Figure 1.

Design and validation of the anti-S7 and anti-L1 peptide antisera. (A) Schematic model of S7- and L1-clade proteins showing position of the designed target peptide motifs. One of the S7 target motifs is located towards the N-terminus of the S7 protein clade, while the other is located further towards the C-terminus. Both the target motifs identified in the L1 PIRs are located towards the N-terminus of the protein sequence. TM, Transmembrane Domain. (B) ELISA reactivity of the individual anti-S7 and anti-L1 peptide sera (anti-S7 motif 1 peptide antiserum, anti-S7 motif 2 peptide antiserum, anti-L1 motif 1 peptide antiserum, anti-L1 motif 2 peptide antiserum) against the target P. chabaudi PIR peptides that were used to immunise rabbits. ELISAs performed by Cambridge Research Biomedicals, and figure adapted from those supplied by Cambridge Research Biochemicals. (C) Western blot analysis of S7, L1 and L4 his-tagged recombinant proteins probed with the individual anti-S7 and anti-L1 peptide antisera. Migration of S7 proteins was between 33-37 kDa markers and of L1 proteins between the 75-150 kDa markers (arrows indicate monomers). Probing against the his-tag served as loading control. Pre-immune sera served as negative controls, where they were found to be unreactive and did not recognise any of the recombinant proteins, confirming the lack of pre-existing reactivity to PIRs in the rabbit serum. ++ denotes a protein that highly matches the sequence of the peptide motif that the sera were raised against; + denotes a protein that contains a similar peptide motif (50-60% alignment) to that used for raising the immune sera; - denotes a protein that it does not contain any of the anti-sera target motifs. The specific recombinant proteins loaded are the PCHAS_0600600 (L4), PCHAS_1100300 (L1), PCHAS_0400300 (S7), PCHAS_1200500 (S7) and PCHAS_1300101 (S7). (D) ELISA reactivity of the pooled anti-S7 and anti-L1 peptide sera (pooled motif 1 and 2 for each antiserum) against WT parasite lysates extracted from MT and SBP acute-stage parasites. Pre-immune serum served as negative control. OD 405 ± standard deviation of three replicate assays is shown. MT, Mosquito Transmitted; SBP, Serially Blood Passaged. (E) Western blot analysis of whole parasite lysates (reducing conditions) prepared from P. chabaudi MT WT parasites at the trophozoite-stage, using the anti-S7 and anti-L1 pooled peptide immune sera. Arrows indicate the estimated average size of the monomers or dimers of each PIR clade (S7 33-50 and 100 kDa; L1 75-150 kDa). Probing with the pre-immune sera served as negative control. Probing against the Plasmodium EXP2 was used as stage-specific and loading control. The molecular weights on the right indicate the positions to which size markers had migrated.