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

Various antigens have been isolated from different constituents of Plasmodium and these antigens have exhibited a protective role against malaria (Newbold 1984). The parasite constituents can be separated by differential centrifugation (Banyai et al. 1979), however, the role of the various cellular organelles in the protection against malaria has not been elucidated. In this study a fraction of P. berghei separated by centrifugation induced protective immune response in mice. MATERIALS AND METHODS Parasite: P. berghei (NK-65) asexual erythrocytic stages were maintained in white Swiss mice (Balb/C), 20-25 g of either sex. When the parasitaemia was 50%-60%, blood fromjR berghei-infectcd mice was collected in citrated saline and passed through CF-1

1 cellulose (Whatmann). Erythrocytes were washed with phosphate buffered saline (0.01 M, PBS, pH 7.2) and cell-free parasites were isolated by saponin lysis (0.2% w/v) followed by centrifugation at 10,000 g for 20 min. The cells were washed twice with PBS, pH 7.2. Cell-free parasites were suspended in an appropriate volume of 0.25 M sucrose solution and homogenised in a Potter Elvehjam precooled homogenizer (Remi, Bombay, India) at 2000 rpm with fifty strokes of pestle. Homogenate (HOM) was centrifuged at 600 g for 15 min at 4 °C. The sediment designated SI was obtained and the supernate was further centrifuged at 10,000 g for 25 min. The sediment (SII) was isolated and the supernate again centrifuged at 24,000 g for 35 min. The sediment (SIII) was separated from the supernate fraction (SIV). The method of Banyai et al. (1979) was followed for differential centrifugation. All centrifugations were carried out at 4 °C in a refrigerated centrifuge. A part of the sediment was suspended in normal saline and used as enzyme for biochemical analysis. Rest of the sediments were suspended in PBS (0.01M, pH 7.2) and used for immunizing mice. Protein was estimated by Folin-phenol reagent (Lowry et al. 1951). Immunization of mice : In a typical experiment ten mice were immunized in each group with HOM, SI, and SIII. Each mouse of a group received 100 fig protein of the respective fraction emulsified in Freund's complete adjuvant (FCA) on day 0 subcutaneously (s.c.) followed by the same amount of protein in PBS, pH 7.2 intraperitoneally (i.p.) on day 14 and day 28. Mice of the control group were injected with PBS, pH 7.2 and FCA on day 0 followed by only PBS, pH 7.2 on day 14 and day 28. On day 35, seven mice from each group were injected i.p. with 1 x 10

6 P. berghei-inîecteâ red cells and the remaining mice of each group were bled to collect immune sera (prechallenge). Postchallenge parasitaemia from each mouse was recorded daily by preparing Giemsa-stained blood smears. Percent infection was calculated as: number of infected red cells x JQQ total number of red cells Mice which survived the challenge were bled to collect postchallenge immune sera after their smears were clear from infection. In a different experiment another four groups of 10 mice each were immunized in the following way. Each mouse of a group (SHI-saponin), was injected i.p. with 100 pg of SIII protein in PBS, pH 7.2 containing 30 pg saponin each on days 0,14 and 28.