Rapid neutralization test based on ELISPOT for detecting antibodies against rhinovirus 1B

Rhinovirus (RV) infections range from mild colds to severe respiratory diseases, posing substantial economic and public health burdens. Detecting neutralizing antibodies (nAbs) efficiently against rhinoviruses is crucial for advancing vaccine development and antiviral research. Rhinovirus 1B (RV-1B), predominantly utilized to model RV-induced disease, holds significant potential for therapeutic development. This study establishes a neutralization testing system utilizing the enzyme-linked immunospot (ELISPOT) assay for RV-1B. The monoclonal antibody (mAb) 4F2, exhibiting high affinity for RV-1B, was selected to detect infected cells specifically and effectively. The optimal conditions for generating distinct spots in RV-1B infection using the ELISPOT assay were established as the application of 0.5 μg/mL of 4F2, with detection performed 18 h post-infection. The neutralization ELISPOT (Nt-ELISPOT) demonstrated high consistency with the traditional cytopathic effect-based neutralization assay (Nt-CPE) when detecting neutralization titers. Using the Nt-ELISPOT method, three nAbs against RV-1B were identified with half maximal inhibitory concentrations (IC₅₀) values of 75 ng/mL, 27 ng/mL, and 8 ng/mL. These findings indicate that the Nt-ELISPOT assay provides a rapid and high-throughput method for screening RV-1B nAbs and may support antiviral research and vaccine development.