Sporozoite Reactivity of Purified IgGs

The reactivity of pools of purified IgGs before immunization (n = 10) and after immunization (n = 10), or those depleted (n = 6), was assessed in a Western blot on sporozoite lysates. To generate the lysates, 1 million NF54 sporozoites were incubated with 100 μL of lysis buffer (150 mM Nacl, 20 mM Tris-Hcl, 1% triton, 1 mM EDTA at pH 7.5 and 1× protease inhibition cocktail; Thermo Fisher Scientific) for 15 minutes on ice followed by a 10-minute centrifugation at 13,000g at 4°C. Protein lysate corresponding to 1 × 10⁵ sporozoites were loaded per well on a 4%–12% Bis-Tris Protein Gels. Proteins were transferred into a nitrocellulose membrane (Bio-Rad), and strips were made. After blocking for 1 h with 5% milk in PBST, the blots were incubated for 3 hours with 5 μg/mL of CIS43 (CSP-specific mAb; ref. 42 (link)) or pre- or postimmunization total or depleted purified IgGs tested at 5-point 1:2 dilution at a maximum concentration of 20 μg/mL. After 3 washes with PBST, we incubated samples with secondary antibody (goat anti–human IgG [H+L], HRP, 1:30,000, Thermo Fisher Scientific catalog A18805; polyclonal). Then, blots were washed 6 times with PBST and incubated with Clarity max ECL substrate (Bio-Rad). The imaging was performed in ImageQuant LAS4000 (Bio-Rad). The intensity of the bands was analyzed using ImageJ (NIH).