Mouse Anti-dTT(6-4)TT Recombinant Antibody (clone 64M-2) (CAT#: PABZ-034)

Figure 1 Determination of cyclobutane pyrimidine dimers and (6-4)photoproducts in single-stranded calf thymus DNA irradiated with physiological 254 nm UV doses.

The direct ELISA with a biotin-streptavidin system was used to increase sensitivity. Each point shows the mean of three independent determinations. The dilution of antibody and the amount of immobilized UV-ssDNA per well of microtiter plate are as follows; TDM-2 (0-2 J/m², 1/3333, 50 ng; 0-20 J/m², 1/1000, 10 ng); TDM-3 (1/100, 750 ng); 64M-2 (1/1000, 150 ng); 64M-3 (1/3333, 150 ng); 64M-4 (1/1000, 1000 ng); 64M-5 (1/1000, 150 ng).

Mori, T., Nakane, M., Hattori, T., Matsunaga, T., Ihara, M., & Nikaido, O. (1991). Simultaneous establishment of monoclonal antibodies specific for either cyclobutane pyrimidine dimer or (6‐4) photoproduct from the same mouse immunized with ultraviolet‐irradiated DNA. Photochemistry and photobiology, 54(2), 225-232.

Figure 2 The first round of affinity maturation of 64M-2.

a Affinity maturation of 64M-2. The spheroplasts derived from the 64M-2 library were labeled with anti-Flag for antibody display and with L-probe1 for 6-4PP binding. The samples "Empty plasmids" and "Anti-digoxigenin" were included for negative controls. A sorting gate was marked on the "Sort 0" sample file. The spheroplasts within this gate were harvested for cloning. b Hot spot mutants were compiled by sequencing 50 VL and 50 VH clones. c The 6-4PP binding abilities of the combinations of VL and VH clones with the most abundant mutations

Kong, B., Cao, Y., Wu, D., An, L., Ran, F., Lin, Y., ... & Hang, H. (2018). Affinity maturation of an antibody for the UV-induced DNA lesions 6, 4 pyrimidine-pyrimidones. Applied microbiology and biotechnology, 1-16.

Figure 3 The sensitivities of 9c3, 64M-2, and 64M-5 in immunofluorescence, ELISA, and flow cytometry.

a HeLa cells covered with porous membrane were irradiated with UVC at designated doses, then labeled with 64M-2 (33 μg/ml), 64M-5 (2 μg/ml), and 9c3 (2 μg/ml), respectively. Two cells in each labeling condition were enlarged to show different antibody binding to cytoplasm (the border line between the nucleus and cytoplasm can be observed by enlarging the figure on a computer screen). b The statistical analysis of numbers of bright spots (DNA lesions in pore regions) derived from randomly analyzing more than 200 UV-irradiated HeLa cells labeled with the three different antibodies separately. c ELISA assays of UV-damaged genomic DNA of HeLa cells using 64M-2 (6.6 μg/ml), 64M-5 (0.83 μg/ml), and 9c3 (0.83 μg/ml). d The flow cytometric analyses of UV-irradiated HeLa cells labeled with 64M-2 (0.5 μg/ml), 64M-5 (0.5 μg/ml), and 9c3 (0.5 μg/ml). e Normal human fibroblasts (GM00637) were irradiated with 10 J/m2 UVC and allowed to repair damaged DNA for designated time, then ELISA assays were performed on genomic DNA using 64M-2 (33 μg/ml) and 9c3 (2 μg/ml). f The experiment similar to that in (e) but using Xpc-deficient human fibroblasts (XP4PA-SV) instead of GM00637 cells. g The experiment similar to that in (e) but using HeLa cells instead of GM00637 cells. Data are presented as mean ± SEM (n = 3), * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001

Kong, B., Cao, Y., Wu, D., An, L., Ran, F., Lin, Y., ... & Hang, H. (2018). Affinity maturation of an antibody for the UV-induced DNA lesions 6, 4 pyrimidine-pyrimidones. Applied microbiology and biotechnology, 1-16.