Figure 4.

Assessing performance of a panel of anti-Jagged-1 antibodies. (a) Flow cytometry calibration beads with varying number of anti-human Fc antibodies were coated with Jagged-1-Fc fusion to yield antigen display levels of 29,000, 83,000, 204,000 and 619,000 Jagged-1 molecules/bead. These were labeled with a panel of different recombinant antibodies raised against Jagged-1 and binding was detected with labeled anti-FLAG antibodies. The resulting histograms are shown, giving different levels of sensitivity. In Jag1_D5 for example, five peaks are visible corresponding to uncoated beads and each of the four antigen coated beads. In the case of Jag1_C5, where there is lower sensitivity, only the two beads with highest density are resolved while the others merge with that of the uncoated bead. Where all four beads are clearly resolved, we have calculated the theoretical limit of detection of receptors per bead (Rec. #). (b) 46C mouse embryonic stem cells were stained with the panel of Jagged-1 antibodies and analyzed by flow cytometry. Staining for three antibodies (Jag1_D5, Jag1_D7 and Jag1_A1.2) is shown. The sensitivity of each corresponds to that seen in the bead assay above. (c) A plot of normalized ELISA scores with performance in the bead-based flow cytometry performance assay for nine Jagged-1 specific antibodies. ELISA was carried out using 1 μg/ml of purified antibody. The mean time resolved fluorescence score (expressed as Eu counts) was plotted against median fluorescent intensity determined for an antigen density of 618,888 antigens/bead. The line represents a linear regression analysis of the data (R² value = 0.8323). (d) Immunohistochemical (Jag1_A6 scFv antibody) and (e) in situ hybridization staining of Jagged-1 in developing kidney of E14.5 mouse embryo, demonstrating localization of staining (dark purple) in developing renal tubules.