Development of Eastern Blotting Technique for Analysis of Baicalin Using Anti-Baicalin Monoclonal Antibody

Scutellariae radix (S. radix) is one of the most important crude drugs used in Kampo medicines (KMs). A part of its pharmaceutical properties is due to flavone glycoside, baicalin (BI). A technique named eastern blotting was developed for the specific and easy identification of BI in the extracts of crude drugs and KMs using anti-BI monoclonal antibody (MAb). BI separated by silica gel thin-layer chromatography (TLC) transferred to a polyethersulfone (PES) membrane was treated with a NaIO4 solution and reacted with bovine serum albumin (BSA) preparing BI-BSA conjugate on the PES membrane. Anti-BI MAb was bound and then antibody labeled with peroxidase directed against anti-BI MAb. Finally, a substrate was added and then BI was detected. As little as 1 mg of BI was still detected on the PES membrane under immunostaining method. Various samples of S. radix and KMs which contain S. radix were qualitatively analyzed, and BI was visually detected by eastern blotting technique. Furthermore, this method was applied for the immunohistochemical study to investigate the distribution of BI in the fresh root of Scutellaria baicalensis using immunoblotting by transferred from fresh root to the PES membrane.