Immunohistochemical detection of VHS virus in paraffin-embedded specimens of rainbow trout (Oncorhynchus mykiss); The influence of primary antibody, fixative, and antigen unmasking on method sensitivity

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    Abstract

    The influence of the primary antibody, the fixative, and the antigen unmasking technique on the method sensitivity of immunohistochemistry as a method for the identification of viral hemorrhagic septicemia (VHS) virus in paraffin-embedded specimens of naturally infected rainbow trout (Oncorhynchus mykiss) was examined. Fish (200-300 g) were collected during an outbreak of VHS. Parallel specimens from liver, spleen, kidney, and brain were fixed by immersion in 10% phosphate-buffered formalin, periodate-lysine-paraformaldehyde (PLP), Bouin's fluid, or absolute ethanol. Virus cultivation was also performed on parallel specimens, and the virus titer (TCID50/ml) was determined. Purified nucleocapsid protein (N-protein) of the virus was incorporated in an artificial antigen substrate (polymerized bovine serum albumin), fixed as described above, and embedded in paraffin wax. Microwave unmasking was performed an formalin-, PLP-, and Bouin's fluid-fixed specimens. The presence of virus peptides in situ or N-protein in the artificial antigen substrates was Visualized using an immunohistochemical method based on alkaline phosphatase or peroxidase and one polyclonal and five monoclonal polypeptide-specific antibodies. VHS virus was identified in situ in specimens with high virus titers (10(7-8) TCID50/ml) regardless of the fixative and without the need of an unmasking procedure. A pronounced masking effect was observed for the cross-linking formalin and PLP fixatives. Regardless of the primary antibodies used, there was a significantly higher epidemiologic sensitivity (the proportion of virus positive samples that tested positive by immunohistochemistry) using ethanol and Bouin's fluid compared with formalin and PLP (P
    Original languageEnglish
    JournalVeterinary Pathology
    Volume34
    Issue number3
    Pages (from-to)253-261
    ISSN0300-9858
    Publication statusPublished - 1997

    Keywords

    • VHS virus
    • sensitivity
    • immunohistochemistry
    • primary antibody
    • antigen unmasking
    • rainbow trout
    • fixation technique

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