Establishment and Application of a Direct Immunofluorescence Assay for Determining Rabies Virus Titer

Abstract

Objective: To establish and validate a detection method for the titration of extracellular rabies virus as an alternative to the intracerebral mouse titration method.

Methods: The direct immunofluorescence method was developed using Vero cells and BHK-21 cells to determine the virus titer of the rabies virus (CTN strain) in vaccine harvest fluid. The precision, relative accuracy, and linear range of the method were validated.

Results: Both cell types exhibited distinct fluorescent infection foci within 24 hours, with the number of infections decreasing as the virus was diluted. This observation indicates the method's applicability, with Vero cells demonstrating greater sensitivity than BHK-21 cells. Method validation revealed that the relative standard deviations (RSD) for repeatability and intermediate precision were both less than 15%. Additionally, the relative bias (RB) for relative accuracy was below 10%. The virus dilution factor, ranging from 5⁰ to 5^-9, displayed a strong linear relationship with the virus titer, further confirming the method's effectiveness.

Conclusion: The direct immunofluorescence method established using Vero cells and BHK-21 cells demonstrates significant potential for quantifying the virus in the harvest fluid of human rabies vaccines derived from Vero cells.