A Method for Preparing Specific Fluorescein-Conjugated Antibody Reagents Using Bentonite Immunoadsorbents

Abstract

Abstract A simple, rapid, and reproducible method is described for the preparation of fluorescein-conjugated specific antibody reagents. Bentonite clay was used to adsorb and insolubilize antigen. Release of antigen from bentonite particles was prevented by treating the adsorbed antigen with a water-soluble carbodiimide reagent. The resulting immunoadsorbent could be used both to isolate homologous antibody and to remove cross-reacting antibodies from heterologous antisera. These immunoadsorbents were reusable, well-adapted to batch techniques, and stable on storage at -70° to 4°C for periods in excess of 1 year. Prior isolation of antibody globulin is not required before fluorescein conjugation when antibody is to be isolated subsequently by immunoadsorbents. The quantity of fluorescein isothiocyanate (FITC) used for conjugation of the antiserum was rigorously controlled by adding 1-µ1 portions of dissolved FITC, so that subsequent fractionation to remove excessively labeled globulin is not required. With these methods highly specific antibody reagents can be prepared in a single working day when homologous antigen is already available. More than 95% of the protein in such conjugates has antibody activity as measured by the ability to bind to homologous antigen. When used to locate antigen in tissue, the antibody reagents exhibit virtually no background fluorescence.