RESUMO
In this research, affinity chromatography have been developed and standardized for production of Neuraminidase antigen of influenza virus for preparation of monospecific antiserum in rabbits. 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 polyethylenglycol 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- [paminophenyl] oxamic acid. Fractions that had high NA-activity and did not show HA activity were pooled and analyzed by neuraminidase inhibition and SDS-PAGE. For preparation of antisera, rabbits were immunized by purified NA and Freund's adjuvant at three weeks interval, and sera collected 7 days after boosting. In SDS-PAGE no viral protein band detected except for single band in the position of NA. NA activity of purified protein was 3.8 x 104 NA units. Enzymatic activity of Neuraminidase purified by this procedure decrease sharply above 48°C. The purified neuraminidase was producing a significant antibody response in agar gel precipitation. No reaction was observed with neuraminidase specific antiserum and H9-HA of the same virus. According to virtual purity and enzymatic activity of purified neuraminidase and highest avidity and specificity of antiserum, it was speculated that optimized protocol can be directly applied to produce antigen and antiserum from all subtypes of virus and can be easily used in commercial diagnostic tests