Heat induced epitope retrieval for rabies virus detection by direct fluorescent antibody test in formalin-fixed dog brain tissues

There is a great need for a chemical method of tissue preservation that would allow storage for extended periods without freezing. This study aimed at retrieving and detecting rabies virus antigen by Direct Fluorescent Antibody Test (DFAT) in formalin-fixed dog brain tissues. Forty fresh dog brain specimens were collected as paired samples from rabies suspected cases that were received for postmortem detection of rabies in the Central Diagnostic Laboratory, National Veterinary Research Institute, Vom. One portion of each paired sample was prepared for fresh fluorescent antibody testing and the other portion was prepared for epitope retrieval and florescent antibody testing following fixation in 10% neutral buffered formalin. DFAT on formalin-fixed tissue exhibited a sensitivity of 100% in comparison to DFAT on fresh-tissue. No false positive result was obtained in formalin-fixed DFAT procedure, demonstrating 100% specificity. There was no apparent difference in the intensity of fluorescence in DFAT on fresh sample and formalin-fixed DFAT following heat induced epitope retrieval (concordance = 98%; 95% C.I. 0.9660 to 0.9903). The strength of agreement between DFAT on formalin-fixed and DFAT on fresh tissue was very good (Cohen’s kappa coefficient value= 1.000; 95% C.I. 1.000-1.000). This study provides new information on the retrieval of rabies antigen by heat induced epitope retrieval for DFAT on formalinized tissues. The heat induced epitope retrieval can be routinely used to retrieve rabies virus antigen for DFAT in cases where only formalin-fixed tissues are available or when preservation by freezing is difficult.