A Highly Sensitive Detection System based on Proximity-dependent Hybridization with Computer-aided Affinity Maturation of a scFv Antibody

Zhiheng Wang,Yan Li,Wenbin Liang,An Chen,Shuhui Li,Chuanmin Hu,Junsong Zheng

doi:10.1038/s41598-018-22111-4

Zhiheng Wang, Yan Li + Show 5 more

Open Access

https://doi.org/10.1038/s41598-018-22111-4

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Journal: Scientific Reports	Publication Date: Mar 1, 2018
Citations: 8	License type: open-access

Affiliation: Army Medical University, Southwest Hospital

Abstract

The hepatitis B virus (HBV) infection is a critical health problem worldwide, and HBV preS1 is an important biomarker for monitoring HBV infection. Previously, we found that a murine monoclonal antibody, mAb-D8, targets the preS1 (aa91-107) fragment of HBV. To improve its performance, we prepared the single-chain variable region of mAb-D8 (scFvD8) and constructed the three-dimensional structure of the scFvD8-preS1 (aa91-107) complex by computer modelling. The affinity of scFvD8 was markedly increased by the introduction of mutations L96Tyr to Ser and H98Asp to Ser. Furthermore, a highly sensitive immunosensor was designed based on a proximity-dependent hybridization strategy in which the preS1 antigen competitively reacts with an antibody labelled with DNA, resulting in decreased proximity-dependent hybridization and increased electrochemical signal from the Fc fragment, which can be used for the quantisation of preS1. The results showed a wide detection range from 1 pM to 50 pM with a detection limit of 0.1 pM. The sensitivity and specificity of this immunosensor in clinical serum samples were 100% and 96%, respectively. This study provides a novel system based on proximity-dependent hybridization and the scFv antibody fragment for the rapid quantisation of antigens of interest with a high sensitivity.

Highlights

Hepatitis B virus (HBV) infection is a prevalent health problem as more than 350 million humans are chronically infected, and nearly one million people die of hepatitis B virus (HBV) infection related liver disease every year[1]
The single chain variable fragment, which consists of variable regions of heavy (VH) and variable regions of light (VL) chains of immunoglobulin connected with a flexible linker is the most interesting antibody derivative
In the presence of HBV preS1 in the detection system, HBV preS1 would competitively react with antibody labelled DNA (Ab-DNA), decreasing the formation of immune complex between the Ab-DNA and Ag-DNA, and more Fc labelled blocking DNA would hybridize with the Capture Linker

Summary

Introduction

Hepatitis B virus (HBV) infection is a prevalent health problem as more than 350 million humans are chronically infected, and nearly one million people die of HBV infection related liver disease every year[1]. Recent studies have shown that preS1/hepatitis B virus large surface protein (LHBs) detection can be more sensitive than HBeAg and can more accurately reflect HBV replication[5]. For the sensitive determination of HBV, an electrochemical immunosensor has been reported recently by Rosa F Dutra et al It was developed for the detection of antibodies to hepatitis B core protein (anti-HBc) with a linear concentration range up to 6 ng/mL and a detection limit of 0.03 ng/mL21. Because of the specific hybridization among the capture probe, Fc-probe, Ag-DNA and Ab-DNA at a predesigned melting temperature, the surface proximity assay was achieved This biosensor was shown to be sensitive, specific, and selective with a low detection limit and a wide linear dynamic range

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Results

Conclusion