Attempt at use of the fluorometric neuraminidase assay system for the enzyme antibody inhibition test.

The fluorometric neuraminidase assay method using 4-methylumbelliferyl-N-Ac-aD-neuraminide as a substrate was investigated for the purpose of ascertaining its validity as a procedure for influenza viral neuraminidase antibody inhibition test. The enzyme antibody inhibition titer obtained by the fluorometric assay system was far weaker than that obtained by the standard colorimetric neuraminidase assay method using fetuin as a substrate. Although solubilization of viral neuraminidase spikes with Triton X-100 increased the enzyme inhibition of antiserum to some extent, there is little possibility of using the fluorometric assay system for the purpose. Previously we reported the fluorometric assay method for neuraminidase activity of influenza viruses using 4-methylumbelliferyl (4-MU)-NAc-a-D-neuraminide as a substrate5>. This method is more sensitive and simple, and less timeconsuming in performance than the colorimetric neuraminidase assay method2> using fetuin as a substrate. The other assay method1> using a synthetic substrate of low molecular weight such as sialyl lactose (MW =640) has been known to have similar advantages; however, the method can not be used for the viral neuraminidase activity inhibition test by the specific antibody against influenza virus1•3>. At present, the colorimetric method is exclusively used as the standard one for the neuram.inidase antibody inhibition test2>. Since the viral :heuraminidase activity assay is generally performed as the preceding procedure for ne.uraminidase inhibition test, the fluorometric neuraminidase assay method was investigated so as to inquire whether it is an effective procedure for the enzyme antibody inhibition test. Influenza virus strains employed were A/ Aichi/2/68 (H3N2), A/USSR/92/77 (RINI) and B/Kanagawa/3/76 (B). Viruses were propagated in MDCK cells according to the method described by Tobita et al8>. Each culture supernatant was centrifuged at 100, 000 x g for 60 min and the precipitate was suspended in PBS (-) at 1 :20 volume of the starting mateial. These virus preparations were used as enzyme sources or immune antigens. Antiinfluenza virus serum was prepared by three serial injections of each virus preparation in to the marginal ear vein of rabbit at one-weekinterval. The whole blood was collected 10 days after the final injection. The neuraminidase antibody inhibition titer was determined according to the method de-