Exposed nucleoprotein inside rabies virus particle as an ideal target for real-time quantitative evaluation of rabies virus particle integrity in vaccine quality control.

Currently, the integrity of rabies virus particle in quality control can only be assessed through electron microscopy. However, its time-consuming nature, operational complexity, limited accuracy and lack of quantification capability no longer meet the needs of modern vaccine production. Based on this, we developed a time-resolved fluoroimmunoassay (TRFIA) to enable real-time quantitative analysis of rabies virus particle integrity in human rabies vaccine, by detecting exposed nucleoprotein. Monoclonal antibodies against rabies glycoprotein and nucleoprotein were prepared using the classical hybridoma technology with the aim of constructing a novel detection approach for assessing particle integrity. A monoclonal antibody against rabies glycoprotein was immobilized on microplate wells to capture rabies virus particles, while a labeled antibody against the nucleoprotein served as the signal tracer. Multiple types of vaccine samples were analyzed to evaluate the effectiveness and accuracy of the developed TRFIA, combined with various virus particle destruction methods to validate its capability to assess particle integrity. The validation results were consistent with those obtained from electron microscopy. Therefore, this novel TRFIA offers a promising solution to address existing gaps in current analytical methods, enabling straightforward real-time, and quantitative evaluation of rabies virus particle integrity in the laboratory.