Validation of two direct immunofluorescence techniques for measuring rabies virus titers
Objective To validate two direct immunofluorescence techniques for measuring rabies virus(RABV)titers:the fluorescence focus unit(FFU)method and the 50％fluorescence focus infection dose(FFD50)method.Methods We used FFU and FFD50 to assess titers from different RABV strains,calculated coefficients of variation(C V)of FFU and FFD50 values to determine reliability(precision and stability),and compared the values with 50％lethal dose(LD50)values using the standard mouse brain titration method to validate accuracy and range of linearity.Results Repeatability and intermediate precision validation showed that CV values of FFU and FFD50 titers of the CVS-11 RABV strain were both less than 30％.Stability validation showed that after changing cell-virus incubation time,acetone fixation time,and staining time,CV values of FFU and FFD50 titers of the CVS-11 strain were all less than 30％.Accuracy validation showed that FFU and FFD50 values of CVS-11,SC16,GD1,and QH2 RABV strains were close to their LD50 values.Both FFU and FFD50 titers had linear relationships with the virus dilution ratio method ranging from 5-1 to 5-7,with linear regression equations of y=-1.350x+7.391(R2=0.991)and y=-1.453x+8.285(R2=0.980).Conclusions The FFU and FFD50 method have good reliability and accuracy for determining RABV titers,providing supportive evidence to use these method to substitute for the mouse brain titration method.
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