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 E. 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 MBP-pfMSP119 antigen surface which is recognized by the anti-pfMSP119 antibody G17.12. We identified three epitope-carrying peptides: 386GRNISQHQCVKKQCPQNSGCFRHLDE411, 386GRNISQHQCVKKQCPQNSGCFRHLDEREE414, and 415CKCLLNYKQE424 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.