Monoclonal antibodies against naturally occurring bioactive compounds

The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate w as determined by matrix-assisted laser desorption/ionization (MALDI) tof ma ss spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produc ed by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-s ensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodi es 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 mu g/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published sep aration methods. The capacity of the immunoaffinity column as determined by ELISA is 9 mu g/ml. Forskolin has been isolated directly from the crude ex tracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against tetrahydrocannabinolic acid (THCA) was produced. The cross-reactio n of anti-THCA antibody against other cannabinoids was very wide. Many cann abinoids and a spiro-compound were reactive, but did not react with other p henolics. It became evident that this ELISA was able to be applied to the b iotransformation experiments of cannabinoids in plant tissue culture system . Anti-ginsenoside Rb1 MAbs were produced. New western blotting method of d etermination for ginsenosides was established. Ginsenosides separated by si lica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membran e. The membrane was treated with NaIO4 solution followed by BSA, resulting in a ginsenoside-BSA conjugate. Immunostaining of ginsenosides was more sen sitive compared to other staining. Immunostaining of ginsenosides in the fr esh ginseng root was succeeded using anti-ginsenoside Rb1 (GRb1) MAb after blotting to PVDF membrane.