Determining the Specific Activity of Anti-Rabies Sera and Immunoglobulin Using Atomic Force Microscopy of Cell Cultures

BACKGROUND: Mouse neutralization test is widely used to determine the level of anti-rabies antibodies, but it is labor-intensive and time consuming. Alternative methods for determining the neutralizing activity of anti-rabies sera and immunoglobulin in cell cultures are also known. Methods such as FAVN and RFFIT involve the use of fluorescent diagnostics. Determination of Cytopathic Effect (CPE) is often complicated due to features of rabies virus replication in cells. Atomic Force Microscopy (AFM) is able to detect the interaction of the virus with the cell at an early stage. Therefore, in this study, a method has been developed for determining the specific activity of anti-rabies sera and immunoglobulin using AFM of cell cultures. METHODS: The method is based on the preliminary interaction of rabies virus with samples of rabies sera or immunoglobulin drug, adding the specified reaction mixture to cell culture (Vero or BHK-21), and then measuring the surface roughness of the cells using AFM. AFM was carried out in the intermittent contact mode by the mismatch method in the semi-contact mode. The results were compared with the values obtained in the mouse neutralization test. The consistency of the results obtained by both methods was evaluated by Bland-Altman method. RESULTS: The increment in the surface roughness of the cells is a consequence of the damaging effect of the virus, which is weakened as a result of its neutralization by rabies antibodies. A dilution allowing 50% suppression of the increase in the surface roughness of cells was selected as the titer of rabies sera or immunoglobulin. In this case, the recommended range for determining the antibody titer is from 1:100 to 1:3000. CONCLUSION: For the first time, a new methodological approach in virology and pharmaceutical research is presented in this study. The use of the proposed methodological technique will reduce the time from 21 to 2 days to obtain results in comparison with the mouse neutralization test; also, fewer laboratory animals are required in this approach which is in agreement with 3 R Principle.