Immunohistochemical localization of Gnathostoma spinigerum larval antigens by monoclonal antibodies: II. Electron microscopy.

Immunocytochemical localization of antigens in advanced third-stage larvae of Gnathostoma spinigerum (GsAL3) was studied by immunogold labeling method using seven G. spinigerum specific monoclonal antibodies (MAbs), FS-3D11, SS-5H5, SK-6C4, SK-4E1, SK-7G6, SK-8D4 and SA-9B5. All these MAbs belong to the IgG1 subclass and only FS-3D11 and SS-5H5 recognize carbohydrate epitopes. The paraformaldehyde-fixed GsAL3 were embedded in Lowicryl K4M medium, and the gold colloidal particles used were 15 nm in size. When the worm sections were probed with FS-3D11, the gold particles appeared to concentrate specifically on the intestinal brush border. When SS-5H5 was applied, the particles were scattered densely over the brush border and in the cytoplasm of epithelial cells. The rest of the MAbs which recognize protein determinants exhibited a lack of labeling. The results suggested that the carbohydrate antigenic determinants recognized by the two MAbs are the most stable and most abundant particularly in the intestine of GsAL3. These results also confirmed the previous finding that the most antigenic site of GsAL3 is the intestine.

Purification of Gnathostoma spinigerum larval antigens by monoclonal antibody affinity chromatography.

An immunoaffinity column was prepared by coupling a partially purified Gnathostoma spinigerum-specific IgG1, MAb SK-6C4 (5 mg/ml) to CNBr-activated Sepharose 4B. Ten milliliters of approximately 0.3 mg/ml of crude soluble G. spinigerum larval antigens (GsAL3) were loaded onto the affinity column at a flow rate of about 5 ml/hour. Elution of the bound antigens was accomplished using 50 mM diethylamine-HCI containing 0.15 M NaCL, pH 11.5. The average amount of eluted antigens obtained by one passage of crude GsAL3 (1-4 mg) through 4 to 8 ml of column matrix was 143 micrograms (range, 67 - 414 micrograms). The minimal amount of purified GsAL3 detectable by ELISA using MAb SK - 6C4 (100 micrograms/ml) was 50 ng/ml. The SK - 6C4 affinity-purified GsAL3 was found to be relatively pure and immunologically specific as determined by SDS-PAGE and Western blotting, respectively.

Immunohistochemical localization of Gnathostoma spinigerum larval antigens by monoclonal antibodies: I. Light microscopy.

Immunohistochemical localization of antigens in advanced third-stage larvae of Gnathostoma spinigerum (GsAL3) was studied by indirect enzyme immunostaining using 7 G. spinigerum specific monoclonal antibodies, FS-3D11, SS-5H5, SK-6C4, SK-4E1, SK-7G6, SD-8D4 and SA-9B5. All these MAb belong to the IgG1 subclass and only FS-3D11 and SS-5H5 recognize carbohydrate determinants. Each MAb exhibited a different reaction pattern and staining intensity in sectioned GsAL3. FS-3D11 bound primarily to the intestinal brush border whereas SS-5H5 reacted with various tissues of the parasite including intestinal epithelium and brush border, lateral cords, muscle, pseudocoel, and cuticle. SK-6C4 predominantly stained muscle, however, SK-4E1 and SK-7G6 exhibited a lack of labeling. SD-8D4 bound to the cuticle and the lateral cords whereas SA-9B5 reacted primarily with the pseudocoel. These results suggest that antigens sharing common epitopes are present in various structures of the larvae with the intestine being the most antigenic site. The present data also suggest that certain GsAL3 antigens recognized by the MAb obtained in this study are sensitive to formalin fixation and/or paraffin embedding since for 2 out of the 7 MAb staining was negative.