| Co-Investigator(Kenkyū-buntansha) |
OZAWA Mika Josai International University, Department of Pharamaceutical Sciences, Research Instructor, 薬学部, 助手 (40398558)
SUZUKI Naoto Josai International University, Department of Pharamaceutical Sciences, Associate Professor, 薬学部, 講師 (60372299)
HISHIMURA Takanori Tohoku, University Hospital, Associate Professor, 病, 助教授 (20199003)
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| Research Abstract |
In order to investigate and/or determine the structure of glycyrrhetic acid (GA) to contribute the binding affinity and specificity against the antibodies, the mouse anti-GA monoclonal antibody (AGA-1) was carefully investigated the binding to the GA-derived compounds, which were 17 compounds tested, with the inhibition ELISA. AGA-1 binding was inhibited by the 16 compounds investigated, except bile acid. We found that the A-ring, the C-3 hydroxyl group, and the C-30 carbonic group for the E-ring, oleanane structure and its C-11 carbonic group were important for the recognition of the AGA-1.
We prepared the recombinant Fab fragment antibodies which were substituted the each amino acid in the CDR-H3 with alanine, and investigated the binding activities against the GA-BSA with ELISA. It suggested that the amino acid-side chains for the root region of the CDR-H3 was contributed to the specific binding.
In order to investigate the reactivity of the AGA-1 and each rFab, GA was fixed on the sensor chip CM5 for BIACORE and tested. The kinetic parameters were calculated. The parameters kon (E4 /M/s), koff (E4 /M/s), and KD (E-9 M) for AGA-1 were 37.4 31.6, and 3.2, respectively. However, the parameters for the rFab were Y99A (0.9, 0.2, 20.8), Y100bA (36.4, 2.3, 6.2), D101A (16.5, 3.7, 25.1), W95A (5.2, 5.1, 98.4), respectively.
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