Peptide aptamer-based time-resolved fluoroimmunoassay for CHIKV diagnosis

Abstract

Background: Chikungunya virus (CHIKV) and Dengue virus (DENV) have similar clinical symptoms, which often induce misdiagnoses. Therefore, an antigen detection diagnostic system that can clearly identify these two viruses is desirable.

Methods: In this study, we developed a novel peptide with high affinity and specificity to CHIKV, and further constructed peptide aptamer-based TRFIA assay to efficiently detect CHIKV. Peptide aptamer B2 (ITPQSSTTEAEL) and B3 (DTQGSNWI) were obtained through computer-aided design and selected as CHIKV-specific peptide aptamers based on their high binding affinity, strong hydrogen bonding, and RMSD of molecular docking. Then, a sandwich-Time-Resolved Fluoroimmunoassay (TRFIA) was successfully constructed for the detection of the interaction between peptide aptamers and viruses.

Results: When using B2 as the detection element, highly specific detection of CHIKV E2 was achieved with detection limits of 8.5 ng/ml in PBS solution. Variation coefficient between inter-assay showed the disturbances received from the detection of clinical fluid specimens (including serum and urine), were also within acceptable limits. The detection limits for 10-fold dilution serum and urine were 57.8 ng/mL and 147.3 ng/mL, respectively. The fluorescent signal intensity exhibited a good linear correlation with E2 protein concentration in the range of 0-1000 ng/mL, indicating the potential for quantitative detection of E2 protein.

Conclusions: These results demonstrate that the construction of peptide aptamers with high affinity and specificity provides an excellent method for rapid diagnostic element screening, and the developed peptide aptamer B2 contributed to better detection of CHIKV viral particles compared to traditional antibodies.

Keywords: Chikungunya Virus; Computer-aided design; Molecular docking; Peptide aptamer; TRFIA.

Fig. 1

Designing of T1-T5 peptide aptamers. Fab sequences of the broadly neutralizing human antibody EDE1 from the Protein Data Bank (PDB ID: 4UTA; resolution: 3.0 Å). (a-b): The complementarity-determining regions (CDRs) and framework regions (FRs) of the heavy and light chains are labelled separately, where CDR-H1 does not meet the requirement for the number of amino acid residues in the peptide aptamer and is removed

Fig. 2

Molecular docking-based screening of CHIKV-specific peptide aptamers (a). The binding energy of the 21 peptide aptamers to the three mosquito-borne virus envelope proteins was selected to be below − 6.5 kcal/mol with CHIKV and above − 6.5 kcal/mol with the other two mosquito-borne viruses; *** P < 0.001. (b) Root Mean Square Deviation (RMSD) of peptide aptamers docked in conformation with three mosquito-borne virus envelope proteins

Fig. 3

Visualization of peptide aptamer docking results with three mosquito-borne virus envelope proteins and analysis of hydrogen bonding interactions. Using a value of 2.5 Å as an endpoint, Pymol analyses all hydrogen bond (H-bond) lengths, the number of hydrogen bonds, and the sum of lengths resulting from the nine docked conformations (a). Examples of the lowest conformations of peptide aptamers B2 (left) and B3 (right) docked to CHIKV binding energy (b), red: domainI; yellow: domainII; blue: domainIII; Short yellow sticks: H-bond

Fig. 4

Analysis of direct peptide aptamer-virus interactions. (a.) Diagram of covalent coupling of peptide aptamers to europium nanospheres (Eu-Nps). (b-c.) Direct and sandwich detection of antigenic protein principles, with analysis of peptide aptamer-antigen interactions by fluorescent signals (excitation: 355 nm and emission:612 nm). (d.) 500 ng/mL antigen encapsulated using peptide aptamers and antibodies for direct detection of fluorescent signal intensity; *** P < 0.001. (e.) Detection of serially diluted (0-1000 ng/mL) viral recombinant proteins using peptide aptamers

Fig. 5

Performance of B2 and B3 as a detection element for CHIKV in fluid specimens. (a.) Detection of different concentrations of CHIKV in healthy serum and urine specimens (n = 12); *** P < 0.001. (b.) Resistance of peptide aptamers to clinical specimens as measured by the coefficient of variation. (c.) Dose-response curves, the limit of detection (LoD), and the limit of blank (LoB) parameters for urine and 10-fold diluted serum