From laboratory quantification to rapid on-site testing: Screening of high-affinity monoclonal antibody pairs...

Monoclonal antibodies against respiratory syncytial virus (RSV) F protein are key reagents for antigen-based diagnosis, yet antibody pairs compatible with multiple assay formats remain limited. In this study, BALB/c mice were immunized with recombinant postfusion RSV F protein, yielding six hybridoma cell lines secreting anti-RSV F monoclonal antibodies. The antibodies were characterized by indirect ELISA, Western blotting, and surface plasmon resonance, and the optimal sandwich pair was selected by cross-pairing ELISA. The mAb-14/mAb-26 pair showed high affinity and the best pairing performance and was used to establish double-antibody sandwich ELISA (DAS-ELISA), magnetic microparticle-based chemiluminescent immunoassay, and Au@Pt nanoparticle-based lateral flow immunoassay (Au@Pt-LFIA). The limits of detection were 0.25, 0.04, and 0.78 ng/mL, respectively. Au@Pt-LFIA achieved an approximately fourfold lower visual detection limit than conventional colloidal gold immunochromatography without additional signal-amplification steps. None of the three methods showed detectable cross-reactivity with the seven tested heterologous respiratory pathogen antigens. In preliminary clinical sample testing, the positive percent agreements of DAS-ELISA, chemiluminescent immunoassay, and Au@Pt-LFIA were 90.0%, 93.3%, and 83.3%, respectively, and the negative percent agreements were 100.0% for all methods. These results support mAb-14/mAb-26 as a versatile antibody pair for RSV antigen detection across complementary laboratory and on-site assay platforms.