Sensitive Immunoassay Detection of Plasmodium Lactate Dehydrogenase by Inductively Coupled Plasma Mass Spectrometry

Abstract

Rapid, reliable, and sensitive detection of Plasmodium infection is central to malaria control and elimination. Many Malaria Rapid Diagnostic Tests (RDTs) developed for this purpose depend upon immunoassays that can be improved by advances in bound antibody sensor technology. In a previous study, immuno-polymerase chain reaction (PCR) was shown to provide highly sensitive detection of Plasmodium falciparum lactate dehydrogenase (PfLDH) in monoclonal antibody (mAb) sandwich assays. Here, we show comparably high immunoassay sensitivity by inductively coupled plasma mass spectrometry (ICP-MS) detection of gold nanoparticles (AuNPs). Following capture of PfLDH with the primary mAb and binding of the AuNP-labeled detection mAb, ICP-MS signals from the AuNPs provided quantitative measures of recombinant PfLDH test dilutions and P. falciparum-infected erythrocytes. A detection limit of 1.5 pg/mL was achieved with the PfLDH protein. Parasitemia in cultures of P. falciparum-infected erythrocytes could be detected to a lower limit of 1.6 parasite/μl (p/μl) for early ring-stage forms and 0.3 p/μl for mixed stages including mature trophozoites and schizont-stages. These results show that ICP-MS detection of AuNPs can support highly sensitive and accurate detection of Plasmodium infection.

Keywords: Gold nanoparticles; ICP-MS; antigen-based detection; diagnostic tests; malaria.

Figure 1

Schematic of the processes involved in the immunoassay detection of PfLDH by ICP-MS. (A) The test tubes are coated with the primary anti-PfLDH monoclonal antibody (mAb). (B) After blocking with BSA and washing, PfLDH antigens are captured by the primary mAb. (C) AuNP-labeled detection mAbs are bound to the capture PfLDH molecules. (D) The AuNP–protein complexes are dissolved in the aqua regia for injection into the spray chamber. ICP, which is a plasma sustained by inductively coupled radio frequency energy via a load coil after generation from a stream of argon passed through a quartz torch, serves as the ionization source. In the vaporization and ionization phase, the droplets of the sample travel through the different heating zones, in which they are dried by the plasma torch, vaporized, atomized, and finally ionized. (E) Ions are separated by the mass analyzer. (F) The detector displays the isotopic fingerprint and the analyte ion count rate, which is proportional to the mass fraction of the analyte in the sample droplets.

Figure 2

ICP-MS immunoassay results showing the elemental gold signal levels from known amounts of recombinant PfLDH protein. PfLDH was captured by the primary mAb from 30 µl volumes of the indicated concentrations of PfLDH. After binding of AuNP-labeled detection mAb and analysis by ICP-MS, the elemental gold levels were used to calculate the linear best-fit calibration line (solid line) and 95% confidence intervals (dotted lines). Average values with standard errors are shown. All experiments were performed in duplicate.

Figure 3

Sensitive detection of the P. falciparum parasites by ICP-MS immunoassay targeting the PfLDH antigen. The ICP-MS method is able to detect AuNPs from different samples of (A) mixed stage and (B) ring stage P. falciparum-infected erythrocytes. Limits of detection (LOD) of the ICP-MS PfLDH immunoassay are estimated to be (C) 0.3 p/µl for the mixed stages, and (D) 1.6 p/µl for the ring stages. The horizontal bars represent the 95% confidence interval. The averages are from experiments repeated separately on different days.