Assay employing a labeled fab-fragment ligand complex

Abstract

Methods and compositions are provided for improved protein binding assays by preparing compositions having indirectly labeled ligands substantially free of label conjugated to materials other than the indirectly labeled ligand. The method for preparing the compositions involves employing a monovalent receptor to which the label is conjugated and combining the monovalent receptor labeled conjugate to the ligand, either in pure or impure form. The mixture is then segregated according to molecular weight and the ligand conjugated to the labeled receptor isolated. This conjugate may then be directly used as a reagent in a protein binding assay, where the assay mixture is substantially free of label other than labeled receptor bound to ligand. The labels will be for the most part of relatively low molecular weight, while the receptor is preferably a Fab fragment. The ratio of receptor to ligand will be chosen so as to provide reasonable molecular weight distinctions between unbound ligand, unbound labeled receptor, ligand bound to labeled receptor, and other materials which may be in the mixture. Various techniques may be used for the separation. The assays are performed in accordance with known methods employing a second receptor composition, which may be labeled or unlabeled.