A rapid and quantitative time-resolved fluorescence immunoassay for chikungunya virus detection based on...

Chikungunya virus (CHIKV), an arthropod-borne alphavirus, poses a severe threat to global public health due to its explosive epidemic potential and severe clinical consequences. The E2 glycoprotein is a key target for diagnostic reagent development, but recombinant monomeric E2 exhibits weak immunogenicity, hindering the preparation of high-affinity antibodies. Time-resolved fluorescence immunoassay (TRFIA) is a highly sensitive detection platform suitable for detecting low-abundance antigens. This study aimed to construct a highly immunogenic CHIKV E2 nanoantigen, generate E2-specific monoclonal antibodies, and establish a sensitive TRFIA for CHIKV detection.

The CHIKV E2 protein and Aquifex aeolicus lumazine synthase (AaLS) were fused with SpyTag and SpyCatcher, respectively, and the AaLS-E2 nanoantigen was prepared by covalent conjugation via the SpyTag-SpyCatcher system. C57BL/6 mice were immunized with AaLS-E2, E2 monomer, or PBS, and hybridoma technology was used to screen monoclonal antibodies against E2. The affinity of monoclonal antibodies was determined by bio-layer interferometry (BLI). A double-antibody sandwich TRFIA was established using the selected monoclonal antibody pair, and its performance was validated with clinical serum samples, with qPCR as the reference method.

Soluble AaLS-SpyCatcher and E2-SpyTag proteins were successfully expressed and purified, and formation of the AaLS-E2 nanoantigen conjugate was confirmed. Immunization with AaLS-E2 elicited significantly higher E2-specific IgG titers (OD450 = 2.42) than the E2 monomer group (OD450 = 1.48, P < 0.05). Two stable monoclonal antibodies (CK8 and CK19) were obtained, with equilibrium dissociation constants (KD) of 6.47 nM and 118 nM, respectively. The optimized TRFIA showed a good linear relationship (R²=0.9813) with a limit of detection (LOD) of 145 ng/mL. In clinical validation with 98 serum samples, the assay achieved a sensitivity of 91.30%, specificity of 98.67%, and overall concordance of 96.94% with qPCR (Kappa = 0.91).

The AaLS nanocage significantly enhances the immunogenicity of CHIKV E2 antigen, enabling the preparation of high-affinity monoclonal antibodies. The established TRFIA exhibits high sensitivity, specificity, and good clinical concordance with qPCR, supporting its potential use as a rapid screening tool for acute CHIKV infection. This strategy also offers a framework for the development of diagnostics for other emerging arboviral diseases.