Pipetting-based immunoassay for point-of-care testing: Application for detection of the influenza A virus

Seungjoo Haam,Sun-Woo Yoon,Daesub Song,Min-Chul Jung,Min-Ju Ahn,Van Phan Le,Thi Van Lo,Hye Kwon Kim,Tran Bac Le,Woonsung Na,Ji Yeong Noh,Youngji Kim,Dae Gwin Jeong

doi:10.1038/s41598-019-53083-8

Abstract

Point-of-care tests (POCT) for pathogens are considered important for low-resource countries and facilities. Although lateral flow immunoassays (LFIA) have many advantages including speed and ease of use, their sensitivity is limited without specific equipment. Furthermore, their response cannot be enhanced through enzymatic reactions. Owing to these limitations, LFIAs have not yet been generally adopted as the standard protocol for in vitro analysis of infectious pathogens. We aimed to develop a novel pipetting-based immunoassay using a removable magnetic ring-coupled pipette tip. The “magnetic bead-capture antibody-targeted protein complex” was simply purified by pipetting and quantified by enzymatic colour development or using a lateral flow system. This pipetting-based immunoassay was applied to detect the nucleoprotein (NP) of the influenza A virus. Using an HRP-conjugated monoclonal antibody as a probe, the assay allowed for specific and sensitive detection. Furthermore, when this assay was applied exclusively for antigen capture in the lateral flow system, the limit of detection improved 100-fold and displayed greater sensitivity than the lateral flow system alone. Therefore, the pipetting-based immunoassay may be potentially used as a sensitive POCT to clinically detect a target antigen.

Highlights

Point-of-care tests (POCT) for pathogens are considered imperative for low-resource countries and facilities[1]
Lateral flow immunoassays (LFIA) have many advantages, including speed and ease of use, their sensitivity is limited without specific equipment and it is not possible to enhance the response through enzymatic reactions[3]
To select the optimal pipette tip, two types of 1 mL pipette tips were compared for their applicability in the pipetting-based immunoassay targeting nucleoprotein (NP) of influenza A virus: a general tip (OHAUS) and a low-binding tip (Bioneer) were compared using 100 μL of 110 μg/mL recombinant influenza NP protein as the positive control and phosphate buffered saline (PBS) as the negative control

Summary

Introduction

Point-of-care tests (POCT) for pathogens are considered imperative for low-resource countries and facilities[1]. LFIAs have many advantages, including speed and ease of use, their sensitivity is limited without specific equipment and it is not possible to enhance the response through enzymatic reactions[3] Due to this limitation, LFIAs have not yet been generally adopted as the standard protocol for the standard in vitro analysis of infectious pathogens. There are still practical barriers to clinical application due to the need for specific equipment and complicated fluidic networks To overcome these barriers, a new POCT device was recently developed based on the volumetric measurement of oxygen generated through an ELISA reaction, called the multiplexed volumetric bar-chart chip[11,12]. A “magnetic bead-capture antibody-targeted protein complex” was purified by pipetting and quantified by an enzyme-based colour reaction and lateral flow test This simplified protocol is easy to develop and is applicable in low-resource settings. Its suitability for the POCT was evaluated with the influenza A virus

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Results

Conclusion