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

incubated at 37 °C for 20 min. The reaction was stopped with 50 pi of 7% (v/v) sulphuric acid and plates were read at 492 nm in an ELISA reader (Eurogenetics, model MPR A4, Torino, Italy). The data were calculated from the mean of duplicate determinations as units/ml as computed by comparison with the standard curve using recombinant IL-1 (Genzyme, U.S.A). Acid protease assay: Acid protease assay was performed using 2.5 ml reaction mixture containing 0.1 M acetate buffer, pH 4.6,0.1% (w/v) haemoglobin (denatured) and 0.1 ml enzyme (Banyai et al. 1982). Tyrosine released in protein-free supernate was measured by Fohn-phenol method (Lowry et al. 1951). One unit of enzyme was the amount of enzyme that liberated one mole of tyrosine in 60 min at 37 °C. Acid phosphatase assay:The 2.5 ml assay mixture of acid phosphatase contained 0.1 ml of enzyme, 18 mM sodium B-glycerophosphate, 20 mM sodium barbitone and 0.1 M acetate buffer, pH 5.0 (Banyai et al. 1980). Phosphorus released after incubation at 37 °C for 60 min was measured by the method of Fiske and Subba Row (1925). Alkaline phosphatase assay: The assay mixture contained similar amount of substrate and sodium barbitone as in the acid phosphatase assay but without acetate buffer and the pH of the substrate solution was raised to 8.9 with 0.1 N NaOH. Phosphorus released was measured as above and one unit of enzyme (acid and alkaline phosphatase) activity was the amount of enzyme which liberated one mole of phosphorus in 60 min at 37 °C. The protein content of samples was estimated by the Folin-phenol method (Lowry et al. 1951). The specific activity of an enzyme was expressed as units of enxyme activity per milligram protein. RESULTS Figure 1 represents the course of parasitaemia in mice immunized with homogenate (Horn) and sediments SI, SIII along with controls in a typical experiment. All the mice of control group and the SI group died because of infection. Mice in the HOM and SIII groups survived but the former group had higher parasitaemia (10-20%) followed by clearing of infection. However, the latter group had very little infection (1-8%) which was subsequently cleared off. Mice were also immunized using SII and SIV sediments. All SIV mice died upon challenge, whereas some of the SII mice survived the challenge after having high parasitaemia (30%) (data not shown). A comparable study using SIII with FCA (SIII - FCA) and saponin (Slll-saponin) adjuvants was also carried out (Fig. 2). All the mice immunized with SIII using both the adjuvants were protected upon challenge. The maximum postchallenge parasitaemia ranged between 5%-7.1% and 0.2%-1.7% in SIII-FCA and Slll-saponin groups respectively. The results depicted in Figs 1 and 2 are of a typical experiment; however, a similar pattern of protection and course of parasitaemia in postchallenge mice was observed in repeated experiments. The level of antimalarial antibodies in immunized mice before (prechallenge) and after challenge (postchallenge) was measured by IFA (Fig. 3). No false reaction was observed because of counterstaining of slides with Evans blue. Prechallenge immune sera of the HOM-FCA and SIII-FCA groups had a high level of antibodies which was further boosted after challenge. However, with SI a low antibody titre was recorded.