Development of time-resolved immunofluorometric assay of vascular permeability factor

We describe a two-site time-resolved immunofluorometric assay for guinea pig vascular permeability factor (VPF) for quantifying VPF in different biological fluids. Antibody against the carboxy terminus (C-IgG) is immobilized on microtiter wells, and antibody against the amino terminus (N-IgG) is labeled with Eu(3+)-chelate. Line 10 tumor culture medium, known to be rich in VPF, is assayed in a two-step incubation. Bound Eu3+ is then quantified by dissociation into a fluorescent enhancement solution, with measurement of the time-resolved fluorescence. The analytical sensitivity is 0.35 VPF unit, and the intra-assay CV is about 20%. The assay is specific for VPF, because pre-treatment with the appropriate C- or N-peptide, or pre-extraction of VPF, greatly decreases fluorescence. The VPF immunoassay is highly correlated (r2 = 0.94) with the Miles permeability assay, the classical bioassay of VPF. In addition, the immunofluorometric assay is about 30-fold more sensitive than the Miles assay.