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- pf MSP1_{19}, 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 MBP- pf MSP1_{19} antigen surface that is recognized by the anti- pf MSP1_{19} antibody G17.12. We identified three epitope-carrying peptides, ^{386}GRNISQHQCVKKQCPQNSGCFRHLDE^{411}, ^{386}GRNISQHQCVKKQCPQNSGCFRHLDEREE^{414}, and ^{415}CKCLLNYKQE^{424}, from the GluC-derived peptide mixture. These peptides belong to an assembled (conformational) epitope on the MBP- pf MSP1_{19} 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.
【저자키워드】 mass spectrometry, Antigen, malaria, epitope mapping, protein-protein interaction, Native MS, Plasmodium falciparum, Plasmodium, conformational epitope, Mass Spectrometry (MS), assembled epitope, malaria screening., pfMSP119 antigen, pfMSP1_19,