ITEM-THREE analysis of a monoclonal anti-malaria antibody reveals its assembled epitope on the pfMSP1₁₉ antigen

Rapid diagnostic tests are first-line assays for diagnosing infectious diseases, such as malaria. To minimize false positive and false negative test results in population-screening assays, high-quality reagents and well-characterized antigens and antibodies are needed. An important property of antigen-antibody binding is recognition specificity, which best can be estimated by mapping an antibody's epitope on the respective antigen. We have cloned a malarial antigen-containing fusion protein, MBP-pfMSP1₁₉, in Escherichia coli, which then was structurally and functionally characterized before and after high pressure-assisted enzymatic digestion. We then used our previously developed method, intact transition epitope mapping-targeted high-energy rupture of extracted epitopes (ITEM-THREE), to map the area on the MBPpfMSP1₁₉ antigen surface that is recognized by the antipfMSP1₁₉ antibody G17.12. We identified three epitope-carrying peptides, ³⁸⁶GRNISQHQCVKKQCPQNSGCFRHLDE⁴¹¹³⁸⁶GRNISQHQCVKKQCPQNSGCFRHLDEREE⁴¹⁴, and ⁴¹⁵CKC-LLNYKQE⁴²⁴, from the GluC-derived peptide mixture. These peptides belong to an assembled (conformational) epitope on the MBP-pfMSP1₁₉ antigen whose identification was substantiated by positive and negative control experiments. In conclusion, our data help to establish a workflow to obtain high-quality control data for diagnostic assays, including the use of ITEM-THREE as a powerful analytical tool. Data are available via ProteomeXchange: PXD019717.