Immunomagnetic capture and colorimetric detection of malarial biomarker Plasmodium falciparum lactate dehydrogenase

Abstract

We report a sensitive, magnetic bead-based colorimetric assay for Plasmodium falciparum lactate dehydrogenase (PfLDH) in which the biomarker is extracted from parasitized whole blood and purified based on antigen binding to antibody-functionalized magnetic particles. Antigen-bound particles are washed, and PfLDH activity is measured on-bead using an optimized colorimetric enzyme reaction (limit of detection [LOD] = 21.1 ± 0.4 parasites/μl). Enhanced analytical sensitivity is achieved by removal of PfLDH from the sample matrix before detection and elimination of nonspecific reductases and species that interfere with the optimal detection wavelength for measuring assay development. The optimized assay represents a simple and effective diagnostic strategy for P. falciparum malaria with time-to-result of 45 min and detection limits similar to those of commercial enzyme-linked immunosorbent assay (ELISA) kits, which can take 4-6 h. This method could be expanded to detect all species of malaria by switching the capture antibody on the magnetic particles to a pan-specific Plasmodium LDH antibody.

Characterization of Plasmodium Lactate Dehydrogenase and Histidine-Rich Protein 2 Clearance Patterns via Rapid On-Bead Detection from a Single Dried Blood Spot

“…Anti- p LDH and anti-HRP2 beads were prepared as reported previously. 19 , 24 Briefly, anti- p LDH (Vista 19g7) or anti-HRP2 (Abcam ab9203) was biotinylated with EZ-Link NHS–PEG4–Biotin, No-Weigh Format (Thermo no. 21329).…”

Simultaneous capture and sequential detection of two malarial biomarkers on magnetic microparticles

“…For p LDH, magnetic microparticles were used to isolate Plasmodium falciparum lactate dehydrogenase ( Pf LDH) from lysed whole blood. The biomarker was then detected using the Malstat assay, and enzymatic turnover assay catalyzed by Pf LDH [15]. The detection limit for this method was 26 pM Pf LDH, an order of magnitude higher than the developed on-bead SCSD ELISA for p LDH and Pf HRPII.…”

“…Target-specific antibody-functionalized beads were prepared as reported previously [15]. Briefly, α- p LDH (Vista, 19g7) or α- Pf HRPII (Abcam, ab9203) antibodies were biotinylated with EZ-Link NHS-PEG4-Biotin, No-Weigh Format (Thermo Pierce #21329) in PBS with a 20× excess of NHS-PEG4 Biotin.…”

Simple, rapid, and accurate malaria diagnostic platform using microfluidic-based immunoassay of Plasmodium falciparum lactate dehydrogenase

“…Therefore, it is necessary to develop an alternative diagnostic biomarker instead of HRP2. In this study, we chose P. falciparum lactate dehydrogenase (PfLDH) as an alternative biomarker for the diagnosis of malaria (P. falciparum), which is a water-soluble enzyme that converts pyruvate to lactate in glycolysis in P. falciparum infection [5,13,14].…”

“…To overcome the limitations of existing diagnostics, several studies demonstrated improved detection efficiency by introducing nanoparticles such as magnetic beads. Markwalter et al successfully detected PfLDH up to 21.1 ± 0.4 parasites/ mL within 45 min using antibody-immobilized magnetic beads as a colorimetric assay [14]. Additionally, they developed a simultaneous capture and sequential detection of two malarial biomarkers (PfLDH and HRP2) on magnetic microparticles [23].…”