Biotin-binding human immunoglobulin G (B-IgG) was quantitatively measured using an F(ab′)₂anti-human IgG-coated multi-well microplate for the first time. B-IgG was caught by F(ab′)₂anti-human IgG and was detected by the following detecting reagents: Peroxidase-labeled streptavidin, avidin and peroxidase-biotin, or avidin-biotinylated peroxidase complex with method A, B, or C, respectively. Commercially available B-IgG was detected by all these three methods. However, method A and B could not detect B-IgG in the human sera used in this study, but we quantitatively measured the B-IgG level using method C. The result is probably due to the fact that the sensitivity of method C was higher than that of methods A or B. Properties of B-IgG detected by method C are discussed in the text.