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

We developed a solid phase immunoassay that measured mucosal and systemic a ntibody responses from mice inoculated with either a staphylococcal enterot oxin B vaccine (SEBv) or noninfectious virus-like particles (VLP) of lentiv iral origin. The assay used time-resolved fluorescence (TRF) with affinity- purified goat anti-mouse IgA and IgG conjugated to samarium, and europium c helates, respectively. By employing these fluorogenic conjugates with diffe rent spectral emissions, IgA and IgG specific for SEB or VLP were readily d etected in serum and saliva from mice inoculated intranasally. The TRF assa y detected anti gen-specific IgA in saliva 10 min after the addition of enh ancement solution, while a conventional alkaline phosphatase-based assay fo r salivary IgA required 18 h after substrate addition. The TRF assay also p rovided 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. (C) 2001 Elsevier Science B.V. A ll rights reserved.