Localization of Antigen within Eggs of Schistosoma japonicum That Participate in Circumoval Precipitin (COP) Reaction

Abstract

The circumoval precipitin (COP) reaction was first reported by Oliver-Gonzales (1954, J. Infect. Dis. 95: 86-91) who observed precipitates around living schistosome eggs when they had been placed in homologous sera from infected humans or experimental animals. Since then, this reaction has been applied as an immunodiagnostic test for human schistosomiasis japonica (Yokogawa et al., 1967, Japan. J. Parasitol. 16: 77-84; Yogore et al., 1968, Am. J. Trop. Med. Hyg. 17: 65-71). Because the COP test requires only minimal equipment and has high specificity, this test is useful both for laboratory diagnosis and field survey in endemic areas (Tanaka, 1976, Southeast Asian J. Trop. Med. Publ. Hlth. 7: 176-179; Yogore et al., 1978, Southeast Asian J. Trop. Med. Publ. Hlth. 9: 344-355). Immunochemically, the COP was shown to be an antigen-antibody complex by demonstrating immunoglobulin in the precipitate (Sala et al., 1962, Proc. Soc. Exp. Biol. Med. 117: 921-923; Hillyer and Marrero, 1980, Am. J. Trop. Med. Hyg. 29: 1249-1253; Demaree and Hillyer, 1981, Am. J. Trop. Med. Hyg. 30: 402-405). Hillyer and Pelly (1980, Am. J. Trop. Med. Hyg. 29: 582-585) and Cruise et al. (1981, AJEBAK 59: 503-514) reported that some specific murine hybridoma-derived antibodies produced the COP reaction with S. mansoni eggs or S. japonicum eggs. However, the chemical and physical nature of the antigen(s) involved in the precipitate formation has not been clearly characterized. It is known the eggs retain reactivity in the COP test even after lyophilization (Sala et al., 1962, Am. J. Trop. Med. Hyg. 11: 199) or heating at relatively high temperature (Kamiya, 1980, Japan. J. Vet. Res. 28: 149-154). Interestingly, von Lichtenberg (1964, Am. J. Pathol. 95:75-93) has demonstrated that the autoclaved eggs possessed the antigenicity and induced egg-granulomas in the lung of injected mice. This may suggest a possible role of heat-stable antigen(s) within the eggs. Kamiya and Kamiya (1980, Japan. J. Vet. Res. 28: 155-1-60) indicated the location of immunofluorescent-stainable antigen in the eggs in paraffin sections of liver tissue. However they did not examine tissue heated at a temperature higher than 58 C. In the present study, we examined the heat-stability of the egg antigen in the COP reaction and attempted to define its localization within the eggs by the indirect immunofluorescent technique. The COP tests were carried out essentially as described by Oliver-Gonzales (1954, loc. cit.). Eggs of Schistosomajaponicum (Philippine strain) were isolated from the intestines of the dd strain of mice infected for 8 to 9 wk and were lyophilized according to the method of Matsuda et al. (1977, Japan. J. Exp. Med. 47: 369-375). Eggs of the same lot were used through the experiment and some of the eggs were autoclaved at 121 C for 15 min. The criteria of positivity and classification of precipitates in COP test proposed by Yokogawa et al. (1967, loc. cit.) were adopted. The titration of serum was done by determining the highest dilution at which positive reaction was still detectable. Cryostat sections of infected mouse liver containing S. japonicum eggs were examined by indirect immunofluorescence (IIF). Livers from the mice infected for 8 to 9 wk with S. japonicum (Philippine strain) were cut into small pieces (5 mm) and were embedded in 1.5% agarose (Sigma, St. Louis, Missouri, low gelling temperature type) in phosphate-buffered saline (PBS). They were then rapidly frozen in n-hexane with an acetone-dry-ice mixture at -70 C and were sectioned on a cryostat microtome at 5 um and placed on microscope slides. The specimens were fixed in 95% ethanol for 5 min and some of the fixed sections were autoclaved at 121 C for 15 min. The liver sections were layered with serial dilutions of test sera from rabbits for 30 min at 37 C in a moist chamber. After three separate,