TANNIC-ACID HEMAGGLUTINATION TEST WITH 7S AND 19S ANTIBODY IN SCHISTOSOMIASIS JAPONICA

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1 THE KURUME MEDICAL JOURNAL Vol. 13, No. 3, 1966 TANNIC-ACID HEMAGGLUTINATION TEST WITH 7S AND 19S ANTIBODY IN SCHISTOSOMIASIS JAPONICA SETSUKO TSUCHIYA* Department of Medical Zoology, Nippon Veterinary and Zootechnical College, Musashino-shi, Japan (Received for publication November 1, 1966) The tannic-acid hemagglutination (HA) test of schistosomiasis mansoni has been described by Kagan (1955)4'. He conducted detailed studies on hemagglutination in animals infected with Schistosoma mansoni. He subsequently found that the HA test was more sensitive than any other applicable test in animals infected with the parasite. In the field of the immunology of parasites, a scrupulous investigation has not been conducted on the mechanism of relationship between antigen and antibody participating in the immunological reaction, particularly the case of antibody. Multiplicity of antibody is a common phenomenon in the immunology of bacteria. It should also be noted in the immunology of parasites. The recent advancement of immunochemistrv has chemically proven the occurrence of multiplicity of antibody. Numerous experiments have been conducted on the assumption that the antibody produced by infection with parasites is contained mainly in the gamma-globulin fraction. The present investigation was performed on animals and human subjects infected with Schistosoma japonicum to clarify the immunological relationship between parasite and host in infection, taking only the limited scope of immunoglobulin into consideration. The immunoglobulin of schistosomiasis was divided into 7S and 19S antibody by column chromatography using DEAE cellulose. The HA test was carried out with the antibodies and red blood cells treated with tannic acid. The immunological relationship between these antibodies and the HA test was then investigated. MATERIALS AND METHODS A dog was infected with about 3, 000 cercariae of Schistosoma japonicum. Serum from the dog was harvested about 3 months later. In addition, serum was collected from persons who had schistosome eggs in their feces. These sera were used for this experiment. DEAF cellulose column chromatography Sober and Peterson'ss' method was applied in this examination. Initially, 10 ml of serum was dialysed against phosphate buffer solution (ph 6. 3, M) and * Introduced by Dr. K. Okabe, Professor of Parasitology, Kurume University School of Medicine. 140

2 HEMAGGLUTINATION TEST IN SCHISTOSOMIASIS 141 spread onto a 10 g DEAE cellulose column. Then stepwise elution was performed. The serum protein was determined by ultraviolet absorption at 280 mƒê and fractionated. The fraction eluted in M phosphate buffer solution, at ph 6. 3, was regarded as 7S antibody and that eluted in 0.4 M phosphate buffer solution, at ph 5.2, as 19S antibody. These antibodies were employed for this experiment. Decomposition with mercaptoethanol The presence of 7S and 19S antibody in the fractions obtained with DEAE cellulose was confirmed by reducing decomposition with 2-mercaptoethanol through the Chan and Deutsch's method (1960). Equal amounts of 0.1 M mercaptoethanol and the antibody were mixed, allowed to stand at room temperature for one hour for decomposition, and subjected to cellophane dialysis at low temperature. The dialysed material was further condensed with polyvinylpyrrolidone and examined for the disappearance of activity in the HA test. Ultracentrif ugation The antiserum was centrifuged at 40, 000 r. p. m. for 7 hours. A Hitachi centrifuge, model 40P, with a roter of was used. The resulting contents of the centrifuge tube were divided into five fractions, which were identified as fraction I, II, III, IV, and V beginning from the top. The HA test was carried out with each fraction. Paper electrophoresis In accordance with the method described by Mori and Kobayashi (1953), paper electrophoresis was applied to both the five fractions after the above-mentioned ultracentrif ugation and the four fractions separated by means of DEAF cellulose. It was continued for 7 hours at ph 8. 6, p=0.05 (Veronal buffer) at 0.3 ma/cm. Toyo Brand No. 51 filter papar was used and subsequently the paper was stained by B.P.B. Serological test A slightly modified Boyden's method was used for the HA test. 1) Red blood cells used : Human O-type red blood cells were washed 3 times with physiological saline solution and made to a 2 % suspension in physiological saline solution, ph ) Treatment of red blood cells with tannic acid : The 2 % red blood cell suspension was mixed with an equal amount of 1:50,000 dilution of tannic acid and placed in the water bath at 37 Ž for 10 minutes. The red blood cells were then washed 3 times with physiological saline solution, at ph 7. 2, and made to a 2 % suspension. 3) Sensitization of red blood cells : The antigen was added as a 1:4 ratio to the 2 % suspension of red blood cells treated with tannic acid. The mixture was permitted to stand at room temperature for 10 minutes for sensitization. Furthermore, the red blood cells were suspended in physiological saline solution, at ph 6. 4, containing 0.05% serum albumin, and centrifuged at 1, 200 r. p. m. for 3 minutes. The packed cells were then washed 3 times and made to a 2 % suspension in physiological saline solution of the same composition as previously mentioned. However, physiological saline solution was added to a suspension of red blood cells treated with tannic acid. The mixture was then subjected to the same steps as mentioned above so that a 2 % suspension of red blood cells may be obtained. 4) Dilution of test serum : Test serum was inactivated in a water bath at 56 Ž

3 142 SETSUKO TSUCHIYA for 30 minutes. Following the addition of 0.05% serum albumin, the serum was diluted in physiological saline solution, at ph 6. 4, so as to make 1:10, 1:20, 1:50, 1:100, 1:200, and 1:500 dilutions. 5) Procedures : The dilutions of serum, mentioned above, were distributed, in 0.5 ml amounts, into small test tubes. Each tube was instilled with 0.05 ml of 2 % suspension of sensitized red blood cells. The contents thoroughly mixed and the tubes were allowed to stand at room temperature. They were then examined for the occurrence of agglutination of red blood cells at the bottom of the tube. Results were read 1, 2, and 3 hours after mixing. RESULTS Fractions I, II, III, IV, and V, which had been obtained by ultracentrifugation, were compared by paper electrophoresis. Results are shown in Fig. 1. The results of HA test on these fractions are shown in Table I. Figure 1. Paper electrophoresis of ultracentrifuged fraction. Electrophoresis was continued for 7 hours at ph 8.6 (p=0.05, Veronal buffer). B. P. B. staining. Fractions III, IV, and V revealed a positive reaction and activity. Fraction V exhibited a positive reaction to a 1:100 dilution of antigen. Ultracentrif ugation of fractions I and II failed to separate the antibody which would take part in the HA test from the fractions.

4 HEM AGGLUTINATION TEST IN SCHISTOSOMIASIS 143 TABLE 1 Results of HA test to ultracentrifuged fractions Agglutination was confirmed after 1 hour. Fractionation was performed by DEAE cellulose column chromatography in stepwise elution. Fig. 2 shows the results obtained. The so-called 7S antibody and Figure 2. HA test of DEAE cellulose fraction. Elution of 5ml of Schistosoma japonicum antigen 2ml of DEAE cellulose, optical density at 280 mĐ

5 144 SETSUKO TSUCHIYA fractions B and C, which had been eluted in M phosphate buffer solution, and those eluted in 0.4 M phosphate buffer solution were used as materials. Canine serum with these materials was subjected to paper electrophoresis. The patterns of electrophoresis were stained with B. P. B. and compared. The results are shown in Fig. 3. Figure 3. Paper electrophoresis of DEAE cellulose fractions obtained from serum of a dog infected with Schistosoma japonicum. Electrophoresis was continued for 7 hours at ph 8.6 (Đ=0. 05, Veronal buffers). B.P.B. staining. Fractions A and D correspond to 7S and 19S antibodies, respectively. The HA test was also carried out on these fractions and the results shown in Table II. Positive reaction was revealed by both 7S and 19S antibody. When compared, the fraction containing 7S antibody was very weak and that containing 19S antibody was rather strong. HA tests were performed with crude antibody before fractionation obtained from 6 human cases with eggs of Schistosoma japonicum in their feces, and with 7S and 19S antibody obtained from these cases after fractionation. The results are shown in Table III.

6 HEMAGGLUTINATION TEST IN SCHISTOSOMIASIS 145 TABLE II Results of HA test to the fractions obtained from serum of a dog infected with Schistosoma japonicum by means of DEAE cellulose TABLE III Results of HA test to the sera of patients infected with Schistosoma japonicum It should be noted that in all cases the 19S antibody fraction gave positive HA test, even in a case which was positive for eggs in the feces but the whole serum TABLE IV Results of HA test to the fractions of serum after treatment by 2-mercaptoethanol

7 146 SETSUKO TSUCHIYA of which gave negative HA test. The 7S and 19S antibody used in this experiment were identified by reducing decomposition with 2-mercaptoethanol and examined for activity in HA test. Results are shown in Table IV, which indicates that the activity of the 19S fraction disappeared. DISCUSSION The relationship between the infection of Schistosoma japonicum and the host as far as immunoglobulin was concerned, has been studied. Antibody could not be separated by ultracentrifugation. The 7S and 19S antibody which had been obtained by DEAE cellulose in stepwise elution were then investigated. Initially it seemed necessary to identify 7S and 19S antibodies in the materials used for fractionation. Chan and Deutsch studied changes caused by dissociation in the reaction between 19S antibody and mercaptoethanol and used them for this discrimination of 19S from 7S antibody. It is generally accepted that 19S antibody loses its activity by treatment with mercaptoethanol. In this experiment, 7S antibody showed activity, but 19S did not. It was confirmed that the initial materials used were both 7S and 19S antibodies. In his analysis of rheumatoid factor, Adachi (1965) fractionated normal type-b human serum containing anti-a agglutinin by DEAE cellulose column chromatography. He carried out the hemagglutination test with 7S and 19S antibody obtained from the fractionation. As a result, he found that hemagglutination had been induced by the 7S antibody fraction at a low titer and by the 19S antibody fraction at a high titer. The present author performed the HA test of schistosomiasis japonica using 7S and 19S antibody which had been obtained from immunoglobulin by fractionation. In this test, it is of interest to note that when sera were collected from human subjects infected with Schistosoma japonicum and showing negative HA tests, and fractionated into 19S antibody, all of them gave positive HA tests with the antibody. This result was brought about by the participation of the antibody in the blood, and the reaction seemed to be induced by the increase of antibody titer. Change such as this is considered to be available for immunodiagnosis of schistosomiasis. Hereafter, it should be noted that emphasis be placed on the fact that the antibody be called gamma-glob ulin collectively when considering it as immunoglobulin. SUMMARY Sera were collected from dogs infected with Schistosoma japonicum. The 7S and 19S antibody were separated by DEAE cellulose column chromatography. Both antibodies were proven to have activity in the tannic-acid hemagglutination (HA) test, especially, 19S antibody which possessed a high activity. A case which had given negative test by the use of crude serum, gave positive test when 19S antibody was used. It has been clarified that the results of the HA test with 19S antibody coincided quite specifically with those of detection of schistosome eggs.

8 HEMAGGLUTINATION TEST IN SCHISTOSOMIASIS 147 ACKNOWLEDGMENTS The author wishes to express her gratitude to Professor Shigeya Shimizu, head of the Department of Medical Zoology, Nippon Veterinary and Zootechnical College, for his guidance in the preparation of this manuscript. The outline of this paper was read before the 25th general meeting of the Eastern Japan Division of the Japanese Society of Parasitology in REFERENCES 1) ADACHI, M.: Reactivity of rheumatoid factor with immune complexes. Japan J. Allergy, 14, , ) BOYDEN, S. V.: The adsorption of proteins on erythrocytes treated with tannic acid subsequent hemagglutination by antiprotein sera. J. exp. Med., 93, , ) CHAN, P.C., AND DEUTSCH, H. F.: Immunochemical studies on human Rh agglutinins. J. Immunol., 85, 37-45, ) KAGAN, I. G.: Hemagglutination after immunization with Schistosome antigen. Science, 122, ) MORI, I., AND KOBAYASHI, M.: (Handbook of paper chromatography. J Nankodo, Tokyo, pp , (In Japanese) 6) SOBER, H. A., GUTTER, F. J., WYCKOFF, M. M., AND PETERSON, E. A. : Chromatography of proteins. II. Fractionation of serum protein on anion-exchange cellulose. J. Amer. chem. Soc., 78, , 1963.