A signal-enhanced competitive ELISA based on peptide screened by solid-liquid phase panning hybrid for sensitive detection of respiratory syncytial virus glycoprotein

Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in infants, young children and immunocompromised individuals, to which effective vaccines and specific antiviral therapies remain elusive. Although several RSV vaccines and long-acting monoclonal antibodies have been approved for specific populations, rapid and sensitive diagnostic tools are still critical for early clinical triage. RSV glycoprotein (GP) is a highly glycosylated adhesion protein in mediating viral adhesion and immune escape, making it an ideal target for antigen detection. Herein a combination of solid phase panning with liquid phase panning strategy was explored to isolate peptide GS24 (sequence: SRTCTPNFCYLG) with high affinity and specificity to RSV GP. Molecular dynamics simulation indicates that Arg 2 and Phe 8 residues on GS24 play a crucial role in binding, due to lower binding energies. Glu232 and Glu284 residues on the RSV GP interact with Arg 2 of the peptide, respectively, to form two pairs of hydrogen bonds (Glu232-Arg2, Glu284-Arg2). Additionally, there exists π-π stacking interaction between Pro234 of RSV GP and Phe8 of the peptide. Then the peptide GS24a was used as a recognition probe, and coordination reaction between L-ascorbic-acid 2-phosphate and Fe³⁺ as colorimetric signal, a signal-enhanced competitive binding of peptide/alkaline phosphatase-conjugated streptavidin complex to magnetic bead-immobilized antigen was constructed. The as-developed signal-enhanced competitive ELISA can detect target RSV GP within the linear range of 0.15-10 ng/mL and detection limit of 0.1 ng/mL. Finally, the proposed method was applied to detect human nasopharyngeal swab samples reproducibly, with the results in agreement well with commercial test strip, suggesting its good accuracy. This proposed method features simplicity, convenience and good sensitivity, which may offer a promising alternative for point-of-care RSV diagnosis. This work also provides guidance for developing similar immunoassay to detect other respiratory diseases.