The CF of T. solium metacestodes was prepared from naturally infected pigs, as was described in a previous study (Yang et al., 1998). For the purification of 150 kDa, purification procedures were carried out using an ammonium sulfate precipitation followed by gel filtration equipped with AKTA-FPLC system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) (Chung et al., 2002). Briefly, CF crude extracts (10 ml) was saturated with ammonium sulfate up to 80%, the precipitated proteins were discarded by centrifugation and the resulting supernatant was collected. After dialysis and concentration, the supernatant was loaded onto a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) that was previously equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl. The column was washed with the above buffer and the fractions of 150 kDa were detected by SDS-PAGE. The 150 kDa fractions were collected and concentrated for further study.

BALB/c mice were used to produce the polyclonal antibody of the purified protein by the methods of a previous study (Chung et al., 2002). For immunohistochemical study, the metacestodes in pork muscle were fixed in 10% neutral formalin, embedded in paraffin, and cut into ribbon with a 6 µm thickness. The paraffin ribbons of T. solium metacestode were incubated in phosphate buffered saline (PBS) for 2 hr at room temperature with 1:100 diluted mouse immune sera sensitized against the purified protein. As the negative control, the pre-immune BALB/c mouse serum was incubated in PBS with 1: 100 diluted for 2 hr. The ribbons were washed twice with PBS and incubated for 1 hr with fluorescein isocyanate (FITC)-labeled antimouse IgG antibody (Jackson immunoresearch laboratory, PA, USA) diluted 1:50, and then, the ribbons were washed twice with PBS. After observation with a fluorescence microscope (Olympus AX 70, Japan), the ribbons were washed thoroughly with PBS, and then, stained with hematoxylin to compare the organs that reacted with the immune sera.

The purified 150 kDa protein, a major constituent molecule in CF crude extracts, migrated at molecular weights of 15, 10 and 7 kDa on a 7.5-15% gradient SDS-PAGE (Fig. 1), and this procedure correlated with the results of a previous study (Cho et al., 1988). The native molecular weight of the protein was estimated to be about 150 kDa by a Superose 6 HR 10/30 gel filtration, and the purified protein was soluble even at high concentration of ammonium sulfate solution (up to 80%).

Immunofluorecence staining on T. solium metacestodes sections using polyclonal immunized serum clearly revealed that the 150 kDa protein was mainly distributed at the bladder walls and calcareous corpuscles of the spiral canal of T. solium metacestodes (Fig. 2 A&C). The positive reactions were also observed at the granules of the CF and at host tissues surrounding the bladder walls of T. solium metacestodes. The inner and outer walls of the bladder reacted equivalently to the polyclonal antibody, although all the calcareous corpuscles of the scolex did not react positively (Fig. 2 A&C). Another secreted diagnostic antigen, so called 'Antigen B', was distributed at tegumentary cytons of the bladder wall and in the lumen of the spiral canal of the metacestode scolex (Laclette et al., 1987). The purified 150 kDa protein was secreted secreted into host tissues surrounding the parasite and it induced immune responses. Also, secreted 150 kDa protein in the CF of T. solium metacestodes may have secretory pathways with the calcareous corpuscles, which may have various cellular physiological processes by regulating mineral components in cestodes (Pawlowski et al., 1988). Its relationship with the calcareous corpuscles should be investigated in further studies.