Dr. Murai Éva - Gubányi András szerk.: Parasitologia Hungarica 31. (Budapest, 1998)

MATERIALS AND METHODS Antigens: Protoscoleces were collected from buffalo lung hydatid cysts and washed in Hank's solution. The protoscoleces were homogenized in 10% phosphate buffered saline (PBS), pH 7.4, and subjected to ultrasonication (5 mm probe) for 1 min at 4 °C and centrifuged at 10,000 g for 15 min. The supernatant was diarysed against distilled water and stored at -20 °C with sodium azide as preservative. The protein concentration was determined by the method of Spector (1978). Phosphate buffer (0.15 M, pH 7.4) was used for washing and equilibrating Sephadex G-200 (Pharmacia, Sweden) gel filtration column (2x90 cm). A calibration curve was prepared for detenriining the molecular weights (Mr) of the elution profile by using known standard Mr markers comprising of bovine serum albumin, egg albumin and cytochrome C. Five ml of concentrated crude antigens (CA) having 20 mg of protein was applied on Sephadex G-200 column. Each 5 ml fraction was collected and monitored at 280 nm for protein concentration. Individual peaks of eluent were collected separately, packed into dialysis cellulose tubing (Mr cut-off 3500, Sigma, USA), dialysed against distilled water and polyethylene glycol (PEG, Mr 20,000, Sisco, India), stored at -20 °C until used. Animals: puppies preferably of the same litter were kept on a control diet and divided into five groups. Experimental ten-week-old puppies were starved and freshly collected protoscoleces (about 28,000) were fed to each of the ten dogs according to the method of Irshadullah and Nizami (1992). A group of two uninfected control puppies was also included. Sampling: The blood of infected and control puppies was collected into 10 ml syringes from jugular veins at days 0, 4, 8, 16, 24, 32, and 40 post infection (PI), and used for the separation of serum and monocytes/macrophages. Finally, on day 40 puppies were sacrificed to verify Echinococcus granulosus infection. Migration Inhibition Test (MIT): The dextran sedimentation technique was employed for the isolation of monocytes/macrophages (Nath 1983). MIT was performed as described by David et al. (1964) with some modifications. The cell concentration was adjusted to 40 X10

5 cells in 0.2 ml tissue culture medium with 10% fetal calf serum (FCS). The cell suspension was packed in capillary tubes of 20 ul capacity (Top Syringes, Bombay) by capillary action and sealed at one end with colourless plasticine. After incubation of cells and antigens the percent migration inhibition was recorded. Immunoblot technique: The dot blot assay was performed as described by Hawkes et al. (1982) with some modifications. The test antigens with a protein concentration of 10 ug were spotted on the polyvinyl difluoride (PVDF) membrane and allowed to dry. The test strips were washed with 10 mM PBS, pH 7.4, for 3 x 10 min, and were incubated in Blotto containing 5% non-fat dried milk powder (Anik Spray, India) in 10 mM PBS, pH 7.4, with 0.02% NaNß for 3 hours to cap the unwanted epitopes. Thereafter the membrane strips were washed with 10 mM PBS, pH 7.4, for 3 x 10 min and were incubated in different PI sera in a dilution of 1:100 in Blotto for 3 hours. The membranes were washed in 10 mM PBS containing 0.05% Tween for 3x10 min. Thereafter the membranes were incubated in alkaline phosphatase labelled anti-dog IgG (Sigma, USA) in a dilution of 1:2000 in Blotto for 3 hours. After incubation in secondary antibodies the membranes were again washed in 10 mM PBS, pH 7.4, for 3x10 min. and the reaction was developed in sodium 1-naphthyl phosphate (Koch-Light, England). The reaction was stopped with 4% formaldehyde in doubledistilled water, and photographed.