China CDC weekly · 2026
Validation of the Rapid Fluorescent Focus Inhibition Test for Rabies Virus Neutralizing Antibodies - China, 2025.
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Fang Zixin, Tao Xiaoyan, Liu Shuqing, Liu Qian, Zhang Minghui, Yang Nuo, Hu Zeheng, Jin Tom, Tsao Eric, Yu Pengcheng, Zhu Wuyang
Аннотация
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) <30%. Dilutability was confirmed, with 95% of positive samples showing geometric mean concentration (GMC) differences within ±30% compared to undiluted controls. The standard and detection values were described by y=1.0091x - 0.1128 (R2=0.9948); 95.56% of the samples showed 70%-130% recovery. Specificity was verified using homologous and heterologous antigen competition and a matrix with no significant cross-reactivity. The assay was robust to variations in cells, reagents, and time, with titer differences within ±30%. Stability of samples and reagents under freeze-thaw and different short-term storage conditions was confirmed.
CONCLUSION: The assay was successfully validated for quantifying RVNA content in serum samples.
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) <30%. Dilutability was confirmed, with 95% of positive samples showing geometric mean concentration (GMC) differences within ±30% compared to undiluted controls. The standard and detection values were described by y=1.0091x - 0.1128 (R2=0.9948); 95.56% of the samples showed 70%-130% recovery. Specificity was verified using homologous and heterologous antigen competition and a matrix with no significant cross-reactivity. The assay was robust to variations in cells, reagents, and time, with titer differences within ±30%. Stability of samples and reagents under freeze-thaw and different short-term storage conditions was confirmed.
CONCLUSION: The assay was successfully validated for quantifying RVNA content in serum samples.