3D8 single chain variable fragment (scFv) antibody has cell-penetrating activity, nucleic acid-binding activity, nucleic acid-hydrolyzing activity, and antiviral activity. As a first step toward development of antiviral reagent using 3D8 scFv, functional stability based on DNA-binding and -hydrolyzing activity of 3D8 scFv was analyzed under biochemical and biophysical conditions such as different temperature, pH, storage period, reducing environment, lyophilization, and repetitive freeze-thaw. The DNA-binding activity and -hydrolyzing activity were maintained for 6 h at 50°C. The optimal pH showing the DNA-binding activity and -hydrolyzing activity was exhibited for 2 h at pH 7-11. The DNA-binding and -hydrolyzing activity were fully retained in the presence of reducing agent, 2 mM dithiothreitol (DTT). Moreover, DNA-hydrolyzing activity of 3D8 scFv was maintained after lyophilization and 30 cycles of freeze-thaw. When the 3D8 scFv was stored in pH 3-12 for a month at 37°C, DNA-hydrolyzing activity was retained at pH 4-8. In addition, we defined the unit for DNA-hydrolyzing activity and investigated antiviral activity against influenza A virus according to the unit. Our study would provide the basic information in developing an antiviral reagent with 3D8 scFv.
