsters infected with Schistosoma mansoni in 1967 (Berggren and Weller 1967). The detection of CA in human schistosomiasis using MAb and a sensitive antigen-capture sandwich ELISA has been recently reported (Deelder et al. 1989). A major CA was shown to be a negatively charged polysaccharide and was designated circulating anodic antigen (Deelder et al. 1976). A second CA has the characteristics of a polysaccharide with a cathodic migration and was designated circulating cathodic antigen (Deelder et al. 1976). Both CA are extracted from adult worms in the TCA-soluble fraction and are also present in the E + S antigen obtained from schistosome adult worms incubated in culture medium (Deelder et al. 1980). They are heat stable and resistant to proteolytic enzymes, DNase, and RNase, but are destroyed by periodate treatment (Gold et al. 1969; Nash 1974; Carlier et al. 1978; Nash and Deelder 1985). In the present study we have developed a sensitive antigen-capture sandwich ELISA using an IgM MAb that detects antigenemia in the serum during murine schistosomiasis mansoni. MATERIALS
AND METHODS
Animals and sera. Female CBA/J mice and BALBlc mice were obtained under the auspices of the Veterans Administration through a National Cancer Institute supply conttact and CFl mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). All animals were housed in facilities approved by the American Association for Accreditation of Laboratory Animal Care. Mice were infected subcutaneously with S. mansoni cercariae (Puerto Rican strain). At various times after infection, mice were bled from the retroorbital chamber while under ether anesthesia and then further euthanized by intraperitoneal injection of sodium pentobarbital and perfused for worm counts. Blood was allowed to clot. Sera obtained by centrifugation were stored at -20°C until used.
AND KAMAL Preparation of E + S antigen. E + S was prepared as described by Rotmans et al. (1981). Briefly, adult worms obtained from mice infected for 8 weeks with S. mansoni were incubated under sterile conditions in petri dishes with RPM1 1640 medium (GIBCO) without serum and with 3% penicillin-streptomycin (GIBCO) for 2U8 hr in 5% CO, at 37°C. Supematant fluids from the cultures were centrifuged at 12OOgfor 10 min, dialysed in Dulbecco’s PBS with two changes of buffer, and concentrated in an Amicon concentrating chamber (Amicon, Danvers, MA) with 10,000 MW cut-off membrane (Pharmacia, Piscataway, NJ). The E + S antigen was filter sterilized, ahquoted, and kept frozen at -20°C. Protein content was determined by the Lowry method (Lowry et al. 1951) and carbohydrate content was determined by the phenolsulfuric acid method (Dubois et al. 1956). Preparation of monoclonal antibody. BALBlc mice were immunized with adult schistosome worm antigen preparation (SWAP) (Colley et al. 1977) in complete Freund’s adjuvant with subsequent injections in incomplete Freund’s adjuvant. Sensitized spleen cells were fused with murine myeloma cells 8653 using polyethylene glycol as described previously (Barsoum et al. 1989). After HAT selection, hybridomas were screened for antibodies against E + S, soluble egg (SEA), and SWAP by ELISA. Hybrids that were highly reactive to E + S, with or without reactivity to SWAP and with minimal reactivity to SEA, were cloned by limiting dilution, and their isotypes were determined using an anti-mouse immunoglobulin subclass kit (Zymed laboratories, San Francisco, CA). Antibody production by the clones was retested by ELISA and hybridoma cells were injected intraperitoneally into BALB/c mice for ascites production. MAb SHll/Bl (an IgM) was purified from ascitic fluids by a simple procedure of euglobulin precipitation in distilled water (Garcia-Gonzalez et al. 1988). Antigen-capture sandwich ELISA. Wells of microtiter plates (Immulon-2) (Dynatech Laboratories, Chantilly, VA) were coated with 50 pl of MAb (10 kg/ml) in carbonate buffer, pH 9.6, and incubated for 2 hr at room temperature (RT) and overnight at 4°C. Plates were washed with PBS-Tween 20 (O.OS%), incubated for 45 min with PBS-milk (5%), and washed. Mouse serum was tested untreated or treated with 2% TCA to precipitate protein and/or dissociate CA from immune complexes as described by De Jonge et al. (1987). Briefly, equal volumes of serum and 4% TCA were mixed at RT for 15 min and centrifuged at 10,OOOgfor 5 min. The supematant was collected, neutralized with carbonate buffer (pH 9.6), added to the wells, and incubated for 3040 min at RT. Plates were washed and an optimum dilution of the same MAb conjugated to biotin (Pierce, Rockford, IL) was added, incubated, and washed as before. Avidin-peroxidase (Sigma Chemical Co., St. Louis, MO) was added to
DETECTION
OF
CIRCULATING
the wells, and plates were incubated and washed as above. ABTS peroxidase substrate (Rirkegaard and Perry Laboratories, Gaithersburg, MD) was added and the blue-green color was read at the 410-nm wavelength using an MR 600 ELISA reader (Dynatech). Readings that were above mean -1- 3 SD of control wells were considered positive. Pools of normal mouse serum and serum from mice infected for 8 weeks were included as controls in each assay. Biotinylation of MAb 5HllIBl. The procedure was done as recommended by the manufacturer (Pierce). Briefly, MAb 5Hll/Bl was dialyzed in bicarbonate buffer, pH 8.5, and mixed with NHS-LC-biotin for 2 hr in ice. The mixture was centrifuged at 1OOOgin Amicon centrifuge tubes (CentriconMicroconcentrators, Amicon), resuspended in bicarbonate buffer and mixed again with NHS-LC-biotin, incubated, and centrifuged as before. Biotinylated 5Hl l/B 1 was washed 2x in PBS and the protein content was determined by absorption at the 280~nm wavelength and adjusted to 2 mg protein/ml. RESULTS
Selection
of h4Ab 5HllIBI
From a panel of nine positive hybridomas, three were selected that strongly reacted with E + S with some crossreactivity with SWAP and minimal reactivity with SEA. These hybrids were cloned and injected into BALB/c mice for ascites production. Two of these MAbs were IgM (4F7/FlO and 5Hl l/Bl) and one was IgG2a (2G2/D9). The reactivity of these MAbs with SEA, SWAP, and E + S is shown in Fig. 1. MAb 5H1 l/B1 showed the strongest reaction with E + S and was selected as both the capture Ab and the second Ab conjugated with biotin. MAb 5HlllBl was tested against E + S untreated or treated with sodium periodate as described by Stewart et al. (1977). Briefly, 0.02 M sodium periodate was dissolved in 0.1 M sodium acetate and the pH was adjusted to 4.5 with glacial acetic acid. Equal volumes of E + S and sodium periodate buffer were mixed and incubated in ice for 2.5 hr and dialyzed in PBS. Both untreated and periodate-treated antigens were diluted in sodium carbonate buffer (pH 9.6) to 10 pg protein/ml and added to the wells. The
ANTIGENS
IN
109
SCHISTOSOMIASIS
@ I 0
4F7/FIO
5Hll/Bl
SEA E+S SWAP
2G2/D9
FIG. 1. Reactivity of MAbs 4F7/FlO (IgM), 5Hl l/B1 (IgM), and 2G2/D9 (IgG,a) (1: 160 dilution) against soluble egg antigen (SEA), excretorysecretory (E + S) antigen, and soluble worm antigen preparation (SWAP) measured by ELISA.
treatment of E + S with sodium periodate completely abrogated the reactivity of 5Hll/Bl with E + S as shown in Fig. 2. The Effect of Immune Complexes on the Detection of E + S in Mouse Serum E + S (40 &ml) was added to normal, 8-week and ldweek-infected mouse sera and each tested either as such (untreated) or after pretreatment with 2% TCA. In untreated samples, normal mouse serum showed the highest E + S level followed by 16-week-infected serum while &weekinfected serum showed the lowest level of detectable E + S (Fig. 3). When the sera were pretreated with TCA, all sera showed parallel levels of E + S which were com-
060: OSa 04z 030.2Ol-
FIG. 2. Reactivity of MAb 5HlllBl against E + S antigen untreated (0) or treated (0) with 0.02 M sodium periodate as measured by ELISA.
110
BARSOUM,
COLLEY,
0.1 -
8
16
32 RECIPROCAL
64
128
256
512
OF DILUTIONS
FIG. 3. Detection of E + S (40 pg protein/ml) in normal mouse serum (ff; NMS), 8-week-infected (0; I-week MS), and lbweek-infected mouse serum (0; ldweek MS) by sandwich ELISA using 5HlllBl without treatment with 2% trichloroacetic acid (TCA).
parable to the added levels seen in untreated normal serum as shown in Fig. 4. When 8- and 16-week-infected sera were tested for anti-E + S Abs (on E + S-coated plates), 8-week-infected serum showed a higher response than 16-week-infected serum (OD readings at I:1000 serum dilution of 0.54 vs 0.18 for 8-week and 16weekinfected sera, respectively). E + S in Sera of CBAIJ Mice before and after Praziquantel Treatment CBA/J mice infected 8 weeks earlier with 45 cercariae of S. mansoni were treated with praziquantel. The drug was given orally in three doses of 100 mg/kg body wt
AND
KAMAL
at 4-hr intervals (Tawfik et al. 1986). A control group was left untreated. At weekly intervals mice were bled, perfused, and the number of worms counted. Mesenteric venules were examined and livers were examined after pressing between glass plates to assure complete perfusions. Each week, sera were pooled from a group of three untreated mice and from a group of three treated mice and tested for CA after TCA exposure. CA was detected in both groups before treatment. A significant drop in CA was observed on the first week after treatment (Fig. 5). This decreased level of CA in the sera of treated mice continued through the end of the experiment 4 weeks after treatment. The level of CA in the sera of untreated mice did not alter over this time period (Fig. 5). The numbers of worms perfused from mice the first week after treatment were 3.3 * 0.6 (mean + SEM) and 0.6 + 0.6, in the control and treated groups, respectively. The mean worm counts of the mice perfused the fourth week after treatment were 5.6 2 2.0 in the control group and 0 in the treated group. Similar results were obtained in a second experiment. Decreases in the levels of CA were observed by the first day after treatment and re0.61 0.5
0.6 -
0.4
NMS
5
-16WkMS
0.5 -
o---o
;
8 Wk MS
0.3
\.
0.4 -
2
E
0.2
$j 0.3 -
01 z
02-
0 01 o-
0
O-Control
GD
.-Treated
Gp
bc-5 Before
Treatment
FIG.
b
1’6
I28 256 i, 6’4 RECIPROCAL OF DILUTIONS
512
FIG. 4. Detection of E + S (40 pg protein/ml) in NMS, 8-week MS, and 16-week MS by sandwich ELISA using 5HlllBl after pretreatment with 2% TCA.
5. Detection
1 WEEKS
2
AFTER
3
4
TREATMENT
of E + S in sera of mice infected
with 45 S. mansoni cercariae 8 weeks before treatment and either treated orally with praziquantel(lO0 m&g body wt, three doses at 4-hr intervals) (0; treated gp) or untreated (0; control gp). Serum was pooled from a group of three mice at each time interval and pretreated with 2% TCA.
DETECTION
OF
CIRCULATING
ANTIGENS
mained low through the next 4 weeks (data not shown). Correlation between Worm Burden and the Level of Antigenemia in Mice CFI mice were divided into four groups of 12 mice each and infected with 200, 100, 50, or 25 S. mansoni cercariae. Eight weeks after infection, mice were bled and sera collected for detection of CA after TCA treatment of serum. Mice were then euthanized using sodium pentobarbital and perfused for worm counts. Figure 6 shows a scatter plot of the number of worms from each mouse, relative to the level of antigenemia detected in the TCA-treated serum of that mouse by sandwich ELISA using MAb 5Hl l/Bl. A strong coefficient of correlation (r = 0.80) was found between the two parameters (n = 42). DISCUSSION
We have developed an antigen-capture sandwich ELISA using a new MAb to an epitope on an E + S antigen of adult schistosome worms. A significant correlation was found between the number of worms perfused from mice and their circulating levels of CA. Recent reports have shown strong correlation between fecal egg counts and detection of a CA (Deelder et al. 1989) 1.0,
I .
0.8 .
WORM
.
.
COUNT
FIG. 6. Correlation between the worm count and the detection of CA in individual mice. Mice were infected with 200, 100, 50, or 25 S. mansoni cercariae 8 weeks earlier. Individual mice were bled and perfused for worm count. Serum was used to detect CA after 2% TCA pretreatment (n = 42).
IN
SCHISTOSOMIASIS
111
and a reduction in the level of CA after treatment with praziquantel in human schistosomiasis (De Jonge et al. 1989). These authors have used an IgG, MAb as the capture Ab and the same MAb conjugated with alkaline phosphatase as the second Ab in a sandwich ELISA or an indirect hemagglutination (IHA) assays. In their studies using the IHA, CA was detectable only in patients excreting >500 eggs/g of feces, while they could detect CA corresponding to a level of 10 eggs/g of feces using the sandwich ELISA. However, Deelder et al. (1989) stated that anti-CAA IgM MAbs did not perform well in the ELISA assay. We have successfully used an IgM MAb (5Hll/Bl) as the capture Ab and the same biotin-conjugated MAb as the second Ab. In a previous study, a direct relation was found between the number of worms in hamsters and the amount of antigen in the serum (Gold et al. 1969). Because the same MAb is used to capture and detect CA, it is likely that a repeating epitope on an E + S is being detected by 5Hl l/Bl. This epitope is almost certainly a carbohydrate because it withstands 2% TCA treatment and is sensitive to sodium periodate treatment (Fig. 2). It is evident from our results (Figs. 3 and 4), as well as those of others (De Jonge et al. 1988; Deelder et al. 1989), that TCA treatment of sera from infected animals or patients dissociates these CA from immune complexes that are formed between antibody and antigen during the infection and allows their detection. In our study, the level of detection of added E + S in 8week-infected mice is presumed to be low before TCA treatment because of the existence of antibodies that form immune complexes (Fig. 3). These levels approximated the same level as in normal serum after TCA treatment of the serum (Fig. 4). Using our MAb in this system, we demonstrated a significant decrease in the level of CA detected after treatment of infected mice with praziquantel (Fig. 5). Previous
112
BARSOUM,
COLLEY,
studies in murine schistosomiasis have also demonstrated a rapid decrease in the level of antigenemia (72-96 hr) following treatment with praziquantel (Weltman 1982; Abdel-Hafez et al. 1983). Similar results were obtained in human schistosomiasis mansoni by De Jonge et al. (1989), using an IgG, MAb alone and conjugated with alkaline phosphatase in a sandwich ELISA. Based on these results, the sandwich ELISA using MAb 5Hl l/B 1 appears to be a sensitive assay for the detection of CA in murine schistosomiasis. This system is currently being evaluated with sera from patients with schistosomiasis and other infections. In preliminary studies it appears to have promise in the immunodiagnosis of human schistosomiasis and in the monitoring of chemotherapy and possible future vaccines. ACKNOWLEDGMENT
The authors gratefully acknowledge the secretarial and administrative assistance of Judith O’Connell. This work was supported by a grant from the Department of Veterans Affairs and the Department of Defense. REFERENCES
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