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Updated: Oct 13, 2025
Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues
Published on: October 26, 2021
Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues
Michael Niezgoda1, Panayampalli Subbian Satheshkumar2
1Poxvirus and Rabies Branch, Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention; man6@cdc.gov.
Insights
Formalin-fixed tissues can be used to detect rabies virus antigen using conventional immunohistochemistry (IHC). This method allows for histological analysis and safer sample handling compared to fresh tissue tests.
Area of Science:
- Veterinary Pathology
- Virology
- Diagnostic Pathology
Background:
- Rabies diagnosis relies on detecting viral ribonucleoprotein (RNP) antigen in tissues.
- Direct fluorescent antibody (DFA) and direct rapid immunohistochemical test (DRIT) are common but require fresh/frozen tissues.
- Formalin fixation is suboptimal for DFA/DRIT but enables other diagnostic methods.
Purpose of the Study:
- To evaluate the utility of conventional immunohistochemistry (IHC) for rabies antigen detection in formalin-fixed, paraffin-embedded tissues.
- To highlight the advantages of formalin fixation for rabies diagnostics.
Main Methods:
- Rabies antigen detection in formalin-fixed tissues using conventional immunohistochemistry (IHC).
- Tissue processing involved paraffin embedding, sectioning, deparaffinization, antigen retrieval, and antibody staining.
- Visualization of antigen using horseradish peroxidase/amino ethyl carbazole and hematoxylin counterstain.
Main Results:
- Conventional IHC successfully detects rabies virus antigen in formalin-fixed tissues.
- Formalin fixation allows for concurrent histological examination of tissue changes.
- Formalin fixation offers improved sample storage, transport, retrospective case analysis, and biosafety.
Conclusions:
- Conventional IHC is a viable method for rabies diagnosis in formalin-fixed tissues.
- Formalin fixation provides significant advantages for rabies sample management and diagnostic flexibility.
Abstract:
One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.
