Abstract: Methods and compositions are disclosed for the qualitative and quantitative identification of specific ligand in a fluid medium suspected of containing the ligand. The method of the present invention involves the formation of a liquid reaction mixture comprising an enzyme, a ligand-specific binding component, a conjugate comprising ligand coupled to a modulator label, and sample fluid medium suspected of containing ligand. Once the reaction mixture is formed, enzyme activity is monitored and the presence and amount of ligand in the sample fluid medium is determined by changes in enzyme activity.Novel compositions useful in practicing the disclosed methods are provided. These include a biotin binding protein enzyme modulator, novel ligand-enzyme modulator conjugates, and biotin containing enzymes.