Detection of rabies virus antigen or antibody using flow cytometry

Abstract

Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. Rabies diagnosis must be rapid and conclusive. Detection and quantification of antirabies antibodies is used for assessment of the effectiveness of rabies vaccines. Hence, computer-automated detection of fluorescence using flow cytometry was attempted to reduce the work time required to undertake the conventional rapid fluorescent focus inhibition test (RFFIT). Pasteur virus (PV)-infected mouse neuroblastoma (MNA) cells were stained with rabies virus antinucleocapsid antibody, fluorescein isothiocyanate (FITC) conjugate, and the percentage of infected cells at 24, 48, and 72 h postinfection (PI) was determined using flow cytometry. Serum samples containing known antibody titres estimated by RFFIT in terms of IU/ml were used to neutralize 50 FFD50 dose of PV. The percentage of MNA cells infected by the un-neutralized virus was estimated by flow cytometry. Using the value of the percentage of cells infected in the presence of known negative serum as 100%, the infection inhibition caused by antibodies at each dilution of positive reference serum was calculated and a regression equation generated for the prediction of rabies virus antibody titres in test sera samples as equivalent units per ml (EU/ml). There was a significant increase in the percentage of infected cells between 24 and 48 h PI from 26.45 to 75.28%. The percentage of cells having high side scatter was also highest at 72 h PI (11.11%). Antibody titres predicted by flow cytometry and those estimated by RFFIT as IU/ml showed a correlation coefficient of 0.74. Thus, flow cytometry could be used to detect rabies virus antigen in infected cells and to predict serum antibody titres from a single dilution of serum tested with the potential advantages of automation, rapidity, and lack of subjectivity. It has the potential to replace the time-tested RFFIT in rabies serology in the years to come.