Immunohistochemical observation on the antigens inducing IgG and IgM antibodies against sparganum

, Kim, C H; , Choi, W S

doi:1991.29.4.339

Korean J Parasito > Volume 29(4):1991 > Article

Original Article


Korean J Parasitol. 1991 Dec;29(4):339-353. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1991.29.4.339
Copyright © 1991 by The Korean Society for Parasitology


Immunohistochemical observation on the antigens inducing IgG and IgM antibodies against sparganum
C H Kim and W S Choi^*
Department of Biology, College of Natural Sciences, Gyeongsang National University Chinju, Kyongsangnam-do 660-701, Korea.


Abstract
Localization and characterization of the antigenic components of sparganum which induced IgG and IgM antibodies in the host were studied by immunohistochemical techniques and SDS-PAGE and Western blotting. The antigen recognized by IgG antibody of rats or mice which were immunized by infection or injection of crude extracts of metacestodes of Spirometra erinacei, was located in the parenchyma of sparganum, especially at the cortex and around the calcareous corpuscles. The immunoreaction was demonstrated not only in the encysted fibrous wall of host but around the arterioles or venules in the connective tissue of host. The antigen recognized by IgM antibody of rats or mice was also observed in the parenchyma of sparganum and in the connective tissue of host. By 5-20% gradient SDS-PAGE and EIBT, we detected antigenic components by IgG and IgM antibodies of the rat or mouse immunized by infection or injection of crude extract of spargana. Twenty-three antigenic bands from crude extracts of spargana were recognized by IgG antibody and 15 components by IgM antibody of immunized rats. Out of the bands recognized by IgG and IgM antibodies, 15 were cross-reacted each other. Twenty components of excretory-secretory proteins from spargana were recognized by IgG, and 5 components by IgM antibody of immunized rats. By IgG and IgM antibodies of immunized mice, 16 components of crude extracts were recognized by IgG antibody and 9 components by IgM antibody. Twenty components of excretory-secretory preparation were recognized by IgG antibody and 5 components by IgM antibody. Thirteen components of crude extracts were cross-reacted by IgG antibody of rats and mice.

Figures


	Fig. 1 Procedure for preparation of crude antigen and excretory-secretory antigen from sparganum.


	Fig. 2 Procedure for preparation of primary antisera from rats and mice.


	Fig. 3 Procedure of immunohistochemical method in immunofluorescent assay and ABC immunoperoxidase assay.


	Fig. 4 Procedure of enzyme linked imunoassay(ABC immunoperoxidase method).

Fig. 5
Localization of antigenic components reacted with IgG antibody from rats by ABC immunoperoxidase assay and IFA. (Ew.: encyst wall, Mu.: muscle of host, T.: tegument, Sp.: sparganum, Etm.: epidermal transverse muscle, Elm.: epidermal longitudinal muscle, Pm.: parenchyme)

1. Antigenic components were demonstrated in parenchyma and subintegument in sparganum and host connective tissue by ABC immunoperoxidase assay (×20). Chocolate brown color indicates positive immunoreaction. Background was stained with hematoxylin.

2. Higher magnification of a portion of No. 1 (×40).

3. By indirect immunofluorescent antibody assay, green color indicates positive immunoreaction and red negative immunoreaction (×20).

4. Higher magnification of a portion No.

5. Positive reation was demonstrated in the host connective tissue(×40).

Fig. 6
Localization of antigenic components reacted with IgG antibody from the mouse by ABC immunoperoxidase assay and IFA. (Sp.: sparganum, T.: tegument, Mu: muscle of host)

1. Antigenic components were demonstrated in the parenchyme and subintegument of sparganum and host connective tissue around sparganum (×20) by ABC immunoperoxidase assay. Chocolate brown color indicates positive immunoreaction, background was stained with hematoxylin.

2. Higher magnification of a portion of No. 1 (×40).

3. By indirect immunofluorescent antibody assay, green color indicates the positive immunoreaction and red negative immunoreaction (×20).

4. Higher magnification of a portion No. 3. Positive reaction was demonstrated in the host connective tissue (×40).

Fig. 7
Localization of antigen in the parenchyme and tegument of a sparganum in the rat by ABC immunoperoxidase assay. Chocolate brown is positive immunoreaction, counterstain hematoxylin. (T.: tegument, Elm.: epidermal longitudinal muscle, Pm.: parenchyme, Etm.: epidermal transverse muscle, Ew.: encyst wall, C.: calcareous corpuscle)

1. Higher magnification of parenchyme of sparganum. An arrow indicates the immunoreacted cells(×100).

2. Higher magnification of a portion in the parenchyme(×200).

3,4. Higher magnification of the portion of tegument of sparganum(×100).

Fig. 8
Distribution of antigen reacted with IgG antibody from rats immunized with sparganum by ABC immunized with sparganum by ABC immunoperoxidase assay, counterstain hematoxylin. (Sp.: sparganum, Ct.: host connective tissue, Ew.: encyst wall, Elm.: epidermal longitudinal muscle, Ar.: arteriole, Mu.: host muscle tissue, Ve.: venule, T.: tegument)

1. Antigen was demonstrated in the parenchyme of sparganum and host connective tissue around sparganum(×20).

2. Higher magnification of a portion in the host connective tissue of 1(×40).

3. Higher magnification of the portion of arteriole and venule of host (×100).

4. Distribution of antigen reacted with IgG antibody from rats immunized for 4 weeks (×20).

5. Higher magnification of the portion of No. 4. An arrow indicates localization of the antigen (×100).

6. Higher magnification of the portion of host muscle tissue (×100).

Fig. 9
Distribution of antigen reacted with IgG antisera from the mouse immunized with sparganum by immunofluorescent antibody assay(IFA). (Ct.: host connective tissue, Elm.: epidermal longitudinal muscle, Mu.: host muscle tissue, Sp.: sparganum, T.: tegument)

1. Localization of antigen reacted with antisera(IgG) from mouse post-infected for 2 weeks. Antigen was found in the parenchyme of sparganum and inside of encyst wall. Green color indicates the positive immunoreaction and red negative immunoreaction (×20).

2. Higher magnification of a part in sparganum and host connective tissue (×40).

3. Higher magnification of host connective tissue localized between the muscle (×100).

4. Localization of antigen in sparganum and host tissue infected for 4 weeks (×20).

5. Higher magnification of the portion of 4. (×100).

6. Higher magnification of a portion of 5. Antigen was located around the host connective tissue and muscle (×100).

Fig. 10
Localization of antigenic components reacted with IgG antibodies by avidin-biotin complex immunoperoxidase method. (Ew.: encyst wall, Mu.: host muscle tissue, SP.: sparganum, T: tegument)

1. Distribution of antigen reacted with IgG antibody of rat immunized with sparganum (×20).

2. Distribution of antigenic components reacted with IgM antibody of rat (×20).

3. Distribution of antigenic components reacted with IgG antibody of mouse immunized with sparganum for 2 weeks (×40).

4. Distribution of antigenic components reacted with IgM antibody of mouse immunized for 2 weeks (×40).


	Fig. 11 Coomassie blue staining and silver staining of the crude extracts and excretory-secretory antigenic components from sparganum by 5~20% gradient SDS-PAGE. CE: crude extracts, ES: excretory-secretory, Co: Coomassie blue R 250, Si: silver stain


	Fig. 12 EITB of crude extracts and excretory-secretory antigenic components from sparganum reacted with antirat sera immunized with crude extracts and with sparganum. Rt G: antirat IgG, Rt M: antirat IgM, Co: control, CE: crude extracts, ES: excretory-secretory


	Fig. 13 EITB of crude extracts and excretory-secretory antigenic components from sparganum reacted with antimouse sera immunized with crude extracts and with sparganum. Mo G: antimouse IgG, Mo M: antimouse IgM, Co: control, CE: crude extracts, ES: excretory-secretory

References


1.	Anders K, Foley K, Stern E, Brown WJ. Intracranial sparganosis: an uncommon infection. Case report. J Neurosurg 1984;60(6):1282–1286.

2.	Araki J, et al. Jpn J Parasit 1976;25(5):1–7.

3.	Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254.

4.	Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981;112(2):195–203.

5.	Rodriguez-Carbajal J, Salgado P, Gutierrez-Alvarado R, Escobar-Izquierdo A, Aruffo C, Palacios E. The acute encephalitic phase of neurocysticercosis: computed tomographic manifestations. AJNR Am J Neuroradiol 1983;4(1):51–55.

6.	Chan ST, Tse CH, Chan YS, Fong D. Sparganosis of the brain. Report of two cases. J Neurosurg 1987;67(6):931–934.

7.	Chang KH, Cho SY, Chi JG, Kim WS, Han MC, Kim CW, Myung H, Choi KS. Cerebral sparganosis: CT characteristics. Radiology 1987;165(2):505–510.

8.	Chi JG, Chi HS, Lee SH. Histopathologic Study On Human Sparganosis. Korean J Parasitol 1980;18(1):15–23.

9.	Cho SY, Bae JH, Seo BS. Some Aspects Of Human Sparganosis In Korea. Korean J Parasitol 1975;13(1):60–77.

*10.*	Cho SY, Kim SI, Kang SY, Choi DY, Suk JS, Choi KS, Ha YS, Chung CS, Myung HJ. Evaluation of enzyme-linked immunosorbent assay in serological diagnosis of human neurocysticercosis using paired samples of serum and cerebrospinal fluid. Korean J Parasitol 1986;24(1):25–41.

*11.*	Cho SY, Kang SY, Kong Y. Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody. Korean J Parasitol 1990;28(3):135–142.

*12.*	Choi SH, Kang SY, Kong Y, Cho SY. Antigenic protein fractions reacting with sera of sparganosis patients. Korean J Parasitol 1988;26(3):163–167.

*13.*	Coker-Vann M, Brown P, Gajdusek DC. Serodiagnosis of human cysticercosis using a chromatofocused antigenic preparation of Taenia solium cysticerci in an enzyme-linked immunosorbent assay (ELISA). Trans R Soc Trop Med Hyg 1984;78(4):492–496.

*14.*	Da Costa Castro JM, Deschamps F, Benbachir M, Henrichsen J, Volle PJ, Guinet RM. Highly sensitive biotin-avidin sandwich ELISA for the rapid detection of pneumococcal capsular polysaccharide antigens. J Immunol Methods 1987;104(1-2):265–270.

*15.*	Dobson SR, Taber LH, Baughn RE. Recognition of Treponema pallidum antigens by IgM and IgG antibodies in congenitally infected newborns and their mothers. J Infect Dis 1988;157(5):903–910.

*16.*	Fan KJ, Pezeshkpour GH. Cerebral sparganosis. Neurology 1986;36(9):1249–1251.

*17.*	Freund J, Thomson KJ, et al. Antibody formation and sensitization with the aid of adjuvants. J Immunol 1948;60(3):383–398.

*18.*	Furuya K, et al. Jpn J Parasitol 1989;38(4):184–193.

*19.*	Gershoni JM, Palade GE. Protein blotting: principles and applications. Anal Biochem 1983;131(1):1–15.

*20.*	Grogl M, Estrada JJ, MacDonald G, Kuhn RE. Antigen-antibody analyses in neurocysticercosis. J Parasitol 1985;71(4):433–442.

*21.*	Guesdon JL, Ternynck T, Avrameas S. The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 1979;27(8):1131–1139.

*22.*	Horii Y, Owhashi M, Ishii A, Bandou K, Usui M. Leukocyte accumulation in sparganosis: demonstration of eosinophil and neutrophil chemotactic factors from the plerocercoid of Spirometra erinacei in vivo and in vitro. Am J Trop Med Hyg 1984;33(1):138–143.

*23.*	Hosoi T. Jpn J Parasitol 1969;18(2):149–158.

*24.*	Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29(4):577–580.

*25.*	Ishii A, et al. Jpn J Parasitol 1969;18(1):1–7.

*26.*	Iwata S, et al. Jpn J Parasitol 1967;16:568.

*27.*	Kamiya H, et al. Jpn J Vet Res 1980;28:155–160.

*28.*	Kim CH, et al. Korean J Zool 1989;32(4):429–440.

*29.*	Kim H, Kim SI, Cho SY. Serological Diagnosis Of Human Sparganosis By Means Of Micro-ELISA. Korean J Parasitol 1984;22(2):222–228.

*30.*	Kim SC, et al. J Korean Neurosurg 1981;10:589–591.

*31.*	Kondo K, et al. Jpn J Parasitol 1984;33(2):99–104.

*32.*	Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–685.

*33.*	Laclette JP, Merchant MT, Willms K. Histological and ultrastructural localization of antigen B in the metacestode of Taenia solium. J Parasitol 1987;73(1):121–129.

*34.*	Lee SH, Chai JY, Seo BS, Cho SY. Two cases of human infection by adult of Spirometra erinacei. Korean J Parasitol 1984;22(1):66–71.

*35.*	Maeno Y, et al. Jpn J Parasitol 1986;35(2) Suppl:11–13.

*36.*	Merril CR, Goldman D, Sedman SA, Ebert MH. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 1981;211(4489):1437–1438.

*37.*	Mineura K, Mori T. Sparganosis of the brain. Case report. J Neurosurg 1980;52(4):588–590.

*38.*	Miller B, Goldberg MA, Heiner D, Myers A, Goldberg A. A new immunologic test for CNS cysticercosis. Neurology 1984;34(5):695–697.

*39.*	Morrissey JH. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 1981;117(2):307–310.

*40.*	Oakley BR, Kirsch DR, Morris NR. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 1980;105(2):361–363.

*41.*	Ohnish Y, et al. Jpn J Parasitol 1986;35(1):25–33.

*42.*	Pack SH, et al. J Korean Neurosurg Asso 1972;1:204–207.

*43.*	Parsons JC, Bowman DD, Grieve RB. Tissue localization of excretory-secretory antigens of larval Toxocara canis in acute and chronic murine toxocariasis. Am J Trop Med Hyg 1986;35(5):974–981.

*44.*	Schantz PM, Shanks D, Wilson M. Serologic cross-reactions with sera from patients with echinococcosis and cysticercosis. Am J Trop Med Hyg 1980;29(4):609–612.

*45.*	Sugiyama H, Sugimoto M, Akasaka K, Horiuchi T, Tomimura T, Kozaki S. Characterization and localization of Paragonimus westermani antigen stimulating antibody formation in both the infected cat and rat. J Parasitol 1987;73(2):363–367.

*46.*	Suzuki N, et al. Jap J Parasitol 1982;31(1):23–26.

*47.*	Takahashi Y, et al. Jpn J Parasitol 1988;37(6):435–440.

*48.*	Towbin H, Gordon J. Immunoblotting and dot immunobinding--current status and outlook. J Immunol Methods 1984;72(2):313–340.

*49.*	Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76(9):4350–4354.

*50.*	Tsang VC, Peralta JM, Simons AR. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol 1983;92:377–391.

*51.*	Weinstein PP, Krawczyk HJ, Peers JH. Sparganosis in Korea. Am J Trop Med Hyg 1954;3(1):112–129.