Abstract
BackgroundDespite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field.MethodsThis report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay.ResultsPurified nHRP2 was identified by SDS-PAGE and western blot as a − 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL.ConclusionsPurified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.Graphical
Highlights
Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field
Binding of anti‐HRP2 antibody to native HRP2 (nHRP2) by Western blot and Enzyme‐linked immunosorbent assay (ELISA) Purified nHRP2 from HB3 culture supernatant was evaluated by SDS-PAGE
Both the Coomassie gel and Western blots show the change in band intensity and clarity during the process of purification via the Nickel nitrilotriacetic acid (Ni–NTA) column, going from the unprocessed cultured supernatant followed by the column flow-through, and lastly, the eluted antigen
Summary
Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field. The HRP2 antigen was first identified in 1974 in the avian parasite Plasmodium lophurae [13], and since three unique histidine-rich proteins have been identified in the P. falciparum genome [8]. These include the knobassociated histidine-rich protein, PfKAHRP ( known as HRP1 [14, 15]), as well as HRP2 and HRP3 [16,17,18], which were all characterized shortly after the methods for P. falciparum culture were first published in 1976 by Trager and Jensen [19]. The two exons of HRP2 encode for a protein with a range of 600–960 amino acids [20] which includes numerous repeating epitopes composed of approximately 34% histidine, 37% alanine, and 10% aspartic acid [16]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
