Authors
Qianli Li, Jinsong Zhang, Kehang Yang, Jiayue Su, Lirun He, Fengxia He, Jian Zhou, Shuai Yuan
Published in
Diagnostic microbiology and infectious disease. Volume 117. Issue 1. Pages 117652. Sep 10, 2026. Epub Sep 10, 2026.
Abstract
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.
PMID:
42748649
Bibliographic data and abstract were imported from PubMed on 17 Sep 2026.
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