AFFINITY THIN-LAYER CHROMATOGRAPHY TEST OF THE IMMUNOREACTIVE FRACTION OF RADIOLABELED ANTIBODIES

A simple, rapid and self-contained system for assaying the immunoreact ive fraction of radiolabeled antibodies was developed using affinity t hin-layer chromatography (ATLC). ATLC combines use of solid-phase-boun d antigen and conventional TLC. The technique is an improvement over e xisting means of measuring immunoreactive fraction (bead-type or cell- type assays) in that it has neither wash steps nor centrifugation step s, yet provides results essentially identical to those obtained with t he more time-consuming assays. ATLC is accomplished using chromatograp hy strips that are coated with antigen material in a discrete region n ear the origin. The antigen-coated strips are then blocked in serum, a ir-dried and stored. For nse, radiolabeled antibody is spotted at the origin, and the strip is developed using a buffered solvent. Immunorea ctive antibody binds to the antigen at or near the origin, while radio activity 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 des cribed in this report uses antigens from colon tumor and is suitable f or use with B72.3 and other colon cancer-reactive antibodies.