Comparison of a Micro-Neutralization Test with the Rapid Fluorescent Focus Inhibition Test for Measuring Rabies Virus Neutralizing Antibodies

The rapid fluorescent focus inhibition test (RFFIT) is routinely used in the United States to measure rabies virus neutralizing antibodies (rVNA). RFFIT has a long history of reproducible and reliable results. The test has been modified over the years to use smaller volumes of reagents and samples, but requires a 50 μL minimum volume of test serum. To conduct pathogenesis studies, small laboratory animals such as mice are regularly tested for rVNA, but the minimum volume for a standard RFFIT may be impossible to obtain, particularly in scenarios of repeated sampling. To address this problem, a micro-neutralization test was developed previously. In the current study, the micro-neutralization test was compared to the RFFIT using 129 mouse serum samples from rabies vaccine studies. Using a cut-off value of 0.1 IU/mL, the sensitivity, specificity, and concordance of the micro-neutralization test were 100%, 97.5%, and 98%, respectively. The geometric mean titer of all samples above the cut-off was 2.0 IU/mL using RFFIT and 3.4 IU/mL using the micro-neutralization test, indicating that titers determined using the micro-neutralization test are not equivalent to RFFIT titers. Based on four rVNA-positive hamster serum samples, the intra-assay coefficient of variability was 24% and inter-assay coefficient of variability was 30.4%. These results support continued use of the micro-neutralization test to determine rabies virus neutralizing antibody titers for low-volume serum samples.