ELISA for the determination of saikosaponin a, an active component of Bupleuri Radix.
In order to quantify saikosaponin a (SSA), one of the major active components of Bupleuri Radix, a competitive and indirect ELISA method was developed. High titer rabbit polyclonal antibodies (pAbs) were raised against a conjugate of SSA and bovine serum albumin, coupled with a periodate oxidation method. SSA competitively inhibited the binding of rabbit anti-SSA pAbs to SSA-ovalbumin on the solid phase, a coated antigen on the well. The quantity of pAbs bound to the well was monitored using a peroxidase-conjugated anti-rabbit IgG as a secondary antibody, and tetramethylbenzidine solution as a substrate. The measuring range extended from 50 pg/ml to 20 ng/ml of SSA, with a detection limit of 40 pg/ml (5.13 pM). Antibodies showed some cross-reactivity with saikosaponin c (12.74%). However, the antibodies showed only slight cross-reactivities with saikosaponin d (0.3%), which differs from SSA only in the stereochemistry of the 16-hydroxyl group, and the artificial saikosaponins, saikosaponin b1 (2.1%) and saikosaponin g (0.53%). The specific and sensitive ELISA is especially suited for determination of SSA in samples when only small quantities of materials can be extracted for analysis.
College of Pharmacy, Chonnam National University, Kwangju, Korea.
D W
DW
Jung
M
M
Shibuya
Y
Y
Ebizuka
K
K
Yoshimatsu
K
K
Shimomura
C K
CK
Sung
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000032:analysis* / Q000493:pharmacokinetics; D000918:Antibody Specificity; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D009828:Oleanolic Acid / Q000031:analogs & derivatives*; D010047:Ovalbumin / Q000737:chemistry; D010946:Plants, Medicinal / Q000737:chemistry*; D011817:Rabbits / Q000276:immunology; D012502:Sapogenins / Q000032:analysis* / Q000493:pharmacokinetics; D012503:Saponins
D000894:Anti-Inflammatory Agents, Non-Steroidal; D012502:Sapogenins; D012503:Saponins; D009828:Oleanolic Acid; D010047:Ovalbumin; C025759:saikosaponin D