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 MBP-pfMSP119 antigen surface that 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.
['Opuni|Kwabena F M|KFM|', 'Koy|Cornelia|C|', 'Russ|Manuela|M|', 'Reepmeyer|Maren|M|', 'Danquah|Bright D|BD|', 'Weresow|Moritz|M|', 'Alef|Astrid|A|', 'Lorenz|Peter|P|', 'Thiesen|Hans-Juergen|HJ|', 'Glocker|Michael O|MO|']
D000595:Amino Acid Sequence D000818:Animals D000911:Antibodies, Monoclonal D000936:Antigen-Antibody Complex D000953:Antigens, Protozoan D015153:Blotting, Western D004591:Electrophoresis, Polyacrylamide Gel D018604:Epitope Mapping D000939:Epitopes D016778:Malaria, Falciparum D020066:Merozoite Surface Protein 1 D021241:Spectrometry, Mass, Electrospray Ionization
assembled epitope pfMSP119 antigen antigen malaria Plasmodium falciparum conformational epitope epitope mapping malaria screening. mass spectrometry mass spectrometry (MS) native MS pfMSP1_19 plasmodium protein-protein interaction