Yukihiro Shoyama
Nagasaki International University, Japan
Title: Fingerprinting of natural product by eastern blotting using monoclonal antibodies
Biography
Biography: Yukihiro Shoyama
Abstract
We prepared many kinds of monoclonal antibodies (MAbs) against natural products and developed a new staining method using MAb named as Eastern blotting. Glycosides like ginsenoside were developed by TLC, and the TLC plate was covered by PVDF or PES membrane and blotted. The membrane was treated with NaIO4, and then with carrier protein resulting in glycoside-carrier protein conjugates on membrane. In the case of non-glycoside natural product like aristolochic acids, the conjugate with carrier protein was synthesized by appropriate pathway on the membrane. Peroxidase labeled secondary MAb and then substrate were added, successively. Several ginsengs were analyzed to find out unknown ginsenosides in American and Japanese ginseng by this fingerprinting. Also we separated ginsenosides using affinity column combined MAb from the crude extract. The other bioactive glycosides like saikosaponins and glycyrrhizin can be stained and applied for fingerprinting analysis. Aristolochic acids having kidney toxicity in Alistolochia spp. were separated by TLC and then blotted onto a PES membrane by employing a modified carbodiimide method. The resulting membrane-bound aristolochic acids-protein conjugates can be stained by Eastern blotting and fingerprinting. The staining of aristolochic acid in mouse kidney tissues was succeeded. Moreover, we detected sequensed and determined a target protein against aristolochic acid in mouse kidney cell lines using anti-aristolochic acid MAb. These related results will be also discussed.