Development of ELISA-analysis methods for the quantification of bioactive natural products in plants, phytomedicines and in humans or similar.

In the course of a program in developing new ELISA-methods for the quantification of bioactive natural products in plants, phytomedicines and animals in a μg and ng scale, monoclonal antibodies against various natural products of medicinal and analytical importance have been developed. The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization (MALDI) of mass spectrometry. A hybridoma secreting monoclonal antibodies (MAb) was produced by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl-forskolin was 5.6%. A very small cross-reaction appeared with other derivatives. The full measuring range of the assay extends from 5 ng to 5 μg/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published separation methods. The capacity of the immunoaffinity column as determined by ELISA is 9 μg/ml. Forskolin has been isolated directly from the crude extracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against Δ(1)-tetrahydrocannabinolic acid (THCA) was produced. The cross-reaction of anti-Δ(1)-THCA antibody against other cannabinoids was very wide. Many cannabinoids and a spiro-compound were reactive, but did not react with other phenolics. It became evident that this ELISA method was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. Anti-solamargine MAbs were produced. A method of determination for solasodine glycosides by using TLC-immunostaining was established. Solasodine glycosides separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO(4) solution followed by BSA, resulting in a solasodine glycoside-BSA conjugate. Immunostaining of solasodine glycosides was more sensitive compared to other staining. Immunostaining of ginsenosides in fresh ginseng root was successful using anti-ginsenoside Rb1 (G-Rb1) MAb after blotting to PVDF membrane.