Proc. Nati. Acad. Sci. USA Vol. 75, No. 11, pp. 5715-5717, November 1978
Medical Sciences
Immunodiagnosis of infection with Schistosoma mansoni: Enzymelinked immunosorbent assay for detection of antibody to circulating antigen (polylysine-polysaccharide binding/worm burden and antibody relationship)
G. H. KELSOE AND T. H. WELLER Departments of Tropical Public Health and Microbiology, Harvard School of Public Health, Boston, Massachusetts 02115
Contributed by Thomas H. Weller, September 7,1978
ABSTRACT A circulating antigen, a negatively charged polysaccharide from the trematode Schistosoma mansoni, was noncovalently bound to the surface of poly(L-lysine).coated wells in polystyrene trays, which were then used in a micro-enzymelinked immunosorbent assay (ELISA) test. The method provides an immunodiagnostic test for schistosomiasis of exceptional sensitivity with a high degree of specificity. Comparison of Bell egg counts and ELISA titers revealed a good correlation (rn 0.80) in young individuals with low to moderate worm burdens, but this relationship was less marked in older individuals or those with high egg counts. Schistosomiasis, a debilitating disease caused by infection with one of several species of the trematode Schistosoma, is spreading due to proliferation of man-made environments suitable for the molluscan intermediate hosts. The concurrent recognition that schistosomiasis is an impediment to the economic growth of nations in the tropics has stimulated research. Morbidity in the individual with schistosomiasis often is directly related to the worm burden. Classically, the specific diagnosis of infection is based upon the microscopic demonstration of eggs, and an assessment of the worm burden is provided by quantification of eggs in the feces or urine. Schistosomes elaborate an anodic antigen (C-Ag) detectable in the serum of infected animals (1), the amount of which is related to the number of worms present (2, 3). This observation suggested that serodiagnosis of infection and determination of the worm burden in man might be assessed by quantification of circulating antigen or of antibodies against it. We report the development of an enzyme-linked immunosorbent assay (ELISA) technique for the detection and quantification of antibody to C-Ag and results obtained with sera from a population living in an area endemic for schistosomiasis. The findings are correlated with the results of fecal egg counts.
MATERIALS AND METHODS Antigen. Antigen was obtained from a Puerto Rican strain of S. mansoni by a modification of the method of Nash et al. (4). Six weeks after hamsters were inoculated intraperitoneally with 300400 cercariae, the worms were harvested (5), washed in 0.15 M NaCI, lyophilized, and stored at -20°C. Antigen(s) was extracted by homogenizing 200-30 mg of lyophilate with 10 ml of 0.1 M NaOH in a cold Tenbroek homogenizer until particulate matter disappeared (a minimum of 10 min). The homogenate was stirred for 2-3 hr at 4°C. After centrifugation at 10,000 X g for 30 min at 4°C, absolute ethanol was added to the supernatant to a final concentration of 75% and the mixture was stored overnight at 40C. The resulting precipitate was recovered by centrifugation, washed with absolute ethanol,
and redissolved in 10 ml of water. An equal volume of 20% trichloroacetic acid was added. The supernatant was separated by centrifugation, neutralized with 5 M NaOH, and dialyzed against water overnight at 4VC. The antigen-containing dialyzate was then Iyophilized and stored at -200C. The product was identical to the C-Ag described from this laboratory (1-3) as characterized by: rapid anodic migration under electrophoresis at pH 8.2; a photospectrometric absorption peak at 258 nm; development of a line of identity in Ouchterlony plates with whole-worm homogenates and with infected hamster serum when reacted against rabbit anti C-Ag and goat antiadult schistosome serum; and destruction of antigenicity by 0.05 M sodium metaperiodate at 40C. Enzyme-Labeled Antisera. Horseradish peroxidase-labeled goat anti-human immunoglobulin (GaHIg-HRP) (Cappel Laboratories, Dowington, PA) had activity against human y, a, and it heavy chain specificities. For use, GaHIg-HRP was diluted 1:50 with phosphate-buffered saline (Pi/NaCl), pH
7.2.
Enzyme Substrate. 5-Aminosalicyclic acid (100 mg) (Aldrich) was dissolved in 100 ml of hot (>900C) water. This solution was cooled and the pH was adjusted to 6 by the addition of 1 M NaOH. Prior to use, H202 (30%, Fisher) was added to
a final concentration of 0.005%. Assay Plates. Wells in polystyrene microtiter plates
(IS-
FB-96, Linbro Chemical, New Haven, CT) were processed by the addition of 0.1 ml (0.25 or 0.50 mg/ml) of poly(L-lysine) (30,000-70,000 daltons; Sigma, type VII-B) as described by Kennedy and Axelrad (6); polystyrene trays intended for tissue culture use do not require pretreatment with H2SO4. Then, 0.05 ml of an optimal concentration of antigen (200-400 jig/ml) diluted in P1/NaCl was introduced into each well; the plates were incubated for a minimum of 24 hr at 36°C and then stored at 4°C. Prior to use, plates coated with poly(L-lysine) and C-Ag were washed three times with Pi/NaCI and each well was filled with Pi/NaCl containing 5% fetal calf serum or bovine serum albumin and held for 24 hr at 4°C. Assay Procedure. P1/NaCl containing 5% fetal calf serum or bovine serum albumin and 0.001% Tween 20 was used as diluent for sera and for subsequent washes. The wells were emptied by suction and 0.05 ml of diluent was added. Sera (0.05 ml of 1:10 dilution) under test were added to one row and doubling dilutions made with a Microtiter diluter. The trays were sealed with cellophane tape and incubated for 1-1.5 hr at 37°C. Then they were emptied by suction, washed three times over a 45-min period at room temperature, and again Abbreviations: C-Ag, schistosomal circulating antigen; ELISA, enzyme-linked immunosorbent assay; GaHIg-HRP, goat anti-human immunoglobulin serum labeled with horseradish peroxidase; HRP, horseradish peroxidase; Pi/NaCl, phosphate-buffered saline, pH
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
7.2.
5715
5716
Proc. Natl. Acad. Sci. USA 75 (1978)
Medical Sciences: Kelsoe and Weller
Table 1. Lack of relationship* between ELISA positivity and presence of nonschistosomal helminthic infections
A 9.0
Individuals, no.
Intestinal helminths presentt H A T H A H T A T A -T H
C-Ag-ELISA-neg.; Bell tech.-neg.
C-Ag-ELISA-pos.;
15 16 5 4 6 2 7
4 2 2 0 0 1 2
4
1
-
7.0 .0
t
=
3.70, df = 7; P > 0.75.
H, hookworm; A, Ascaris lumbricoides; T, Trichuris trichiura.
emptied. GaHIg-HRP (0.05 ml) was added to each well. The trays were reincubated and washed as before. Finally, 0.1 ml of 5-aminosalicylic acid (1 mg/ml) was added to each well and allowed to react with bound GaHIg-HRP for 35-45 min; the reaction then was stopped by addition of 0.05 ml of 5 M NaOH. Readings (peroxidase activity results in a reddish brown color) were made within 45 min of the addition of NaOH by using an inverted mirror. Known negative and positive control sera were run in each test. Occasionally, a faint reaction occurred at a 1:20 dilution of a control serum. Therefore, reactivity at a 21:40 dilution was considered specific. Optimal concentrations of reagents were determined by box titration of each component, as outlined elsewhere (7). The concentration of antigen was particularly important; on a weight basis, batches of antigen varied 2 to 4-fold in reactivity. Test Sera. Human sera were provided by the HarvardWellcome/Federal University of Brazil/Pan American Health Organization project from Castro Alves, Bahia, Brazil, an area where Manson's schistosomiasis is highly endemic (8). Ascariasis, trichuriasis, and hookworm are also common. As described (8), data on the donors included the results of physical examinations, of fecal examinations for helminth eggs, and of S. nmnsoni egg counts by the Bell technique (9). Sera from patients with other tissue-invasive parasitic infections were coded and tested blindly. Control sera also were obtained from residents and patients in the Boston, MA, area. Analysis of ELISA Results. In examining the relationship between titers of anti C-Ag and worm burdens as reflected by Table 2. Results of C-Ag ELISA test on sera from patients with other parasitic diseases ELISA results No. tested Negative Reactive Diagnosis 1 1 0 Hydatid disease 2 2 0 Tropical eosinophilia 0 2 2 Clonorchiasis 8 8 0 Trichinosis 1 1 0 Eosinophilic meningitis 8 8 0 Amebiasis 2 2 0 Bancroftian filariasis 1 1 0 Onchocerciasis 1* 8 7 Visceral larval migrans 2 2 0 Strongyloidiasis It S. haematobium 2 1 1 1 S. japonicum 0 * Reactive at 1:40; Toxocara ELISA titer, 1:4096.
t Reactive at 1:640.
J
.:
p~~~~~
0 6.0
..--0) *; 5.0c,,
4.0'
j
*
00
8.0
Bell tech.-neg.
6n
0
1.0
2.0
3.0
4.( I
C
I
0
', 9.0 0.
Qi 8.0 7.0
6.0.
9
0
5.0 4.0 0
A
(6)
1.0
2.0
1.0 3.0 4.0 0 Bell count, loglo
2.0
3.0
4.0
FIG. 1. Linear regression analysis of the relationship between C-Ag ELISA titers (log2) and Bell counts (loglo) by age group. (A) 0-9 yr; b = 5.04; m = 0.92; r = 0.79. (B) 10-19 yr; b = 5.06; m = 0.92; r = 0.78. (C) 30-39 yr; b = 5.77; m = 0.22; r = 0.33. (D) > 40 yr; b = 5.71; m = 0.25; r = 0.21. Value below broken line indicates the number of seronegative individuals. Points enclosed by solid lines represent individuals with Bell counts ' 1265 eggs per g of feces and are excluded from regression analysis. Not illustrated is the 20-29 age group (b = 5.10; m = 0.62; r = 0.70; N = 37). The slope and ordinate intercept of the regression line are m and b, respectively.
counts, individuals were grouped into 10-year age cohorts with the exception of those over 40 years in age. For linear regression analysis, the reciprocal of the ELISA titer (log2) was plotted versus the Bell egg count (loglo). Individuals that were ELISA-positive but Bell count-negative were analyzed for a possible correlation between ELISA positivity and the presence of other helminthic infections. Statistical analyses were performed as described by Snedecor and Cochran (10). RESULTS All sera from the 134 Brazilian individuals who were excreting schistosome eggs were positive in the C-Ag ELISA test. Seventeen percent (12/71) of sera from Bell test-negative individuals in the endemic area also were C-Ag ELISA-positive (X2 = 156.4; P < 0.005). All 24 control sera from individuals residing in a nonendemic area were negative. To determine if ELISA seroreactivity of individuals who were Bell test-negative reflected the presence of other helminthic infections (ascariasis, trichuriasis, or hookworm), those negative in both tests were compared to those who were -Bellnegative but ELISA-positive (Table 1). No correlation with a specific infection or combination of infections could be demonstrated. The specificity of the C-Ag ELISA was further investigated by testing sera from individuals infected with various parasitic egg
Medical Sciences: Kelsoe and Weller agents (Table 2). Of 38 sera tested, 36 were nonreactive and 2 exhibited C-Ag ELISA reactivity. These two were from an individual infected with another species of schistosome and one with visceral larva migrans (Toxocara ELISA titer, 1:4096). Serum from an individual with an acute Salmonella typi infection reacted at 1:40. Fig. 1 illustrates the relationship between the anti C-Ag antibody titer and the Bell egg count in individuals by age group. The striking correlation in the two youngest age groups (r = 0.79 for 1-9 yr; r = 0.78 for 10-19 yr) decays significantly by the fourth decade of life (r = 0.70 for 20-29 yr; r = 0.33 for 30-39 yr; r = 0.21 for >40 yr). The correlation also is less marked in younger individuals who are excreting eggs in excess of 1265 eggs per g of feces. However, the basic capacity of the test to discriminate between schistosome-infected and uninfected individuals is unaltered by age or by egg output. DISCUSSION We describe an ELISA method for the recognition and quantification of antibodies against a specific circulating schistosomal antigen. ELISA methods for diagnosis of parasitic infections, and for schistosomiasis in particular, have been reviewed (7). In the past these have utilized crude worm or egg extracts as antigens. Because C-Ag is a polysaccharide, it does not adhere to polystyrene. We chose to bind C-Ag to polystyrene indirectly by utilizing poly(L-lysine). In our hands, ionic bonding is simple and reproducible, and this approach should broaden the spectrum of antigens applicable to the ELISA method. The C-Ag utilized here is secreted by cells lining the gut of the schistosome (11, 12), is elaborated by developing schistosomules (13) as well as adult worms, and is demonstrable in the serum of animals in advance of maturation of worms and egg desposition (3). Antigenic stimulation of the host, therefore, is initiated with cercarial invasion, and its duration and degree reflect the worm burden (2, 3). The difficulties of detecting schistosome eggs in light infections by the classical methods of fecal examination are recognized. A sensitive technique for the detection of specific C-Ag antibody-provided that specificity was not negated by the presence of like antigens in other pathogens-would greatly improve the capacity to detect light infections and should be particularly useful in the field in assessing changes in incidence of infection in the course of control operations. The C-Ag ELISA test appears to meet these criteria. A high order of sensitivity is indicated because all schistosome eggexcreters examined were positive; it is noted that 29 had low egg counts. Furthermore, 12 of 71 "non-egg-excreters" living in the endemic area also were positive; it is probable that repetitive fecal examinations would have detected eggs in many of these individuals, particularly because analysis indicated that the specificity of the test was not influenced by intestinal helminthic infections common in rural Brazil. The limitations of the test in terms of specificity require further definition. Crossreactivity in other human schistosomal infections is to be expected (4). Of 27 sera from patients with nine other tissue-invasive helminthic infections plus 8 sera from patients with amebiasis, only 1 reacted at a minimal diagnostic level-Le., a 1:40 titer obtained with serum from a patient with visceral larval migrans who had a high specific Toxocara ELISA titer. Serum from a patient with typhoid fever reacted at a 1:40
Proc. Nati. Acad. Sci. USA 75 (1978)
5717
dilution, perhaps reflecting a common antigenic determinant. Because the C-Ag stimulus to the host is proportional to the worm burden, it was important to ascertain if worm burdens might be assessed immunologically rather than by laborious egg counts. A concordantly proportional specific antibody response
was demonstrable within certain limits of age and worm burden. Under the hyperendemic conditions prevailing in the Brazilian population studied, the correlation between antibody and egg output decayed after the third decade of life, an observation consistent with a state of hyporesponsiveness due to prolonged antigen stimulation (14). The decrease in ELISA reactivity in individuals excreting more than 1265 eggs per g of feces may be associated with antigenic overloading (15) or may reflect antigen-antibody complexing. Immunological hyporesponsive states associated with other schistosomal antigens have been described (16). The diminished response has been suggested as a factor in the pathology and epidemiology of schistosomiasis (17). The use of several antigens to map and analyze the onset of hyporesponsive states in chronic schistosomiasis would be an important contribution to knowledge of its immunopathology. A full understanding of the humoral response to C-Ag must await the availability of sensitive techniques to assay specific circulating antigens per se and antigen-antibody complexes. The Harvard-Wellcome/Federal University of Brazil/Pan American Health Organization project in Bahia, Brazil, provided sera from a defined population living in an area endemic for schistosomiasis; these were collected and quantitative fecal examinations were carried out by Drs. J. Lehman, K. Mott, and R. Hoff. Sera from individuals infected with other parasitic entities were received from Drs. F. Neva (National Institutes of Health), L. Glickman (Cornell University), and I. Kagan (Center for Disease Control). G.H.K. is the recipient of a Rockefeller Foundation Fellowship. 1. Berggren, W. L. & Weller, T. H. (1967) Am. J. Trop. Med. Hyg.
16,606-612.
2. Gold, R., Rosen, F. S. & Weller, T. H. (1969) Am. J. Trop. Med.
Hyg. 18,545-552. 3. Bawden, M. P. & Weller, T. H. (1974) Am. J. Trop. Med. Hyg. 23, 1077-1084. 4. Nash, T. E., Prescott, B. & Neva, F. A. (1974) J. Immunol. 112, 1500-1507. 5. Radke, M. G., Berrios-Duran, L. A. & Moran, K. (1961) J. Parasitol. 47, 366-368. 6. Kennedy, J. C. & Axelrad, M. A. (1971) Immunology 20,253257. 7. Voller, A., Bidwell, D. E. & Bartlett, A. (1976) Bull. W. H. 0. 53, 55-65. 8. Lehman, J. S., Jr., Mott, K. E., Morrow, R. H., Jr., Muniz, T. M. & Boyer, M. H. (1976) Am. J. Trop. Med. Hyg. 25,285-294. 9. Bell, D. R. (1963) Bull. W. H. 0. 29,525-530. 10. Snedecor, G. W. & Cochran, W. G. (1967) Statistical Methods (The Iowa State University Press, Ames, IA), 6th Ed. 11. Nash, T. E. (1974) Am. J. Trop. Med. Hyg. 23, 1085-1087. 12. Lichtenberg, F. v., Bawden, M. P. & Shealey, S. H. (1974) Am. J. Trop. Med. Hyg. 23,1088-1091. 13. Andrade, Z. A. & Sadigursky, M. (1978) Trans. R. Soc. Trop. Med. Hyg. 72, 316-318. 14. Dresser, D. W. (1962) Immunology 5,378-388. 15. Dresser, D. W. (1963) Immunology 6,345-355. 16. Boros, D. L., Pelley, R. P. & Warren, K. S. (1975) J. Immunol. 114, 1437-1441. 17. Capron, A., Camus, D., Dessaint, J. P. & LeBoubennec-Fischer, E. (1977) Ann. Immunol. (Paris) 128,541-556.
Medical Sciences
Immunodiagnosis of infection with Schistosoma mansoni: Enzymelinked immunosorbent assay for detection of antibody to circulating antigen (polylysine-polysaccharide binding/worm burden and antibody relationship)
G. H. KELSOE AND T. H. WELLER Departments of Tropical Public Health and Microbiology, Harvard School of Public Health, Boston, Massachusetts 02115
Contributed by Thomas H. Weller, September 7,1978
ABSTRACT A circulating antigen, a negatively charged polysaccharide from the trematode Schistosoma mansoni, was noncovalently bound to the surface of poly(L-lysine).coated wells in polystyrene trays, which were then used in a micro-enzymelinked immunosorbent assay (ELISA) test. The method provides an immunodiagnostic test for schistosomiasis of exceptional sensitivity with a high degree of specificity. Comparison of Bell egg counts and ELISA titers revealed a good correlation (rn 0.80) in young individuals with low to moderate worm burdens, but this relationship was less marked in older individuals or those with high egg counts. Schistosomiasis, a debilitating disease caused by infection with one of several species of the trematode Schistosoma, is spreading due to proliferation of man-made environments suitable for the molluscan intermediate hosts. The concurrent recognition that schistosomiasis is an impediment to the economic growth of nations in the tropics has stimulated research. Morbidity in the individual with schistosomiasis often is directly related to the worm burden. Classically, the specific diagnosis of infection is based upon the microscopic demonstration of eggs, and an assessment of the worm burden is provided by quantification of eggs in the feces or urine. Schistosomes elaborate an anodic antigen (C-Ag) detectable in the serum of infected animals (1), the amount of which is related to the number of worms present (2, 3). This observation suggested that serodiagnosis of infection and determination of the worm burden in man might be assessed by quantification of circulating antigen or of antibodies against it. We report the development of an enzyme-linked immunosorbent assay (ELISA) technique for the detection and quantification of antibody to C-Ag and results obtained with sera from a population living in an area endemic for schistosomiasis. The findings are correlated with the results of fecal egg counts.
MATERIALS AND METHODS Antigen. Antigen was obtained from a Puerto Rican strain of S. mansoni by a modification of the method of Nash et al. (4). Six weeks after hamsters were inoculated intraperitoneally with 300400 cercariae, the worms were harvested (5), washed in 0.15 M NaCI, lyophilized, and stored at -20°C. Antigen(s) was extracted by homogenizing 200-30 mg of lyophilate with 10 ml of 0.1 M NaOH in a cold Tenbroek homogenizer until particulate matter disappeared (a minimum of 10 min). The homogenate was stirred for 2-3 hr at 4°C. After centrifugation at 10,000 X g for 30 min at 4°C, absolute ethanol was added to the supernatant to a final concentration of 75% and the mixture was stored overnight at 40C. The resulting precipitate was recovered by centrifugation, washed with absolute ethanol,
and redissolved in 10 ml of water. An equal volume of 20% trichloroacetic acid was added. The supernatant was separated by centrifugation, neutralized with 5 M NaOH, and dialyzed against water overnight at 4VC. The antigen-containing dialyzate was then Iyophilized and stored at -200C. The product was identical to the C-Ag described from this laboratory (1-3) as characterized by: rapid anodic migration under electrophoresis at pH 8.2; a photospectrometric absorption peak at 258 nm; development of a line of identity in Ouchterlony plates with whole-worm homogenates and with infected hamster serum when reacted against rabbit anti C-Ag and goat antiadult schistosome serum; and destruction of antigenicity by 0.05 M sodium metaperiodate at 40C. Enzyme-Labeled Antisera. Horseradish peroxidase-labeled goat anti-human immunoglobulin (GaHIg-HRP) (Cappel Laboratories, Dowington, PA) had activity against human y, a, and it heavy chain specificities. For use, GaHIg-HRP was diluted 1:50 with phosphate-buffered saline (Pi/NaCl), pH
7.2.
Enzyme Substrate. 5-Aminosalicyclic acid (100 mg) (Aldrich) was dissolved in 100 ml of hot (>900C) water. This solution was cooled and the pH was adjusted to 6 by the addition of 1 M NaOH. Prior to use, H202 (30%, Fisher) was added to
a final concentration of 0.005%. Assay Plates. Wells in polystyrene microtiter plates
(IS-
FB-96, Linbro Chemical, New Haven, CT) were processed by the addition of 0.1 ml (0.25 or 0.50 mg/ml) of poly(L-lysine) (30,000-70,000 daltons; Sigma, type VII-B) as described by Kennedy and Axelrad (6); polystyrene trays intended for tissue culture use do not require pretreatment with H2SO4. Then, 0.05 ml of an optimal concentration of antigen (200-400 jig/ml) diluted in P1/NaCl was introduced into each well; the plates were incubated for a minimum of 24 hr at 36°C and then stored at 4°C. Prior to use, plates coated with poly(L-lysine) and C-Ag were washed three times with Pi/NaCI and each well was filled with Pi/NaCl containing 5% fetal calf serum or bovine serum albumin and held for 24 hr at 4°C. Assay Procedure. P1/NaCl containing 5% fetal calf serum or bovine serum albumin and 0.001% Tween 20 was used as diluent for sera and for subsequent washes. The wells were emptied by suction and 0.05 ml of diluent was added. Sera (0.05 ml of 1:10 dilution) under test were added to one row and doubling dilutions made with a Microtiter diluter. The trays were sealed with cellophane tape and incubated for 1-1.5 hr at 37°C. Then they were emptied by suction, washed three times over a 45-min period at room temperature, and again Abbreviations: C-Ag, schistosomal circulating antigen; ELISA, enzyme-linked immunosorbent assay; GaHIg-HRP, goat anti-human immunoglobulin serum labeled with horseradish peroxidase; HRP, horseradish peroxidase; Pi/NaCl, phosphate-buffered saline, pH
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
7.2.
5715
5716
Proc. Natl. Acad. Sci. USA 75 (1978)
Medical Sciences: Kelsoe and Weller
Table 1. Lack of relationship* between ELISA positivity and presence of nonschistosomal helminthic infections
A 9.0
Individuals, no.
Intestinal helminths presentt H A T H A H T A T A -T H
C-Ag-ELISA-neg.; Bell tech.-neg.
C-Ag-ELISA-pos.;
15 16 5 4 6 2 7
4 2 2 0 0 1 2
4
1
-
7.0 .0
t
=
3.70, df = 7; P > 0.75.
H, hookworm; A, Ascaris lumbricoides; T, Trichuris trichiura.
emptied. GaHIg-HRP (0.05 ml) was added to each well. The trays were reincubated and washed as before. Finally, 0.1 ml of 5-aminosalicylic acid (1 mg/ml) was added to each well and allowed to react with bound GaHIg-HRP for 35-45 min; the reaction then was stopped by addition of 0.05 ml of 5 M NaOH. Readings (peroxidase activity results in a reddish brown color) were made within 45 min of the addition of NaOH by using an inverted mirror. Known negative and positive control sera were run in each test. Occasionally, a faint reaction occurred at a 1:20 dilution of a control serum. Therefore, reactivity at a 21:40 dilution was considered specific. Optimal concentrations of reagents were determined by box titration of each component, as outlined elsewhere (7). The concentration of antigen was particularly important; on a weight basis, batches of antigen varied 2 to 4-fold in reactivity. Test Sera. Human sera were provided by the HarvardWellcome/Federal University of Brazil/Pan American Health Organization project from Castro Alves, Bahia, Brazil, an area where Manson's schistosomiasis is highly endemic (8). Ascariasis, trichuriasis, and hookworm are also common. As described (8), data on the donors included the results of physical examinations, of fecal examinations for helminth eggs, and of S. nmnsoni egg counts by the Bell technique (9). Sera from patients with other tissue-invasive parasitic infections were coded and tested blindly. Control sera also were obtained from residents and patients in the Boston, MA, area. Analysis of ELISA Results. In examining the relationship between titers of anti C-Ag and worm burdens as reflected by Table 2. Results of C-Ag ELISA test on sera from patients with other parasitic diseases ELISA results No. tested Negative Reactive Diagnosis 1 1 0 Hydatid disease 2 2 0 Tropical eosinophilia 0 2 2 Clonorchiasis 8 8 0 Trichinosis 1 1 0 Eosinophilic meningitis 8 8 0 Amebiasis 2 2 0 Bancroftian filariasis 1 1 0 Onchocerciasis 1* 8 7 Visceral larval migrans 2 2 0 Strongyloidiasis It S. haematobium 2 1 1 1 S. japonicum 0 * Reactive at 1:40; Toxocara ELISA titer, 1:4096.
t Reactive at 1:640.
J
.:
p~~~~~
0 6.0
..--0) *; 5.0c,,
4.0'
j
*
00
8.0
Bell tech.-neg.
6n
0
1.0
2.0
3.0
4.( I
C
I
0
', 9.0 0.
Qi 8.0 7.0
6.0.
9
0
5.0 4.0 0
A
(6)
1.0
2.0
1.0 3.0 4.0 0 Bell count, loglo
2.0
3.0
4.0
FIG. 1. Linear regression analysis of the relationship between C-Ag ELISA titers (log2) and Bell counts (loglo) by age group. (A) 0-9 yr; b = 5.04; m = 0.92; r = 0.79. (B) 10-19 yr; b = 5.06; m = 0.92; r = 0.78. (C) 30-39 yr; b = 5.77; m = 0.22; r = 0.33. (D) > 40 yr; b = 5.71; m = 0.25; r = 0.21. Value below broken line indicates the number of seronegative individuals. Points enclosed by solid lines represent individuals with Bell counts ' 1265 eggs per g of feces and are excluded from regression analysis. Not illustrated is the 20-29 age group (b = 5.10; m = 0.62; r = 0.70; N = 37). The slope and ordinate intercept of the regression line are m and b, respectively.
counts, individuals were grouped into 10-year age cohorts with the exception of those over 40 years in age. For linear regression analysis, the reciprocal of the ELISA titer (log2) was plotted versus the Bell egg count (loglo). Individuals that were ELISA-positive but Bell count-negative were analyzed for a possible correlation between ELISA positivity and the presence of other helminthic infections. Statistical analyses were performed as described by Snedecor and Cochran (10). RESULTS All sera from the 134 Brazilian individuals who were excreting schistosome eggs were positive in the C-Ag ELISA test. Seventeen percent (12/71) of sera from Bell test-negative individuals in the endemic area also were C-Ag ELISA-positive (X2 = 156.4; P < 0.005). All 24 control sera from individuals residing in a nonendemic area were negative. To determine if ELISA seroreactivity of individuals who were Bell test-negative reflected the presence of other helminthic infections (ascariasis, trichuriasis, or hookworm), those negative in both tests were compared to those who were -Bellnegative but ELISA-positive (Table 1). No correlation with a specific infection or combination of infections could be demonstrated. The specificity of the C-Ag ELISA was further investigated by testing sera from individuals infected with various parasitic egg
Medical Sciences: Kelsoe and Weller agents (Table 2). Of 38 sera tested, 36 were nonreactive and 2 exhibited C-Ag ELISA reactivity. These two were from an individual infected with another species of schistosome and one with visceral larva migrans (Toxocara ELISA titer, 1:4096). Serum from an individual with an acute Salmonella typi infection reacted at 1:40. Fig. 1 illustrates the relationship between the anti C-Ag antibody titer and the Bell egg count in individuals by age group. The striking correlation in the two youngest age groups (r = 0.79 for 1-9 yr; r = 0.78 for 10-19 yr) decays significantly by the fourth decade of life (r = 0.70 for 20-29 yr; r = 0.33 for 30-39 yr; r = 0.21 for >40 yr). The correlation also is less marked in younger individuals who are excreting eggs in excess of 1265 eggs per g of feces. However, the basic capacity of the test to discriminate between schistosome-infected and uninfected individuals is unaltered by age or by egg output. DISCUSSION We describe an ELISA method for the recognition and quantification of antibodies against a specific circulating schistosomal antigen. ELISA methods for diagnosis of parasitic infections, and for schistosomiasis in particular, have been reviewed (7). In the past these have utilized crude worm or egg extracts as antigens. Because C-Ag is a polysaccharide, it does not adhere to polystyrene. We chose to bind C-Ag to polystyrene indirectly by utilizing poly(L-lysine). In our hands, ionic bonding is simple and reproducible, and this approach should broaden the spectrum of antigens applicable to the ELISA method. The C-Ag utilized here is secreted by cells lining the gut of the schistosome (11, 12), is elaborated by developing schistosomules (13) as well as adult worms, and is demonstrable in the serum of animals in advance of maturation of worms and egg desposition (3). Antigenic stimulation of the host, therefore, is initiated with cercarial invasion, and its duration and degree reflect the worm burden (2, 3). The difficulties of detecting schistosome eggs in light infections by the classical methods of fecal examination are recognized. A sensitive technique for the detection of specific C-Ag antibody-provided that specificity was not negated by the presence of like antigens in other pathogens-would greatly improve the capacity to detect light infections and should be particularly useful in the field in assessing changes in incidence of infection in the course of control operations. The C-Ag ELISA test appears to meet these criteria. A high order of sensitivity is indicated because all schistosome eggexcreters examined were positive; it is noted that 29 had low egg counts. Furthermore, 12 of 71 "non-egg-excreters" living in the endemic area also were positive; it is probable that repetitive fecal examinations would have detected eggs in many of these individuals, particularly because analysis indicated that the specificity of the test was not influenced by intestinal helminthic infections common in rural Brazil. The limitations of the test in terms of specificity require further definition. Crossreactivity in other human schistosomal infections is to be expected (4). Of 27 sera from patients with nine other tissue-invasive helminthic infections plus 8 sera from patients with amebiasis, only 1 reacted at a minimal diagnostic level-Le., a 1:40 titer obtained with serum from a patient with visceral larval migrans who had a high specific Toxocara ELISA titer. Serum from a patient with typhoid fever reacted at a 1:40
Proc. Nati. Acad. Sci. USA 75 (1978)
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dilution, perhaps reflecting a common antigenic determinant. Because the C-Ag stimulus to the host is proportional to the worm burden, it was important to ascertain if worm burdens might be assessed immunologically rather than by laborious egg counts. A concordantly proportional specific antibody response
was demonstrable within certain limits of age and worm burden. Under the hyperendemic conditions prevailing in the Brazilian population studied, the correlation between antibody and egg output decayed after the third decade of life, an observation consistent with a state of hyporesponsiveness due to prolonged antigen stimulation (14). The decrease in ELISA reactivity in individuals excreting more than 1265 eggs per g of feces may be associated with antigenic overloading (15) or may reflect antigen-antibody complexing. Immunological hyporesponsive states associated with other schistosomal antigens have been described (16). The diminished response has been suggested as a factor in the pathology and epidemiology of schistosomiasis (17). The use of several antigens to map and analyze the onset of hyporesponsive states in chronic schistosomiasis would be an important contribution to knowledge of its immunopathology. A full understanding of the humoral response to C-Ag must await the availability of sensitive techniques to assay specific circulating antigens per se and antigen-antibody complexes. The Harvard-Wellcome/Federal University of Brazil/Pan American Health Organization project in Bahia, Brazil, provided sera from a defined population living in an area endemic for schistosomiasis; these were collected and quantitative fecal examinations were carried out by Drs. J. Lehman, K. Mott, and R. Hoff. Sera from individuals infected with other parasitic entities were received from Drs. F. Neva (National Institutes of Health), L. Glickman (Cornell University), and I. Kagan (Center for Disease Control). G.H.K. is the recipient of a Rockefeller Foundation Fellowship. 1. Berggren, W. L. & Weller, T. H. (1967) Am. J. Trop. Med. Hyg.
16,606-612.
2. Gold, R., Rosen, F. S. & Weller, T. H. (1969) Am. J. Trop. Med.
Hyg. 18,545-552. 3. Bawden, M. P. & Weller, T. H. (1974) Am. J. Trop. Med. Hyg. 23, 1077-1084. 4. Nash, T. E., Prescott, B. & Neva, F. A. (1974) J. Immunol. 112, 1500-1507. 5. Radke, M. G., Berrios-Duran, L. A. & Moran, K. (1961) J. Parasitol. 47, 366-368. 6. Kennedy, J. C. & Axelrad, M. A. (1971) Immunology 20,253257. 7. Voller, A., Bidwell, D. E. & Bartlett, A. (1976) Bull. W. H. 0. 53, 55-65. 8. Lehman, J. S., Jr., Mott, K. E., Morrow, R. H., Jr., Muniz, T. M. & Boyer, M. H. (1976) Am. J. Trop. Med. Hyg. 25,285-294. 9. Bell, D. R. (1963) Bull. W. H. 0. 29,525-530. 10. Snedecor, G. W. & Cochran, W. G. (1967) Statistical Methods (The Iowa State University Press, Ames, IA), 6th Ed. 11. Nash, T. E. (1974) Am. J. Trop. Med. Hyg. 23, 1085-1087. 12. Lichtenberg, F. v., Bawden, M. P. & Shealey, S. H. (1974) Am. J. Trop. Med. Hyg. 23,1088-1091. 13. Andrade, Z. A. & Sadigursky, M. (1978) Trans. R. Soc. Trop. Med. Hyg. 72, 316-318. 14. Dresser, D. W. (1962) Immunology 5,378-388. 15. Dresser, D. W. (1963) Immunology 6,345-355. 16. Boros, D. L., Pelley, R. P. & Warren, K. S. (1975) J. Immunol. 114, 1437-1441. 17. Capron, A., Camus, D., Dessaint, J. P. & LeBoubennec-Fischer, E. (1977) Ann. Immunol. (Paris) 128,541-556.
