Protein expression and purity were verified by Coomassie blue staining analysis following SDS-PAGE on 10% and/or 12% gels run under both reducing and nonreducing conditions. Immunoblots were probed with an anti-His antibody (Sigma). Following electrophoresis, the proteins were blotted onto polyvinylidene difluoride (PVDF) membranes and incubated with blocking buffer (5% skim milk in PBST) for 2 h at room temperature. The membranes were then incubated overnight at 4°C with anti-His antibody (Sigma), and secondary goat anti-mouse (Sigma) antibodies were used to detect His-tagged recombinant proteins. Results were visualized using a chemiluminescence detection assay (ECL; New Cell and Molecular Biotech, China), and images were merged using ImageJ software. For immune serum and antibody specificity, mouse immune sera, rabbit immune sera, and antibodies against rPvMSP8, rPvMSP1-19, or rPvMSP8+1 were used as primary antibodies for incubation. The following steps were performed as described above.

To test whether specific antibodies against rPvMSP8, rPvMSP1-19, or rPvMSP8+1 could be recognized by P. cynomolgi-infected erythrocyte lysates or monkey healthy erythrocyte lysates, P. cynomolgi-infected erythrocyte lysates were collected and mixed with SDS sample buffer for proper separation by SDS-PAGE. After immunoblots were transferred to PVDF membranes, antibodies against rPvMSP8, rPvMSP1-19, or rPvMSP8+1 were used as primary antibodies for incubation. The following steps were performed as described above.

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