title: Development of an Enzyme-Linked Immunosorbent Assay and Immunoaffinity Column Chromatography for Saikosaponin d Using an Anti-Saikosaponin d Monoclonal Antibody.
abstract: This work developed a novel immunochemical approach for the quality control of saikosaponin d using an enzyme-linked immunosorbent assay. Splenocytes from mice immunized with the saikosaponin d-bovine serum albumin conjugate were fused with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line, and a hybridoma secreting monoclonal antibody against saikosaponin d was successfully obtained. The prepared anti-saikosaponin d monoclonal antibody 1E7F3 has a novel characteristic, showing weak reactivity with compounds that are structurally related to saikosaponin d. Using monoclonal antibody 1E7F3, a specific and reliable enzyme-linked immunosorbent assay was developed to detect saikosaponin d. The system shows a full measurement range from 156.25 to 5000.00 ng × mL(-1). Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10.00%. The recovery rates ranged from 92.36% to 101.00%, meeting the requirements for biological samples. There was a good correlation between the enzyme-linked immunosorbent assay and high-performance liquid chromatography analyses of saikosaponin d, and the saikosaponin d levels in formulated Chinese medicines were successfully determined. Furthermore, immunoaffinity column chromatography was established using this anti-saikosaponin d monoclonal antibody, and the elution profile of saikosaponin d was detected by a Bio-Rad QuadTec UV/Vis detector at 203 nm. The results demonstrate that we generated a reliable and more efficient assay system for measuring saikosaponin d and provide a potential approach for purifying and separating saikosaponin d.
authors.affiliation: The Third Affiliated Hospital, Beijing University of Chinese Medicine, Beijing, China.
authors.forename: Jiayang
authors.initials: J
authors.lastname: Sai
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China.
authors.forename: Wenchao
authors.initials: W
authors.lastname: Shan
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China.
authors.forename: Baoping
authors.initials: B
authors.lastname: Qu
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China.
authors.forename: Jinjun
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Center of Scientific Experiment, Beijing University of Chinese Medicine, Beijing, China.
authors.forename: Huihua
authors.initials: H
authors.lastname: Qu
authors.affiliation: School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China.
authors.forename: Qingguo
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology; D012503:Saponins / Q000032:analysis* / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D012503:Saponins; D009828:Oleanolic Acid; C025759:saikosaponin D