Detection of rabies virus antigen or antibody using flow cytometry
Article first published online: 31 MAY 2006
Copyright © 2006 International Society for Analytical Cytology
Cytometry Part B: Clinical Cytometry
Volume 70B, Issue 5, pages 335–343, 15 September 2006
How to Cite
Vengatesan, D., Raj, G. D., Raja, A., Ramadass, P. and Gunaseelan, L. (2006), Detection of rabies virus antigen or antibody using flow cytometry. Cytometry, 70B: 335–343. doi: 10.1002/cyto.b.20104
- Issue published online: 15 SEP 2006
- Article first published online: 31 MAY 2006
- rabies virus;
- PV strain;
- flow cytometry;
- equivalent units;
- side scatter
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. © 2006 International Society for Analytical Cytology