Development of a time-resolved immunofluorometric assay for quantitation of mucosal and systemic antibody responses.

ABSTRACT

We developed a solid phase immunoassay that measured mucosal and systemic antibody responses from mice inoculated with either a staphylococcal enterotoxin B vaccine (SEBv) or noninfectious virus-like particles (VLP) of lentiviral origin. The assay used time-resolved fluorescence (TRF) with affinity-purified goat anti-mouse IgA and IgG conjugated to samarium and europium chelates, respectively. By employing these fluorogenic conjugates with different spectral emissions, IgA and IgG specific for SEB or VLP were readily detected in serum and saliva from mice inoculated intranasally. The TRF assay detected antigen-specific IgA in saliva 10 min after the addition of enhancement solution, while a conventional alkaline phosphatase-based assay for salivary IgA required 18 h after substrate addition. The TRF assay also provided a significantly higher signal-to-noise ratio and exhibited greater sensitivity. TRF assays detected both IgA and IgG in the same well, thereby reducing sample and reagent requirements.