Abstract
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.
Highlights
Malaria, one of the most significant parasitic diseases in the world, is endemic in 91 countries and territories
All of the MSP1nn polypeptides are shed from the parasite surface with the exception of MSP119, which remains attached to the merozoite surface and, upon the parasite’s entering of red blood cells, is presented on the cell surfaces [9, 10]
The amylose affinity-purified maltose-binding protein (MBP)-pfMSP119 fusion protein was cloned and expressed in Escherichia coli as a starting material for mapping the epitope, which is recognized by a monoclonal anti-pfMSP119 antibody
Summary
One of the most significant parasitic diseases in the world, is endemic in 91 countries and territories. The clinical manifestation of acute malaria due to P. falciparum is normally associated with replication of merozoites, asexual blood-stage parasites, in circulating red blood cells [4]. The antigens that are employed in immunoanalytical assays are histidine-rich protein 2 of P. falciparum (pfHRP2), Plasmodium aldolase, and parasite lactate dehydrogenase (pLDH) [4]. The merozoits' surface proteins are responsible for initial interaction between the parasite and the hosts red blood cells. All of the MSP1nn polypeptides are shed from the parasite surface with the exception of MSP119, which remains attached to the merozoite surface and, upon the parasite’s entering of red blood cells, is presented on the cell surfaces [9, 10]. Together with a specific antibody to be applied as positive control, an improved immunoanalytical assay is envisaged for accurate malaria diagnosis. Toward epitope peptide–based malaria screening of false positive and/or false negative test results when assaying patient sera
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