Measurement of anti-influenza neuraminidase antibody using a peroxidase-linked lectin and microtitre plates coated with natural substrates.
Neuraminidase-induced removal of sialic acid from natural substrates (desialylation) unmasks saccharides that are specifically recognized by the lectin peanut agglutinin (PNA). We demonstrate that, when a neuraminidase substrate is coated on to the wells of a microplate, it is possible to quantitate the binding of PNA to the desialylated substrate using a peroxidase-conjugated PNA (Po-PNA). The amount of bound PNA correlated directly with the amount of sialic acid removed from the substrate and therefore with the neuraminidase activity. By reacting with specific epitopes that are located near to the enzyme active site, anti-neuraminidase antibodies are capable of inhibiting the virus-induced desialylation of the substrate. Such antibodies therefore reduce the binding of Po-PNA. The advantage of this assay is that since different natural substrates for neuraminidase (erythrocytes, fetuin or gangliosides) can be used to coat the microplates, the capacity of anti-neuraminidase antibody to inhibit the neuraminidase activity towards different types of sialoglycoconjugates can be evaluated. Anti-hemagglutinin or non-specific anti-neuraminidase antibody have no interfering reactivity.
Immunopathologie Pulmonaire, INSERM U 139, Hôpital H. Mondor, Créteil, France.
C R
CR
Lambré
H
H
Terzidis
A
A
Greffard
R G
RG
Webster
D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis*; D000918:Antibody Specificity; D001665:Binding Sites; D005732:Gangliosides; D006801:Humans; D007158:Immunologic Techniques; D009980:Influenza A virus / Q000201:enzymology / Q000276:immunology; D009981:Influenza B virus / Q000201:enzymology / Q000276:immunology; D007252:Influenza Vaccines / Q000008:administration & dosage / Q000276:immunology; D037102:Lectins; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000276:immunology*; D009975:Orthomyxoviridae / Q000201:enzymology / Q000276:immunology*; D019887:Peanut Agglutinin; D009195:Peroxidase; D012794:Sialic Acids; D013858:Thiobarbiturates; D000509:alpha-Fetoproteins
D000907:Antibodies, Bacterial; D005732:Gangliosides; D007252:Influenza Vaccines; D037102:Lectins; D019887:Peanut Agglutinin; D012794:Sialic Acids; D013858:Thiobarbiturates; D000509:alpha-Fetoproteins; D009195:Peroxidase; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid; C029684:thiobarbituric acid