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A magnetoelastic biosensor based on E2 glycoprotein for wireless detection of classical swine fever virus E2 antibody.

ABSTRACT: A wireless magnetoelastic (ME) biosensor immobilized with E2 glycoprotein was first developed to detect classical swine fever virus (CSFV) E2 antibody. The detection principle is that a sandwich complex of CSFV E2 - rabbit anti-CSFV E2 antibody - alkaline phosphatase (AP) conjugated goat anti-rabbit IgG formed on the ME sensor surface, with biocatalytic precipitation used to amplify the mass change of antigen-antibody specific binding reaction, induces a significant change in resonance frequency of the biosensor. Due to its magnetostrictive feature, the resonance vibrations and resonance frequency can be actuated and wirelessly monitored through magnetic fields. The experimental results show that resonance frequency shift increases with the augmentation of the CSFV E2 antibody concentration. Scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS) and fluorescence microscopy analysis proved that the modification and detection process were successful. The biosensor shows a linear response to the logarithm of CSFV E2 antibody concentrations ranging from 5 ng/mL to 10??g/mL, with a detection limit (LOD) of 2.466 ng/mL and the sensitivity of 56.2 Hz/?g·mL-1. The study provides a low-cost yet highly-sensitive and wireless method for selective detection of CSFV E2 antibody.

SUBMITTER: Guo X

PROVIDER: S-EPMC5688166 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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A magnetoelastic biosensor based on E2 glycoprotein for wireless detection of classical swine fever virus E2 antibody.

Guo Xing X Sang Shengbo S Guo Jinyu J Jian Aoqun A Duan Qianqian Q Ji Jianlong J Zhang Qiang Q Zhang Wendong W

Scientific reports 20171115 1

A wireless magnetoelastic (ME) biosensor immobilized with E2 glycoprotein was first developed to detect classical swine fever virus (CSFV) E2 antibody. The detection principle is that a sandwich complex of CSFV E2 - rabbit anti-CSFV E2 antibody - alkaline phosphatase (AP) conjugated goat anti-rabbit IgG formed on the ME sensor surface, with biocatalytic precipitation used to amplify the mass change of antigen-antibody specific binding reaction, induces a significant change in resonance frequency ...[more]

PMID: 29142249

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Project description:E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions, including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on a Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains an 872GG873 dipeptide, as well as two aromatic residues (871W and 875W) accessible to solvent. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICv in vitro and virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage primary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days postvaccination. Supporting a role for the E2 the loop from residues 864 to 881 in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for insertion into model membranes and perturbation of their integrity, and this functionality was lost in synthetic peptides harboring amino acid substitutions W871T, W875D, and V878T in FPII.2. IMPORTANCE:This report describes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identification was performed by functional structural analysis of E2. We characterized the functional significance of this FP by using artificial membranes. Replacement of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced the same mutations into the viral sequence, there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuated in vivo efficiently protected pigs against wild-type virus. These results provide the proof of principle to support as a strategy for vaccine development the discovery and manipulation of FPs.

Project description:Vaccination is an effective method to control the spread of classical swine fever virus (CSFV), which is a major cause of economic losses to the swine industry. Although serological detection assays are commonly used to assess immune status, current methods for monitoring of antibodies (Abs) are time-consuming, expensive, and require cell culture and virus manipulation. To address these problems, the E2 protein of CSFV was expressed in transgenic rice seeds as a labeled antigen for the development of an immunochromatographic test strip (ICTS) for rapid, precise, and cost-effective detection of Abs. The ICTS has a reasonable sensitivity of 1:128,000 for detection of serum Abs against CSFV and no cross-reactivity with Abs of other porcine viruses. The similarity of the results between the proposed ICTS and a commercial enzyme-linked immunosorbent assay was 94.1% (128/136) for detection of serum Abs from immunized animals and 92.3% (72/78) for detection of maternally derived Abs. The proposed assay was successfully used to monitor Abs against E2 of both pigs and rabbits immunized with a live attenuated vaccine or an E2 subunit vaccine. The results confirmed that the ICTS can be applied to detect Ab levels in animals with different immunological backgrounds. The ICTS based on plant-derived E2 is a relatively inexpensive, rapid, and accurate assay for detection of Abs against CSFV and avoids the risk of contamination by animal products. IMPORTANCE The E2 protein of classical swine fever virus (CSFV) was expressed in transgenic rice endosperms as a diagnostic antigen for use with a rapid colloidal gold assay for the detection of antibodies (Abs) against CSFV. This improved test was used to monitor Abs against the E2 protein in both pigs and rabbits immunized with a live attenuated vaccine or E2 subunit vaccine. The assay successfully detected Ab levels in serum samples from piglets with different immunological backgrounds. In contrast to current E2 protein-based diagnostic methods using Escherichia coli or insect cells as expression systems, plant-derived E2 avoids the limitations of low immunogenicity of eukaryotic expression systems and potential contamination of fetal bovine serum with bovine viral diarrhea virus in cell culture.

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A magnetoelastic biosensor based on E2 glycoprotein for wireless detection of classical swine fever virus E2 antibody.

Publications

A magnetoelastic biosensor based on E2 glycoprotein for wireless detection of classical swine fever virus E2 antibody.

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