Multiple Fluorescent Labeling of Silica Nanoparticles with Lanthanide Chelates for Highly Sensitive Time-Resolved Immunofluorometric Assays

Abstract

Time-resolved immunofluorometric assays (TrIFA) using lanthanide-labeled nanoparticles have greatly increased the sensitivity of immunoassays. Current labeling strategies, however, use either physical doping of lanthanide chelates into preformed nanoparticles or covalent linking of lanthanide chelates to precursors used for making nanoparticles; both these strategies have drawbacks. Luminescent Eu(III) and Tb(III) chelates were covalently coated on the surface of preformed silica nanoparticles to which detection antibodies or bridging proteins for antibody binding were conjugated. We used the resulting conjugates in TrIFA for detection of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), both individually and simultaneously. We compared the results of the newly established method with results of an ELISA for serum samples. Positive samples identified by TrIFA but not by ELISA were confirmed by additional assays, including real-time PCR detection of viral DNA. The prepared nanoparticle conjugates were homogeneous in size, at approximately 55 (5) nm in diameter [mean (SD)], were stable for long-time storage (>2 years), and contained more chelates [6.86 x 10(5) for Eu(III), 4.73 x 10(4) for Tb(III)] per nanoparticle than particles made as previously reported. The TrIFA established for HBsAg had a comparable or lower detection limit (0.0092 microg/L) than existing nanoparticle-based TrIFA or ELISA. The TrIFA for HBeAg had a much lower detection limit [10.0 National Centre Unit (NCU)/L] than ELISA and detected HBeAg in 5 samples missed by the ELISA method. Simultaneous TrIFA for both HBsAg and HBeAg was achieved with detection limits (0.033 microg/L for HBsAg and 27.0 NCU/L for HBeAg) close to those of the individual assays. Covalent surface labeling of silica nanoparticles with lanthanide chelates provides good fluorescent labels that can be used in TrIFA for highly sensitive and robust detection of clinical targets.