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.
Jung
D W
DW
College of Pharmacy, Chonnam National University, Kwangju, Korea.
Shibuya
M
M
Ebizuka
Y
Y
Yoshimatsu
K
K
Shimomura
K
K
Sung
C K
CK
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