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. 2022 Sep 22;13:1005332. doi: 10.3389/fimmu.2022.1005332

Figure 5.

Figure 5

Parasite Neutralizing Ability of Antibodies Targeting Tag- Free CyRPA. (A) Antibodies raised against tag-free CyRPA were evaluated for specificity by their ability to recognize native CyRPA protein. Schizont extracts from 3D7 parasites were probed with anti-CyRPA immune (I) and pre-immune (PI) rabbit polyclonal sera through immunoblotting. Immune sera specifically detected a band at ~40 kDa corresponding to CyRPA, while no signal was detected with the pre-immune sera. (B) To confirm the ability of CyRPA rabbit antibodies to detect the native protein in its native form, co-immunoprecipitation experiment was performed with 3D7 schizont lysate. As shown, the co-immunoprecipitated components of the multi-protein complex (RH5, Ripr and CyRPA) were successfully detected using their respective specific rabbit antibodies in immunoblotting. Pre-immune sera, used as a negative control, could not coimmunoprecipitate the multiprotein complex. (C) Purified rabbit total IgG against tag-free CyRPA were tested in the standard in vitro Growth Inhibitory Assay (GIA) against 3D7 clone in a one-cycle assay. The antibodies exhibited highly potent dose-dependent parasite neutralization reaching saturation at 2.5 mg/ml with ~90% inhibition levels. The antibody efficacy was similar to those against (6-His) tagged CyRPA that was used as a positive control except at low concentrations of 0.125 and 0.5 mg/ml. Data represents the average of 2 independent experiments conducted in duplicate. The error bars represent the standard error between the 2 independent GIA experiments. The P values were calculated by two-way ANOVA with Bonferroni post-hoc testing. (D) Total IgG against tag-free CyRPA was tested against four P. falciparum clones- 7G8, Dd2, FVO and HB3, in a one-cycle GIA. A robust strain transcending parasite neutralization activity was observed by the antibodies across the four strains with inhibition levels reaching to ~80% to 96%. Data represents the average of 2 independent experiments conducted in duplicate. The error bars represent the standard error between the 2 independent GIA experiments. I, Immune; PI, Pre-immune; IP, Immunoprecipitate.