Rapid fluorescent focus inhibition test optimization and validation: Improved detection of neutralizing antibodies to rabies virus

Abstract The rabies rapid fluorescent focus inhibition test (RFFIT) is the most widely used cell-based assay for detecting and quantitating rabies virus neutralizing antibodies (RVNA) in human serum. However, it is a complex, labor intensive, and somewhat subjective manual assay, the performance of which may be affected by a number of factors including the quality of cells and virus, variability of assay reagents and the skill and expertise of analysts. This study sought to identify and evaluate conditions that may impact RFFIT performance and RVNA detection by evaluating assay parameters including: different serial dilution scheme of serum samples in a 96-well microplate using semi-automated pipetting systems, the range of dose of challenge virus standard (CVS-11) strain of rabies virus, the effect of complement (C′), the effect of cell seeding density and passage number, the effect of diethylaminoethyl (DEAE) dextran concentration on virus infectivity, and the assay incubation period prior to immunostaining. In addition the evaluation of counting fluorescent foci using a microscope versus using scanned images from a cell imaging reader was performed in an effort to ease the reading of slides and have permanent records of the raw data. The results from optimization of each parameter are presented along with subsequent assay validation in accordance with the International Conference on Harmonization (ICH) guidelines. The improved and optimized RFFIT accuracy, linearity and sensitivity was demonstrated by testing World Health Organization (WHO)-1 and WHO-2 Standard Rabies Immune Globulins (SRIGs) and complete assay development and validation was performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines.