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

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.

Optimization and validation of a plaque reduction neutralization test for the detection of neutralizing antibodies to four serotypes of dengue virus used in support of dengue vaccine development.

A dengue plaque reduction neutralization test (PRNT) to measure dengue serotype-specific neutralizing antibodies for all four virus serotypes was developed, optimized, and validated in accordance with guidelines for validation of bioanalytical test methods using human serum samples from dengue-infected persons and persons receiving a dengue vaccine candidate. Production and characterization of dengue challenge viruses used in the assay was standardized. Once virus stocks were characterized, the dengue PRNT(50) for each of the four serotypes was optimized according to a factorial design of experiments approach for critical test parameters, including days of cell seeding before testing, percentage of overlay carboxymethylcellulose medium, and days of incubation post-infection to generate a robust assay.