Validation of the Rapid Fluorescent Focus Inhibition Test for Rabies Virus Neutralizing Antibodies — China, 2025

Introduction The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay and the gold standard for quantifying rabies virus neutralizing antibodies (RVNA) in serums. It is used to assess the biological efficacy of rabies vaccines and evaluate protective immunity in both humans and animals. Despite its broad application, RFFIT requires thorough validation to ensure reliability. Methods RFFIT was validated in this study using the third World Health Organization international standard for anti-rabies immunoglobulin (WHO-3 SRIG) and negative human sera. The validation followed the guidelines outlined by the Food and Drug Administration Guidance for Industry and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH)Q2 (R1) guidelines and included the assessment of intra-assay and intermediate precision, dilutability, linearity, range, accuracy, specificity, robustness, and stability. Results The RFFIT method demonstrated good precision, with intra-assay and intermediate-precision geometric coefficient of variation (GCV)