Rapid concentration and elution of malarial antigen histidine-rich protein II using solid phase Zn(II) resin in a simple flow-through pipette tip format

Abstract

Rapid diagnostic tests (RDTs) designed to function at the point of care are becoming more prevalent in malaria diagnostics because of their low cost and simplicity. While many of these tests function effectively with high parasite density samples, their poor sensitivity can often lead to misdiagnosis when parasitemia falls below 100 parasites/l. In this study, a flow-through pipette-based column was explored as a cost-effective means to capture and elute more histidine-rich protein II (HRPII) antigen, concentrating the biomarker available in large-volume lysed whole blood samples into volumes compatible with-specific RDTs. A systematic investigation of immobilized metal affinity chromatography divalent metal species and solid phase supports established the optimal design parameters necessary to create a flow-through column incorporated into a standard pipette tip. The bidirectional flow inherent to this format maximizes mixing efficiency so that in less than 5 min of sample processing, the test band signal intensity was increased up to a factor of twelve from HRPII concentrations as low as 25 pM. In addition, the limit of detection per sample was decreased by a factor of five when compared to the RDT manufacturer's suggested protocol. Both the development process and commercial viability of this application are explored, serving as a potential model for future applications.

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

“… 16 , 19 In addition, HRP2 in the 43,600 parasites/μL stock was previously determined to be 2.2 pM per parasite/μL. 23 The p LDH concentration of in-house D6 P. falciparum culture (stock 43,600 parasites/μL) was determined to be 4.4 pM per parasite/μL using a standard well-plate ELISA, N = 6 ( Supplemental Text 1 ).…”

Inorganic Complexes and Metal-Based Nanomaterials for Infectious Disease Diagnostics

“…This field-deployable sample isolation and concentration tool displayed up to a 4-fold signal enhancement in commercial rapid diagnostic tests (Figure 7). This study 99 demonstrated that optimizing the IMAC metal-ligand combination for a particular application is critical for efficient biomarker capture and elution; the initial binding cannot be too tight so as to prevent biomarker elution with a competing ligand (e.g., imidazole) nor too weak so as to attain suboptimal biomarker enrichment. 88,99…”

“…The authors found that Co 2+ and Zn 2+ exhibited the fastest binding kinetics (highest k on ) and the highest HRP2 binding affinity (lowest K D ) on BLI biosensors. 99 When the same IMAC complexes were incorporated into a loose solid phase resin, Zn(II)-NTA resulted in the fastest complete binding of HRP2 per mole of divalent metal while also allowing for the most efficient elution of HRP2 upon the addition of imidazole. Once a metal had been selected, HRP2’s binding and elution behavior was evaluated against a variety of ligands and solid supports.…”

“…This small volume sample was then deposited onto a commercially available rapid diagnostic test which enhanced sensitivity for HRP2 detection in a POC setting. Adapted with permission from ref (99). Copyright 2017 American Institute of Physics.…”

Nickel (II) and Cobalt (II) Alginate Biopolymers as a “Carry and Release” Platform for Polyhistidine-Tagged Proteins

“…One example of a successful non-incorporating large-scale use strategy for biopolymer protein immobilization is used in a type of affinity chromatography termed IMAC (immobilized metal affinity chromatography) [ 18 , 19 ]. IMAC uses agarose beads with grafted chelating moieties (iminodiacetic acid—IDA, nitrilotriacetic acid—NTA).…”