Application of a licorice root specific protein to a general method for the assay of licorice root components in traditional Chinese medicines.

A new fuzzy immunoassay method generally applied to ten Glycyrrhizae Radix (GR) preparations of four different botanical origins was studied. Four kinds of antisera were elicited in rabbits immunized with GRs of different botanical origins. The presence of the characteristic GR protein (GRP) was shown using Western blot analyses and selected antibody enzyme immunoassay (SAEIA) methods. A GRP was isolated from one of the GR specimens which was selected using SAEIA methods. The isolated GRP was heated to reduce its binding activity to an anti-GR serum. A new fuzzy SAEIA method generally applicable for assay of the extract of the ten GR specimens was developed using heat-treated GRP as the solid-phase antigen. The fuzzy SAEIA method was successfully applied for the detection and quantitative analysis of the GR component contained in traditional Chinese medicines.

A general enzyme immunoassay for the licorice root component contained in traditional Chinese medicines.

The development and application of a new enzyme immunoassay for general assay of the Glycyrrhizae radix (GR) component in Chinese traditional medicines is described. Three commercial GR-based medicines, Tohoku kanzo (GRTK), Seihoku Kanzo (GRSEK) and Sinkyo kanzo (GRSK) were used as GR specimens. Anti-GRSK serum was elicited from rabbits immunized with GRSK fragments. The presence of common proteins as specific antigens of GR was first established by Western blot analysis of extracts of GRSK, GRSEK or GRTK using anti-GRSK. The specific antigens were applied to develop an ELISA for the assay of GR extract. Anti-GRSK was put in competition with a sample or standard GR extract and immobilized GRSK components in microtiter plate wells. The proportion of antibody binding to the solid-phase GRSK component was detected using an enzyme-labeled second antibody. The ELISA method was specific to GRSK extract and showed low sensitivity for the assay of GRTK extract. The technique of selected antibody enzyme immunoassay (SAEIA) was applied to develop a sensitive general assay method. Solid-phase GRTK extract, rather than immobilized GRSK extract, was used in the SAEIA. The SAEIA possessed the same quantitative working range of between 1 and 100 micrograms/ml for the assay of each extract of GRTK, GRSK and GRSEK. The SAEIA was successful in the detection and quantitative measurement of GR component contents in Chinese traditional medicines.

A new method for the detection and quantitative measurement of the contents of trichosanthes root component composing Chinese traditional medicines.

A new method was developed to estimate the content of Trichosanthes root (TR) component in two Chinese traditional medicines. Characteristic antigens of TR were separated from TR extract using rabbit antiserum specific for TR, a dried root tuber of Trichosanthes kirilowii. Two selected antibody enzyme immunoassay (SAEIA) methods, the SAEIA A for assay of TR extract and the SAEIA B for assay of karasurin A were used as detection methods for the separation of TR antigens. Using several column chromatographies, two kinds of TR antigens, a protein component and a glycan component, were separated from TR extract. A SAEIA C method for assay of TR glycan component was developed using biotinylated second antibody and peroxidase-labeled avidin as the detection method. The SAEIA C was also found applicable for specific assay of TR extract as well as for the contents of TR component present in two Chinese traditional medicines, prescriptions of which contained TR. Content of trichosantin, an abortifacient protein component of T. kirilowii, in both the medicines were also measured by applying the SAEIA B for assay of karasurin A. Little trichosantin was found in either medicine and the reason for this was determined.

A new enzyme immunoassay for a solid Chinese crude drug, pinellia tuber.

A new immunoassay for a solid Chinese crude drug was studied. An antiserum specific for Pinellia tuber was elicited in two rabbits. Using the antiserum and powdered Pinellia tuber-coated microtiter plate as the immunological reagents, and beta-D-galactosidase-labeled goat anti-rabbit immunoglobulin G (IgG) as the tracer, a new enzyme immunoassay for a solid Pinellia tuber with a working range between 0.1 and 1000 micrograms/ml was developed. The assay was specific for a solid Pinellia tuber and showed low cross-reaction values on other Chinese crude drugs and the extract of Pinellia tuber. The specificity of the assay was compared with the selected antibody enzyme immunoassay (SAEIA) for the extract of Pinellia tuber recently developed. Both methods utilized the same immunological reagents such as the serum and the enzyme-labeled goat anti-rabbit IgG, and the only difference between them was the solid-phase antigen used. The assay results of several antigens determined by them were quite different, showing that selective measurements of different antigens, either solid or the extract of Pinellia tuber, were possible using the same antiserum, when the tracing reaction in the immunoassay was adequately selected.