A rapid, simple immunofluorometric assay: Development and characterization

A novel two‐site immunofluorometric assay, which includes only one incubation step and one separation step, is described. The assay is based on using small‐sized beads (0.5 μm diameter) as a soild support and measuring the unbound fraction of labeled antibody in the liquid. The use of a mixture of the solid‐phase and labeled antibodies at different concentrations makes it possible to determine antigen concentration over a wide range, without an initial sample dilution. Two assays were developed: one using anti‐IgG polyclonal antibodies and the other using antibovine serum albumin monoclonal antibodies. The detection range of the polyclonal antibody assay using 30‐minute incubation was 0.2 to 40 μg/mL for human IgG standard. The detection range of the monoclonal antibody assay was 0.2 to 14 μg/mL for bovine serum albumin (BSA) standard with 2 minutes required for the incubation. The interassay variability for the BSA measurement was 1.9% at 4.0 μg/mL of BSA and intra‐assay variability was 2.3% at 3.2 μg/mL of BSA. The principles of this assay can be applied in the measurement of any protein in a rapid and reproducible way. © 1992 John Wiley & Sons, Inc.