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

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 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.

Intact Transition Epitope Mapping—Serological Inspection by Epitope EXtraction (ITEM—SIX)

“…Acetone precipitation of proteins has been found generally applicable for concentrating proteins from aqueous solutions of all kinds [ 45 ], and protein precipitation yields were found to depend on ionic strengths of the solvents [ 46 ]. The third step of our IgG preparation protocol, i.e., filtration, followed a well-established buffer exchange procedure that has been amply applied by us in earlier ITEM projects [ 22 , 27 , 47 ].…”

“…At this point, 2.5 µL of the retentate R1 (solution 0) or 2.5 µL of anti-Ovalbumin antibody (solution 1a) were transferred into a gold-coated capillary needle and analyzed by offline nanoESI-MS using the Q-TOF II mass spectrometer (Waters, Manchester, UK) [ 47 ]. Likewise, 2.5 µL of IgG from converted serum (solution 1b) were diluted with 29 µL of 0.2 M ammonium acetate, pH 6.7, to yield a protein concentration of 0.17 µg/µL.…”

Mass Spectrometric ITEM-ONE and ITEM-TWO Analyses Confirm and Refine an Assembled Epitope of an Anti-Pertuzumab Affimer

“…Contrary to surface plasmon resonance or related methods [50] for determining affinities, ITEM-TWO requests no chemical immobilization of one of the binding partners, making quantitative binding analysis very simple and perhaps least error prone. In addition, performing ITEM-ONE and ITEM-TWO is fast and typically a series of measurements including blanks and negative controls is done within a few hours [51]. Also, compared to all the other above mentioned methods, ITEM-ONE and ITEM-TWO need very little material.…”

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti‐HpTGEKP Antibody and Zinc Finger Protein Linker Phospho‐Hexapeptides

“…Nanospray offline mass spectrometric ITEM‐TWO analyses : NanoESI offline mass spectrometric measurements and ITEM‐TWO analyses[ 23 , 35 ] were performed as previously described. For details see Supporting Information.…”