Figure 1 Immunocytochemistry Selleckchem TPCA-1 and immunohistochemical staining of Sp17 in a human carcinoma cell line and xenograft
tumor tissues. A, B. In vitro cultured cell lines staining with anti-Sp17-mAb; A: Sp17+ SMMC-7721 cells, B: Sp17- HO8910 cells (original magnification, 20×); C, D. Sp17+ SMMC-7721 cell tumor xenograft tissue slices staining with: C: anti-Sp17-mAb, D. unrelated monoclonal antibody (original magnification, 40×). Characterization of anti-Sp17-ICG-Der-02 The anti-Sp17 antibody was conjugated with ICG-Der-02 for in vivo tracing of the dynamics of anti-Sp17- ICG-Der-02 in nude mice subjects. The NHS ester of the NIR fluorescence dyes is reacted with the amino group of the amino acid residue in anti-Sp17 and purified
by dialysis. The absorption and fluorescence emission spectra of the complex were characterized, as shown in Figure 2. The antibody activity of anti-Sp17-ICG-Der-02 was tested with ELISA, and the result showed that the antibody on the conjugate retained major biological activity compared with naked antibody (Figure 3). Figure KU55933 2 Optical characterization of ICG-Der-02-labled anti-Sp17. Figure 3 The antibody activity of anti-Sp17-ICG-Der-02 tested with ELISA. A. naked anti-Sp17 antibody; B. anti-Sp17-ICG-Der-02 conjugate. In vivo targeting capability of anti-Sp17-ICG-Der-02 The in vivo dynamic processes of anti-Sp17-ICG-Der-02 and corresponding blank samples in tumor-bearing nude mice were evaluated with an NIR fluorescence imaging system. For the experimental group, ICG-Der-02 had apparent accumulation in tumor sites at 2 h post-injection. The fluorescence Verubecestat research buy intensity in the region of interest (ROI) was persistently enhanced and reached the maximum at 24 h post-injection. Strong fluorescence was observed even at 7 days post-injection for mice in this group. Images of group B (the control group) indicated that free ICG-Der-02, without the help of anti-Sp17, had little accumulation in tumor tissue at 24 h post-injection. The targeting capability of anti-Sp17-ICG-Der-02 for tumors
was observed both in vivo imaging and ex vitro imaging (Figure 4 and Figure 5) after the process of entrapment. ICG-Der-02 accumulated in the liver then cleared through urine, so the liver and kidneys showed the strongest fluorescence after injection but the intensity tapered with time. From Bcl-w our results, we know that free ICG-Der-02 was excreted faster than anti-Sp17-ICG-Der-02. Figure 4 Iv vivo images of tumor-bearing mice show the tumor targeting effect of anti-Sp17-ICG-Der-02 (dose for each group was 0.2 μg, calculated as the amount of ICG-Der-02). A. Systemic injection of anti-Sp17-ICG-Der-02 (n = 5). Images were obtained in one mouse; bright fluorescent in the tumor region is due to probe accumulation. B. Systemic injection of free ICG-Der-02 (n = 3), images were obtained in one mouse, fluorescent signal in tumor is virtually absent.