Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate.
Antigens, Protozoan
/ immunology
Blotting, Western
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
Erythrocytes
/ chemistry
Humans
Immunoassay
Malaria, Falciparum
/ diagnosis
Microspheres
Plasmodium falciparum
/ chemistry
Protozoan Proteins
/ immunology
Quality Control
Time Factors
Antigen
Histidine-rich protein 2 (HRP2)
Plasmodium falciparum
Protein purification
Journal
Malaria journal
ISSN: 1475-2875
Titre abrégé: Malar J
Pays: England
ID NLM: 101139802
Informations de publication
Date de publication:
17 Oct 2021
17 Oct 2021
Historique:
received:
30
07
2021
accepted:
06
10
2021
entrez:
18
10
2021
pubmed:
19
10
2021
medline:
14
1
2022
Statut:
epublish
Résumé
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. This 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. Purified 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. Purified 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.
Sections du résumé
BACKGROUND
BACKGROUND
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.
METHODS
METHODS
This 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.
RESULTS
RESULTS
Purified 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.
CONCLUSIONS
CONCLUSIONS
Purified 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.
Identifiants
pubmed: 34657602
doi: 10.1186/s12936-021-03946-1
pii: 10.1186/s12936-021-03946-1
pmc: PMC8522059
doi:
Substances chimiques
Antigens, Protozoan
0
HRP-2 antigen, Plasmodium falciparum
0
Protozoan Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
405Informations de copyright
© 2021. The Author(s).
Références
Malar J. 2020 Aug 26;19(1):300
pubmed: 32843041
J Clin Microbiol. 2019 Feb 27;57(3):
pubmed: 30626660
Am J Trop Med Hyg. 2017 Oct;97(4):1159-1162
pubmed: 28722629
Am J Trop Med Hyg. 2008 May;78(5):733-5
pubmed: 18458305
J Biol Chem. 1974 Jul 25;249(14):4650-5
pubmed: 4843147
PLoS One. 2016 Aug 12;11(8):e0157949
pubmed: 27518538
Anal Chem. 2012 Jul 17;84(14):6136-42
pubmed: 22734432
Mol Biochem Parasitol. 1989 Jun 15;35(2):149-60
pubmed: 2671720
Am J Trop Med Hyg. 2010 Oct;83(4):834-7
pubmed: 20889875
J Cell Biol. 1984 Apr;98(4):1256-64
pubmed: 6371019
Malar J. 2020 Mar 4;19(1):108
pubmed: 32131841
Methods Mol Biol. 1994;32:5-8
pubmed: 7951748
Malar J. 2013 Jan 24;12:34
pubmed: 23347727
Malar J. 2014 Jul 22;13:283
pubmed: 25052298
Malar J. 2020 Jul 16;19(1):256
pubmed: 32678034
Sci Transl Med. 2017 Jul 5;9(397):
pubmed: 28679662
J Clin Microbiol. 1991 Aug;29(8):1629-34
pubmed: 1761684
Southeast Asian J Trop Med Public Health. 2003 Dec;34(4):739-43
pubmed: 15115081
J Infect Dis. 2005 Sep 1;192(5):870-7
pubmed: 16088837
Proc Natl Acad Sci U S A. 1986 Aug;83(16):6065-9
pubmed: 3016741
J Exp Med. 1980 Jun 1;151(6):1534-8
pubmed: 6991629
J Infect Dis. 2018 Feb 14;217(5):685-692
pubmed: 29220497
Trends Parasitol. 2020 Feb;36(2):112-126
pubmed: 31848119
Southeast Asian J Trop Med Public Health. 1998 Mar;29(1):35-40
pubmed: 9740265
J Clin Microbiol. 2019 Aug 26;57(9):
pubmed: 31270184
Cell. 1987 Jun 5;49(5):633-42
pubmed: 2884039
J Med Microbiol. 2013 Oct;62(Pt 10):1491-1505
pubmed: 24048274
Malar J. 2014 Jan 29;13:34
pubmed: 24472178
Nucleic Acids Res. 1985 Nov 11;13(21):7837-46
pubmed: 2415925
J Cell Biol. 1986 Oct;103(4):1269-77
pubmed: 3533951
Infect Immun. 1991 Nov;59(11):3909-16
pubmed: 1937750
Am J Trop Med Hyg. 2019 May;100(5):1202-1203
pubmed: 30915959
Science. 1976 Aug 20;193(4254):673-5
pubmed: 781840
Sci Rep. 2020 May 21;10(1):8443
pubmed: 32439948