Preparation of Neuraminidase-Specific Antiserum from the H9N2 Subtype of Avian Influenza Virus


Abstract: Avian influenza virus stocks (A/chicken/Iran/259/1998/(H9N2)) were propagated in the allantoic cavities of 10-day-old embryonated chicken eggs. The harvested suspension was concentrated by polyethylene glycol 6000. Concentrated samples were layered onto sucrose gradient (30%-60%). Both hemagglutinin and neuraminidase were solubilized from purified viruses with Triton X-100 across 30% sucrose gradient. NA was isolated from HA and other viral proteins by affinity chromatography on N-(p-aminophenyl)oxamic acid. Fractions that had high NA activity and did not show HA activity were pooled and analyzed by neuraminidase inhibition and SDS-PAGE. No viral protein bands were detected, except for a single band in the position of NA. NA activity of purified protein was 3.8 x 10^4 NA units. Enzymatic activity of neuraminidase purified by this procedure decreased sharply above 48 °C. For preparation of antisera, rabbits were immunized with purified NA and Freund's adjuvant at 3-week intervals, and sera were collected 7 days after boosting. The purified neuraminidase produced a significant antibody response in agar gel precipitation. No reaction was observed with neuraminidase-specific antiserum or H9-HA of the same virus.

Keywords: Avian influenza, neuraminidase, antiserum

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