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

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.

Figure 1

Construction and expression of anti-preS1 scFvD8 and scFvD8-M. (A) Total RNA from hybridoma. Lanes: M: marker 2000; 1 and 2: total RNA from mAb D8 hybridoma. (B) PCR amplification of VH and VL with 87 pairs of primers. (C) Enlarged inset from picture B showing the amplification of VL. (D) PCR amplification of scFv gene. Lanes: M: marker 2000; 1 and 2: the scFv fragment of anti-preS1 D8 assembled with a (G₄S₁)₄ linker. (E) SDS-PAGE identification of the purified protein, which was expressed with the pET28a-SUMO vector in the BL21 host. (F) SDS-PAGE identification of the purified protein, which was expressed with the pcDNA3.4 vector in CHO-S cells.

Figure 2

Amino acid sequence of scFvD8 from VL (below the light blue arrow) and VH (below the brown arrow). The linker region is indicated in yellow. CDR regions are highlighted in red. The binding site of scFvD8 was boxed under the “” symbol with Kabat numbering.

Figure 3

Binding complex of scFvD8 and preS1 (aa91-107). (A) Pocket of scFvD8 and preS1 (aa91-107) interaction. (B) Docking model of the amino acid residues of preS1 (aa91-107) inside the binding pocket of scFvD8. The distance between interacting residues is not greater than 0.3 nm with the opposite charge. (C) Point mutation site interaction interface. The model of preS1 (aa91-107) shows the surface electrostatic potential. The positive charge region is shown in red, and the negative charge region is in green. The positively charged amino acid residue L96Tyr of scFvD8 falls into the positive charge region of preS1 (aa91-107). (D) The relative affinities of scFvD8 and scFvD8-M were measured by competitive ELISA. The concentrations of preS1 recombinant protein were 1000, 500, 100, 50, 10, 5, 1, 0.1 and 0.01 nM. At 50% protein binding saturation, the concentration of scFvD8 was 50 nM, and the concentration of scFvD8-M was 5 nM.

Figure 4

Detection strategy based on the proximity-dependent hybridization.

Figure 5

The CV characteristics of the electrode assembly process in the presence of buffer solution (0.1 M KCl and 2.5 mM Fe(CN)₆^4−/3−). The labels denote: a, blank glassy carbon electrode; b, after AuNP modified glassy carbon electrode; c, after the modification of the Capture Linker; d, after the hybridization of the Fc labelled blocking DNA; e, after blockade with HT.

Figure 6

The optimization of DNA hybridization conditions. (A) Effects of the Ab-DNA/Ag-DNA concentration (10 to 70 nM) on the detection of 50 pM preS1 recombinant protein. Reaction conditions: TBE buffer (pH 7.4), 0.4 M Mg²⁺, incubation at 37 °C for 1 h. (B) Effects of temperature (25 to 50 °C) on the detection of 50 pM preS1 recombinant protein. Reaction conditions: 50 nM Ab-DNA/Ag-DNA, TBE buffer (pH 7.4), 0.4 M Mg²⁺, incubation for 1 h. (C) Effects of the Mg²⁺ concentration (0 to 0.8 M) on the detection of 50 pM preS1 recombinant protein. Reaction conditions: 50 nM Ab-DNA/Ag-DNA, TBE buffer (pH 7.4), incubation at 37 °C for 1 h.

Figure 7

Electrochemical signals as a function of the concentration of preS1 recombinant protein. (A) Responses of the electrochemical biosensor at different concentrations of preS1 recombinant protein (curves a-g, at concentrations of 0, 0.01, 0.1, 1, 10, 20, and 50 pM, respectively). (B) Calibration curve for the relationship between electrochemical signal and concentration of preS1 recombinant protein.