Hapten-mediated identification of cell membrane antigens using an anti-FITC monoclonal antibody

A monoclonal anti-FITC antibody (F4/1) was produced and demonstrated to be specific for both the free and protein-conjugated (either soluble or cell-bound) form of fluorescein, or carboxyfluorescein. When mouse thymocytes were labelled with a novel fluorescein derivative 5(6)-carboxyfluorescein succinimidyl ester (CFl-NSE), the incorporation of fluorescein was predominantly membrane-bound as demonstrated immunohistochemically. The coupling of CFl-NSE to cells displays a random distribution pattern as shown by immunoblotting of cell extracts prepared by detergent solubilization of CFl-NSE-labelled thymocytes. In addition, the Thy-1.2 antigen immunoprecipitated from a CFl-NSE-labelled thymocyte lysate with a rat monoclonal antibody (Mab) could be detected using the anti-FITC Mab. The molecular weight of the immunoprecipitated material could be estimated immediately by reference to the FITC-labelled molecular weight markers electrophoresed simultaneously.