Abstract
We have developed a rapid magnetic microparticle-based detection strategy for malarial biomarkers Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein II (PfHRPII). In this assay, magnetic particles functionalized with antibodies specific for pLDH and PfHRPII as well as detection antibodies with distinct enzymes for each biomarker are added to parasitized lysed blood samples. Sandwich complexes for pLDH and PfHRPII form on the surface of the magnetic beads, which are washed and sequentially re-suspended in detection enzyme substrate for each antigen. The developed simultaneous capture and sequential detection (SCSD) assay detects both biomarkers in samples as low as 2.0parasites/µl, an order of magnitude below commercially available ELISA kits, has a total incubation time of 35min, and was found to be reproducible between users over time. This assay provides a simple and efficient alternative to traditional 96-well plate ELISAs, which take 5–8h to complete and are limited to one analyte. Further, the modularity of the magnetic bead-based SCSD ELISA format could serve as a platform for application to other diseases for which multi-biomarker detection is advantageous.
Highlights
Enzyme-linked immunosorbent assays (ELISAs) are the gold standard laboratory technique for quantitative and qualitative protein detection, serving as both powerful research tools and clinical diagnostics
For Plasmodium falciparum histidine-rich protein II (PfHRPII) assays, C1–13 capture and MPFG-55P, a detection antibody conjugated to horseradish peroxidase (HRPx), have been previously validated as an appropriate pair for ELISA formats [16]
To perform the simultaneous capture and sequential detection (SCSD) assay, magnetic particles functionalized with capture antibodies for Plasmodium lactate dehydrogenase (pLDH) and PfHRPII were incubated in lysed whole blood samples along with the detection antibodies for each biomarker
Summary
Enzyme-linked immunosorbent assays (ELISAs) are the gold standard laboratory technique for quantitative and qualitative protein detection, serving as both powerful research tools and clinical diagnostics. There are several disadvantages to current multiplexed immunoassays Both planar and bead-based immunoassays require laboratory infrastructure beyond that needed to perform singleplex conventional ELISAs; planar micro-array assays require highresolution fluorescence scanners, and bead-based immunoassays require flow cytometric instrumentation for detection [1]. Available bead-based suspension assays often require 3–4 h for completion, up to 1 h dedicated to the detection step [8] To address these pitfalls, we have developed a magnetic bead-based ELISA in which two biomarkers are simultaneously captured and sequentially detected in less than 1 h with no laboratory infrastructure beyond what is required to perform a conventional singleplex well-plate ELISA. The presented assay design is modular and can be applied to any set of two biomarkers provided validated antibody pairs are available
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