Biotin-binding IgG in human sera was quantitated using F(ab')(2)anti-human IgG-coated multiwell microplates (Muratsugu, M. et al. 2003, Biol. Pharm. Bull., 26, 1605-1608). The biotin-protein ratio of biotinylated IgG, which was used as standard in the assay, was very important to quantitate the level of biotin-binding IgG. We investigated a synthesis method of biotinylated human immunoglobulins, how to determine the biotin-protein ratio of the biotinylated proteins, and their stability to prepare standards for measuring biotin-binding IgG, IgA, and IgM.