ABSTRACT
A simple, rapid and self-contained system for assaying the immunoreactive fraction of radiolabeled antibodies was developed using affinity thin-layer chromatography (ATLC). ATLC combines use of solid-phase-bound antigen and conventional TLC. The technique is an improvement over existing means of measuring immunoreactive fraction (bead-type or cell-type assays) in that it has neither wash steps nor centrifugation steps, yet provides results essentially identical to those obtained with the more time-consuming assays. ATLC is accomplished using chromatography strips that are coated with antigen material in a discrete region near the origin. The antigen-coated strips are then blocked in serum, air-dried and stored. For use, radiolabeled antibody is spotted at the origin, and the strip is developed using a buffered solvent. Immunoreactive antibody binds to the antigen at or near the origin, while radioactivity not associated with immunoreactive antibody migrates with the solvent front. Antigen-negative strips (serum-blocked only) are used to measure "nonspecific" binding. The ATLC development time is about 16 min, and the results can be obtained in about 30 min. The assay described in this report uses antigens from colon tumor and is suitable for use with B72.3 and other colon cancer-reactive antibodies.
