Antigen-capture enzyme immunoassay for detection of avian influenza virus in turkeys

S. Kodihalli From the Departments of Veterinary PathoBiology (Kodihalli, Sivanandan, Nagaraja, Halvorson) and Veterinary Diagnostic Investigation (Goyal), College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108.

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V. Sivanandan From the Departments of Veterinary PathoBiology (Kodihalli, Sivanandan, Nagaraja, Halvorson) and Veterinary Diagnostic Investigation (Goyal), College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108.

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K. V. Nagaraja From the Departments of Veterinary PathoBiology (Kodihalli, Sivanandan, Nagaraja, Halvorson) and Veterinary Diagnostic Investigation (Goyal), College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108.

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S. M. Goyal From the Departments of Veterinary PathoBiology (Kodihalli, Sivanandan, Nagaraja, Halvorson) and Veterinary Diagnostic Investigation (Goyal), College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108.

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D. A. Halvorson From the Departments of Veterinary PathoBiology (Kodihalli, Sivanandan, Nagaraja, Halvorson) and Veterinary Diagnostic Investigation (Goyal), College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108.

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Summary

A double-antibody sandwich elisa (das-elisa) was developed for detection of avian influenza virus (aiv) antigen. A monoclonal antibody to the viral nucleoprotein (np) was used to coat the elisa plates. A direct das-elisa and an indirect das-elisa were evaluated. In the direct das-elisa, monoclonal antibody to the aiv np conjugated with horseradish peroxidase was used. The direct das-elisa was evaluated for its sensitivity to detect purified np; this procedure detected as little as 0.1 ng. In the indirect das-elisa, rabbit np antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobin were used as primary and secondary antibodies, respectively. The indirect das-elisa was evaluated for its ability to detect the aiv antigen in tracheal and cloacal specimens from turkeys inoculated with aiv. Results of indirect das-elisa were compared with those of conventional virus isolation. Percentage agreement between indirect das-elisa and virus isolation in aiv-positive samples was found to be 76.1% and, in aiv-negative samples, it was found to be 82.1%. These results in dicate that the das-elisa might be a viable alternative to virus isolation because of its rapidity, compared with virus isolation.

Summary

A double-antibody sandwich elisa (das-elisa) was developed for detection of avian influenza virus (aiv) antigen. A monoclonal antibody to the viral nucleoprotein (np) was used to coat the elisa plates. A direct das-elisa and an indirect das-elisa were evaluated. In the direct das-elisa, monoclonal antibody to the aiv np conjugated with horseradish peroxidase was used. The direct das-elisa was evaluated for its sensitivity to detect purified np; this procedure detected as little as 0.1 ng. In the indirect das-elisa, rabbit np antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobin were used as primary and secondary antibodies, respectively. The indirect das-elisa was evaluated for its ability to detect the aiv antigen in tracheal and cloacal specimens from turkeys inoculated with aiv. Results of indirect das-elisa were compared with those of conventional virus isolation. Percentage agreement between indirect das-elisa and virus isolation in aiv-positive samples was found to be 76.1% and, in aiv-negative samples, it was found to be 82.1%. These results in dicate that the das-elisa might be a viable alternative to virus isolation because of its rapidity, compared with virus isolation.

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