2.9. In Vitro Invasion Assay

The effect of anti-CpTIPH IgG on the invasion of C. parvum into HCT-8 cells was evaluated using an in vitro assay as described with minor modifications [20]. Briefly, HCT-8 cells were cultured in 96-well plates to ~90% confluence. Oocysts of C. parvum (2 × 10⁴/well) were pre-incubated with an anti-CpTIPH antibody at different concentrations for 30 min in RPMI 1640 medium containing 10% FBS at 37 °C, and then added to the wells to infect host cells for 2 h at 37 °C. Oocyst walls and free sporozoites were removed by exchanging the culture medium. Parasite-infected cells were then incubated at 37 °C for additional 16 h (i.e., total 18 h infection time). Mouse pre-immune IgG was used in parallel as a control. After incubation, cells were washed 3 times with RPMI 1640.

Parasite loads were evaluated by qRT-PCR detection of the relative levels of parasite 18S rRNA as described [11]. Briefly, total RNA was isolated from HCT-8 cells infected with C. parvum using an iScript qRT-PCR sample preparation reagent (lysis buffer) (Bio-Rad Laboratories, Hercules, CA); qRT-PCR reactions were performed using a one-step SYBR Green qRT-PCR kit (DBI Bioscience, Germany) with the following pairs of primers: Cp18S-1011F (5′-TTG TTC CTT ACT CCT TCA GCA C-3′) and Cp18S-1185R (5′-TCC TTC CTA TGT CTG GAC CTG-3′) for C. parvum 18S rRNA (Cp18S), and Hs18S-1F (5′-GGC GCCCCC TCG ATG CTC TTA-3′) and Hs18S-1R (5′-CCC CCG GCC GTC CCT CTT A-3′) for host cell 18S rRNA (Hs18S). Reaction mixtures were incubated at 42 °C for 30 min for synthesizing cDNA, heated at 95 °C for 2 min to deactivate the reverse transcriptase and then subjected to 40 thermal cycles of PCR amplification (95 °C for 10 s, 55 °C for 34 s and 72 °C for 30 s). At least three technical replicates were performed for each sample. The parasite loads were calculated using the empirical 2^−ΔΔCT formula.