Detection of antibody to the PreS2 sequence of the hepatitis B virus envelope protein using an immuno-ligand assay with a silicon sensor detection system.
Sensitive immunoassays are essential for establishing the efficacy of recombinant vaccines to hepatitis B virus (HBV). These experimental vaccines include the PreS2 and S domains of the HBV envelope protein. To facilitate measurement of antibody against HBV PreS2, we employed the immuno-ligand assay with silicon sensor-based detection. Labeling of immune reagents with the haptens biotin and fluorescein allows adaptation to the immunofiltration light addressable potentiometric sensor (LAPS) system. A biotinylated monoclonal anti-PreS2 antibody and anti-PreS2 in clinical serum samples competitively bind in liquid phase to a fluorescein labeled PreS2 + S antigen. Streptavidin mediates the immobilization on biotinylated nitrocellulose membranes. Fluorescein mediates binding of an anti-fluorescein urease conjugate to the immune complex. Urease serves as the signal-generating component which subsequently is measured in the LAPS reader. In comparison to a competitive RIA, the immuno-ligand assay demonstrated a four-fold improved sensitivity using a smaller sample volume. The higher sensitivity resulted in earlier detection of seroconversion during a clinical vaccine study.
Hurni
W M
WM
Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Miller
W J
WJ
Zuk
R F
RF
Kung
V T
VT
D000911:Antibodies, Monoclonal / Q000276:immunology; D001710:Biotin / Q000737:chemistry; D004306:Dose-Response Relationship, Immunologic; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D006510:Hepatitis B Antibodies / Q000032:analysis*; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008024:Ligands; D011498:Protein Precursors / Q000276:immunology*; D011863:Radioimmunoassay; D012825:Silicon; D014614:Vaccines, Synthetic / Q000276:immunology
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D008024:Ligands; D011498:Protein Precursors; D014614:Vaccines, Synthetic; C050807:presurface protein 2, hepatitis B surface antigen; D001710:Biotin; D019793:Fluorescein; D012825:Silicon