To investigate if the free ZT-2
peptide maintained its binding affinity to renal carcinoma cells, we made a synthetic peptide ZT-2 (QQPPMHLMSYAG) labeled with fluorescein isothiocyanate. (A) Immunohistochemical staining of renal AZD2281 research buy carcinoma tissues when bound with phage ZT-2-FITC. The specific binding sites on tumor cells fluoresced green (B) Immunohistochemical staining of nontumorous renal tissues when bound with phage ZT-2 (C) a negative control section stained with random peptide-fluorescein isothiocyanate in renal carcinoma tissues. Magnification × 200. Competitive Inhibition Assay A peptide-competitive inhibition assay was performed to discover whether the synthetic peptide ZT-2 and the corresponding phage clone competed for the same binding site. When the synthetic peptide ZT-2 was pre-incubated with A498 cells, phage ZT-2 binding to A498 cells decreased in a dose-dependent manner. When the peptide ZT-2 concentrations increased, the titer of phages recovered from A498 cells was decreased and the inhibition was increased gradually. When the concentrations of peptide ZT-2 increased above 5 μM, the inhibition reached a flat phase. The control peptide (EAFSILQWPFAH) had no effect on the binding of the phage ZT-2 to A498 cells (Figure 4). Figure 4 Competitive inhibition of binding of the phage ZT-2 to A498 cells by the synthetic peptide ZT-2 Adriamycin QQPPMHLMSYAG. The average inhibition rates
at different concentrations of the peptide are shown. When the concentration of the peptide ZT-2 reached more than 0.001 μM, a significant inhibition occurred. Discussion Targeting specific ligand binding on specific AZD3965 in vivo tumor antigens is an efficient way to increase the selectivity of therapeutic targets in clinical oncology and helpful for the early detection and therapy of RCC. Tumor cells often display certain cell surface antigens such as tumor-associated antigens
or tumor-specific antigens in high quantity, which are different from the antigens on normal tissues. To develop more biomarkers for the diagnosis of RCC, we used peptide phage Guanylate cyclase 2C display technology to identify potential molecular biomarkers of A498 carcinoma cells. After panning for three rounds, 20 clones were selected for further characterization. First, a cell-based ELISA assay was used to confirm the specific binding of the phage clones to A498 cells in vitro. ZT-2 was the best candidate phage clone with the highest specificity. Second, immunocytochemical and immunohistochemical staining were performed to confirm the selectivity of the phage ZT-2 to bind to A498 cells. Third, the results of the competitive inhibitory assays suggest that the peptide displayed by the phage M13-ZT-2, not other parts of this phage, can bind to the renal carcinoma cell surface. Under the same conditions, the normal renal cell line HK-2 did not show significant fluorescence when stained with ZT-2 peptide-FITC, which confirmed the targeting of ZT-2 to be A498 cells.