Construction and evaluation of the TR1-IgMs. (A) Western blot analysis of the recombinant purified TR1-IgMs. Ten nanograms of each of the biotinylated-TR1-IgM(+/−)s (TR1-IgM(J+/−)-401, 404, 419, 422, and 438) and control-IgM(+/−) was immunoblotted with horseradish peroxidase-conjugated streptavidin. (B) Reactivity of the TR1-IgM(J+)s with the recombinant human TRAIL-R family. The binding of the TR1-IgM(J+)s to human TRAIL-R1-Fc, TRAIL-R2-Fc, TRAIL-R3-Fc, TRAIL-R4-Fc, or OPG was examined by ELISA. Control-IgM(J+) was used as a control. The data are shown as the mean ± SD of triplicate cultures. (C) The binding of the TR1-IgM(J+)s to the Colo205 cell line was analyzed by flow cytometry. The cells were stained with 5 μg/mL of biotinylated-TR1-IgM(J+)s and control-IgM(J+) as indicated, followed by R-PE-conjugated streptavidin, and analyzed with flow cytometry. X-axis, fluorescence intensity of PE; Y-axis, relative cell number. Representative data of three independent experiments in which similar results were obtained are shown.