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Korean J Parasitol > 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

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