N. fowleri trophozoites (Cater NF69 strain, ATCC No.30215) were axenically cultured at 37℃ in Nelson' media (Willaert, 1971). CHO cells were cultured with EMEM (Earle's Minimum Essential Medium) containing 10% fetal bovine serum (complete EMEM) at 37℃ in a 5% CO₂ incubator (Im and Shin, 2003).

To obtain a rNfa1 fusion protein, the expression and purification of the nfa1 gene product were performed accordingly by the method mentioned in the previous paper (Shin et al, 2001). The purified DNA (5 µg/µl) obtained from PCR-T7/NT TOPO expression vector (Invitrogen, Grohingen, Netherlands) containing the nfa1 gene was subsequently transformed into the BL21(DE3)pLysS E. coli strain by the heat-shock method. Cells were cultured at 37℃ in the LAC (Luria-Bertani media containing 100 µg/ml of ampicillin and 34 µg/ml of chloramphenichol) plates for selection. A transformed-colony was selected and cultured at 37℃ in the LAC broth until the absorbance reached 0.5-0.8 at 600 nm; then 1 mM IPTG was added to the media. After 4 hrs of incubation, the cells were harvested by centrifugation (6,000 × g for 15 min). Cell extracts were compared with those of non-transformed BL21(DE3)pLysS by SDS-PAGE, and the presence of the expressed gene product was confirmed by Western blotting using both the immune and the infected sera, and anti-His and Xpress antibodies (Invitrogen).

For the production of anti-Nfa1 polyclonal serum, the rNfa1 protein (50 µg/mouse) was mixed with an equal volume of Freund's complete adjuvant (Sigma), and the mixture was injected intraperitoneally into an 8-week-old female BALB/c mice (purchased from the Korea Institute of Science and Technology, Daejeon, Korea). The mouse was boosted biweekly for another 4 weeks with the rNfa1 protein (25 µg/mouse) containing an equal volume of incomplete Freund's adjuvant (Sigma). After the third boosting, the rNfa1 protein (5 µg/mouse) was injected intravenously without the adjuvant. Four days later, anti-Nfa1 polyclonal serum was collected from the mouse blood by centrifuging at 2,500 × g for 30 min at 4℃. ELISA was performed with a purified Nfa1 protein (5 µg/ml) and with a rabbit anti-mouse whole immunoglobulin (1:10,000 dilution) conjugated with alkaline phosphate (Sigma). Western blotting for the recombinant Nfa1 protein was performed according to the method in a previous paper (Cho et al., 2003).

N. fowleri trophozoites (1 × 10⁴ cells) were put in triplet culture tubes containing 1 ml of Nelson's medium. Then an anti-Nfa1 polyclonal antibody (1:200, 1:100 and 1:50 dilution) obtained from mice was added in each tube. Cultivating in a 37℃ incubator, the number of trophozoites in each tube was counted with an haematocytometer up to 48 hrs post-incubation.

N. fowleri trophozoites containing CHO cells cultured in complete EMEM were put in each well of a 96-well plate (group I). For the experimental group (group II), an anti-Nfa1 polyclonal antibody (1:200, 1:100 and 1:50 dilution) obtained from mice was added in the triplet wells refer to the 96-well plate. After incubating at 37℃ for 24 hrs and 48 hrs, respectively, the effect of the anti-Nfa1 polyclonal antibody was observed with a light microscope and LDH (lactate dehydrogenase) release assay. The cytotoxicity from the LDH release assay was observed with the CytoTox 96™ Non-Radioactive Cytotoxicity Assay Kit (Promega, WI, USA). About 1 × 10⁴ CHO cells were prepared with 100 µl of EMEM in a round-bottomed 96-well plate. The amoeba trophozoites (1 × 10⁵) and/or an anti-Nfa1 polyclonal antibody (1:200, 1:100 and 1:50 dilution) were added to each well, and five sets of triplet wells for controls including spontaneous releasing from medium and naive cells were prepared. The casted plate was incubated in a humidified chamber at 37℃ and in a 5% CO₂ incubator for 45 min consecutively, and then centrifuged at 250 × g for 4 min. The supernatant (50 µl) from each well was transferred to a fresh flat-bottomed 96-well plate. Equal volumes of the reconstituted Substrate Mix were added to each well. The plate was incubated at room temperature for 30 min. Fifty µl of Stop Solution was added, and after 1 hr, the absorbance was recorded at 490 nm.

To obtain a purified recombinant Nfa1 protein, a fusion protein of 17 kDa from the BL21 E. coli strain containing the nfa1 gene was purified using a nickel resin column and enzyme digestion. Finally the purified protein eluted from the gels had a molecular weight of 13.1 kDa, which corresponded well to the size in the previous research (Cho et al., 2003) (data not shown). An anti-Nfa1 polyclonal antibody was collected from mice immunized with a recombinant Nfa1 protein, and it's specific reactivity against the Nfa1 protein was observed with ELISA and Western blotting. Values of anti-Nfa1 polyclonal antibodies by ELISA ranged from 0.212 ± 0.021 (1:10,000 dilution, M ± SD, N=5) to 1.213 ± 0.024 (1:200 dilution), which showed a difference from the 0.112 ± 0.005 (1:200 dilution) of the normal sera and also the 0.109 ± 0.005 of the PBS control.

N. fowleri trophozoites (1 × 10⁴ cells) cultured from the beginning with an anti-Nfa1 polyclonal antibody (1:200 dilution) proliferated to 1.1 × 10⁴ cells in 8 hrs, 2.9 × 10⁴ cells in 24 hrs, 3.9 × 10⁴ cells in 32 hrs and 15.1 × 10⁴ cells in 48 hr post-incubation (Fig. 1). When an anti-Nfa1 antibody (1:100 dilution) was treated in cultivating system, N. fowleri trophozoites (1 × 10⁴ cells) proliferated to 1.0 × 10⁴ cells in 8 hrs, 2.5 × 10⁴ cells in 24 hrs, 3.2 × 10⁴ cells in 32 hrs and 6.7 × 10⁴ cells in 48 hrs post-incubation (Fig. 1). In the control group, without the anti-Nfa1 antibody, the amoeba proliferated to 1.7 × 10⁴ cells in 8 hrs, 4.9 × 10⁴ cells in 24 hrs, 8.5 × 10⁴ cells in 32 hrs and 21.7 × 10⁴ cells in 48 hrs post-incubation (Fig. 1). Consequently, the addition of an anti-Nfa1 polyclonal antibody showed an inhibition on the proliferation of N. fowleri in a dosedependent manner (p < 0.05).

CHO cells co-cultured with N. fowleri trophozoites (group I) for 24 hrs showed severe morphological destruction (Fig. 2B). On the contrary, CHO cells cocultured with N. fowleri trophozoites and anti-Nfa1 polyclonal antibody (group II) showed less destruction than above experimental group I (Fig. 2C). In the LDH release assay results, when an anti-Nfa1-antibody (1:200 dilution) was added in the co-cultivating system, group I showed 55.3% cytotoxicity and group II showed 43.7% in the LDH release assay 24 hrs postincubation. After 48 hrs of incubation, group I showed 59.0% cytotoxicity, and group II showd 48.0% (Table 1). When an anti-Nfa1-antibody (1:100 dilution) was added in the co-cultivating system, group I showed 50.6% cytotoxicity 24 hrs post-incubation, and group II showed 39.3% (Table 1). After 48 hr of incubation, group I showed 53.3% cytotoxicity and group II 41.3%, respectively (Table 1). When an anti-Nfa1 antibody (1:50 dilution) was added in the co-cultivating system, group I showed 54.3% cytotoxicity 24 hrs post-incubation, and group II showed 34.0%. After 48 hrs of incubation, group I showed 66.3% cytotoxicity, and group II showed 42.0% (Table 1). Consequently, the addition of an anti-Nfa1 polyclonal antibody showed a decreasing effect of in vitro cytotoxicity of N. fowleri in a dose-dependent manner (Fig. 3).