Immunodiagnosis of infection with Schistosoma mansoni: Enzyme-

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 (ISFB-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 7.2. 5715 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. 5716 Medical Sciences: Kelsoe and Weller Table 1. Lack of relationship* between ELISA positivity and presence of nonschistosomal helminthic infections