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 language | English |
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Journal | Veterinary Pathology |
Volume | 34 |
Issue number | 3 |
Pages (from-to) | 253-261 |
ISSN | 0300-9858 |
Publication status | Published - 1997 |
Keywords
- VHS virus
- sensitivity
- immunohistochemistry
- primary antibody
- antigen unmasking
- rainbow trout
- fixation technique