High-Sensitivity Assays for Plasmodium falciparum Infection by Immuno-Polymerase Chain Reaction Detection of PfIDEh and PfLDH Antigens

Abstract

Background: Rapid diagnostic tests based on Plasmodium falciparum histidine-rich protein II (PfHRP-II) and P. falciparum lactate dehydrogenase (PfLDH) antigens are widely deployed for detection of P. falciparum infection; however, these tests often miss cases of low-level parasitemia, and PfHRP-II tests can give false-negative results when P. falciparum strains do not express this antigen.

Methods: We screened proteomic data for highly expressed P. falciparum proteins and compared their features to those of PfHRP-II and PfLDH biomarkers. Search criteria included high levels of expression, conservation in all parasite strains, and good correlation of antigen levels with parasitemia and its clearance after drug treatment. Different assay methods were compared for sensitive detection of parasitemia in P. falciparum cultures.

Results: Among potential new biomarkers, a P. falciparum homolog of insulin-degrading enzyme (PfIDEh) met our search criteria. Comparative enzyme-linked immunosorbent assays with monoclonal antibodies against PfLDH or PfIDEh showed detection limits of 100-200 parasites/µL and 200-400 parasites/µL, respectively. Detection was dramatically improved by use of real-time immuno-polymerase chain reaction (PCR), to parasitemia limits of 0.02 parasite/µL and 0.78 parasite/µL in PfLDH- and PfIDEh-based assays, respectively.

Conclusions: The ability of PfLDH- or PfIDEh-based immuno-PCR assays to detect <1 parasite/µL suggests that improvements of bound antibody sensor technology may greatly increase the sensitivity of malaria rapid diagnostic tests.

Keywords: PfHRP-II; enzyme-linked immunosorbent assay; immuno-PCR; insulin-degrading enzyme; lactate dehydrogenase enzyme.

Figure 1.

Map of Plasmodium falciparum insulin-degrading enzyme homolog (PfIDE_h) plasmid inserts and plots of enzyme-linked immunosorbent assay (ELISA) results with monoclonal antibody (mAb) pairs. A, Plasmid inserts were amplified from the PfIDE_h sequence by indicated primer pairs. Schematic of the PfIDE_h primary structure shows relative locations of the KQQDNV and DDDNTS repeats. B, Plots show signal levels obtained from horseradish peroxidase (HRP)–labeled 3B4D12, 5A2G2, and 3A3 mAbs in ELISAs of recombinant PfIDE_h (rc-PfIDE_h) or of PfIDE_h captured by rabbit mAb AID-1-1-9 from 3D7 parasites. C, Plots show signal levels from HRP-labeled goat antimouse antibody applied to unlabeled 3B4D12, 5A2G2, and 3A3 mAbs in ELISAs of rc-PfIDE_h or of PfIDE_h captured by rabbit mAb AID-1-1-9 from 3D7 parasites.

Figure 2.

Parasite detection in Plasmodium falciparum cultures by enzyme-linked immunosorbent assay based on P. falciparum histidine-rich protein II (PfHRP-II), P. falciparum lactate dehydrogenase (PfLDH), and P. falciparum insulin-degrading enzyme homolog (PfIDE_h). A, PfHRP-II in 3D7 lysates was captured by unlabeled MPFG-55A and detected by horseradish peroxidase (HRP)–labeled MPFM-55A. Signal levels decreased with dilutions of HRP-labeled-MPFM-55A. B, PfLDH in 3D7 lysates was captured by MBS498007 and detected by HRP-labeled-MBS498008. Signal levels decreased with dilutions of HRP-labeled-MBS498008. C, PfIDE_h in 3D7 lysates was captured by rabbit monoclonal antibody (mAb) AID-1-1-9 and detected by HRP-labeled mAb 3B4D12. Signal levels decreased with dilutions of HRP-labeled mAb 3B4D12. D, PfIDE_h in 3D7 lysates was captured by mAb 3B4D12 and detected by HRP-labeled AID-1-1-9. Signal levels decreased with dilutions of HRP-labeled AID-1-1-9. Dashed line in the panels represents the absorbance signal cutoff. Results are the average ± standard deviation of 3 independent experiments.

Figure 3.

Immuno–polymerase chain reaction (i-PCR) detection of Plasmodium falciparum lactate dehydrogenase (PfLDH) and P. falciparum insulin-degrading enzyme homolog (PfIDE_h) antigens. A, i-PCR curves of PfLDH in 2-fold serially diluted lysates from a culture of P. falciparum 3D7. MBS498007 was employed as the capture antibody and streptavidin-conjugated MBS498008 was used for i-PCR detection of PfLDH. Signal levels were proportional to the lysate dilution. Results from negative controls (NC; no added biotinylated DNA) and no lysate (water only) are shown in black and dark green, respectively. Final PCR products were run on 2% agarose gel to confirm the specific amplification of targeted region (161 bp). Late signals in water control samples (dark green) were from primer dimers, also confirmed by gel electrophoresis. Light-green curve shows the delta cycle threshold (∆Ct) of the tested sample concentration (0.02 parasite/μL) closest to the limit of detection (LOD), calculated as the value of the NC plus 3 standard deviations (SD). B, i-PCR curves of PfIDE_h in 2-fold serially diluted lysates from a culture of P. falciparum 3D7. Mouse monoclonal antibody (mAb) 3B4D12 was employed as the capture antibody and streptavidin-conjugated rabbit mAb AID-1-1-9 was used for detection of PfIDE_h. Signal levels were proportional to the lysate dilution. Results from negative controls of no added biotinylated DNA and no lysate (water only) are shown in black and dark green, respectively. Light-green curve shows the ∆C_t of the tested sample concentration (0.78 parasite/μL) closest to the LOD, which is calculated as the value of the NC plus 3 SD and is limited by the background signal from NC. C, i-PCR curves of 2-fold serially diluted recombinant PfIDE_h (rc-PfIDE_h) protein captured by 3B4D12 and detected by AID-1-1-9. Results from negative controls of no added biotinylated DNA and no lysate (water only) are shown in black and dark green, respectively. Light-green curve shows the ∆Ct of the tested rc-PfIDE_h concentration (3 fg/μL) closest to the LOD, calculated as the value of the NC plus 3 SD. All experiments were performed in duplicate. Abbreviations: iRBC, infected red blood cell; Norm. Fluoro., normalized fluorescence.

Figure 4.

Persistence of detectable Plasmodium falciparum insulin-degrading enzyme homolog (PfIDE_h), P. falciparum lactate dehydrogenase (PfLDH), and P. falciparum histidine-rich protein II (PfHRP-II) after treatment of P. falciparum culture with chloroquine. A, Enzyme-linked immunosorbent assay (ELISA) optical density (OD) values from protein in pelleted infected erythrocytes. B, ELISA OD values from protein in supernatants. Monoclonal antibody pairs used to detect PfIDE_h, PfLDH, and PfHRP-II were as follows: 3B4D12 and horseradish peroxidase (HRP)–labeled AID-1-1-9; MBS498007 and HRP-labeled MBS498008; and MPFG-55A and HRP-labeled MPFM-55A, respectively. Abbreviations: iRBC, infected red blood cell.