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

The other group of mice (SIII-FCA) was immunized with SIII protein emulsified in FCA as explained above. The third and fourth groups were used as controls for saponin and FCA respectively. Each mouse of these last two groups received an equal amount of saponin or FCA as expérimentais without any parasite protein on days 0,14 and 28. On day 35 mice (experimental as well as control) were inoculated with 1 x 10

6

JR berghei parasitized red cells and the postchallenge course of parasitaemia was recorded as explained earlier. Four immune mice of the SHI-saponin group were rechallenged with l x 10

6 P. berghei infected erythrocytes after the first challenge infection was cleared from their blood. Antibody titration by indirect immunofluorescence assay (IFA): Immune serum (pre- and postchallenge) samples were diluted in 2-fold steps starting at 1:16, in PBS pH 7.2. These sera were titrated on acetone-fixed P. berghei antigen slides according to Collins and Skinner (1972). The second antibody employed was rabbit anti-mouse IgG conjugated to FITC (Sigma, USA). Slides were counterstained in 0.5% (w/v) Evans blue in PBS, pH 7.2. The end point was defined as the highest serum dilution showing no fluorescence. Merozoite invasion inhibition assay: The short-term in vitro culture of P. berghei was carried out in 24-well culture plates. Each well contained 1.0 ml of complete medium (RPMI-1640) supplemented with gentamycin (50 pg/ml), penicillin (100 IU/ml), streptomycin (100 pg/ml), sodium bicarbonate (5%, w/v) and N-2-hydroxyethyl piperazine N

;- 2 ethane sulphonic acid (HEPES) (0.6% w/v) and 10% (v/v) heat-inactivated (56 °C, 30 min) fetal calf serum. Normal mouse serum (5%, control) or immune mouse serum (5%, experimental) and normal and schizont/trophozoite infected (l%-2% parasitaemia) erythrocytes (2% haematocrit) were added to the medium. Cultures were placed in a candle jar and incubated at 37 °C. After 19 h of incubation smears were prepared using the well contents, fixed in methanol, stained in Giemsa's stain, and the percent inhibition of merozoite invasion was calculated as follows: . nn number of rings in experimental culture _ t nn % inhibition = 100 • f-. :—: x 100 number of rings in control culture Detection of interleukin (IL-1): Sandwich enzyme immunoassay (Ferma et al. 1988) was performed to measure the level of IL-1 in pre- and postchallenge immune sera raised against sediment (SIII). For the assay, 96-welI microtitre plates were coated with 50 pi of rabbit anti-mouse IL-1 (5 pg/ml) and incubated overnight at 4 °C. After washing with PBS, 0.01 M, pH 7.2 - Tween 20, unbound sites were saturated with 100 pi of 2% (w/v) bovine serum albumin (BSA) in PBS, pH 7.2 for 1 h at 37 °C. Thereafter, plates were washed three times with PBS-Tween 20. Fifty pi aliquots standards (recombinant IL-1) and different dilutions (multiples of log 5) of immune sera (diluted in 0.5% BSA in PBS) were dispensed into duplicate wells of microtitre plates and incubated overnight at 4 °C. After aspirating the unbound material and washing with PBS-Tween 20, 50 pi of goat anti-mouse IL-1 was added to the wells and plates were incubated at 37 °C for 2 h. This was followed by washing of plates three times with PBS-Tween 20. Thereafter, 50 pi of anti-goat Ig-HRP (horseradish peroxidase) labelled antibody (1 : 2500) was added and incubated at 37 °C for 2 h. After aspirating unbound conjugate, plates were washed five times. Fifty pi of orthophenylenediamine (OPD) was added in dark to each well and the plates were