DEVELOPMENT OF THE METHOD FOR STRUCTURAL ANALYSIS OF DOMAIN OF ANTIHAPTENIC ANTIBODY BY HYPHENATED MASS SPECTROMETRY

抗半抗原抗体结构域联用质谱分析方法的建立

• 批准号: 06557120
• 负责人: GOTO Junichi
• 金额: $ 5.57万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557120/
• 关键词: Enzyme immunoassay; Electrospray ionization; LC; MS; 11-Deoxycortisol; Capillary electrophoresis; Monoclonal antibody; リゾチーム; エレエレクトロスプレーイオン化

Recently, many interests have been focused on the usefulness of mass spectrometry (MS) heyphenated with chromatographic methods such as liquid chromatography (LC) and capillary electrophoresis for the structural elucidation of proteins. On the other hand, it is keen needed elucidation of mechanisms of the intermolecular interaction between proteins and biologically active molecules. The purpose of this project was the development of the method for the structural elucidation of the domain of the anti-haptenic antibody bounded to the hapten.Initially, the monoclonal antibody for 11-deoxycortisol was prepared using the steroid having a bridge at C-4 on the nucleus as a hapten. Next, lysosome as a model enzyme for enzyme immunoassay was reacted with 11-deoxycortisol and the structure of the conjugated lysozyme was analyzed with electrospray ionization (ESI) mass spectrometry on the flow injection mede. Before and after reductive alkylation, the conjugated lysosome has been further subjected to hydrolysis with various enzyme followed by the LC/ESI-MS analysis to clarify the structure of the domain.

近年来，质谱与液相色谱、毛细管电泳等色谱方法联用，用于蛋白质的结构分析，引起了人们的广泛关注。另一方面，人们迫切需要阐明蛋白质与生物活性分子之间的分子间相互作用机制。本课题的目的是开发与半抗原结合的抗半抗原抗体的结构域的结构解析方法。最初，使用在细胞核的C-4处具有桥的类固醇作为半抗原，制备11-脱氧皮质醇的单克隆抗体。以溶菌酶作为酶免疫分析的模型酶，与11-脱氧皮质醇反应，用流动注射电喷雾质谱（ESI MS）分析了结合溶菌酶的结构。在还原性烷基化前后，缀合的溶酶体已经进一步经受各种酶的水解，随后通过LC/ESI-MS分析来澄清结构域的结构。

项目成果

後藤順一: "臨床化学診断学" ソフトサイエンス社, 466 (1995)

后藤淳一：《临床化学诊断》软科学出版社，466（1995）

Toshifumi Niwa, Chiaki Yoshida, Toyoaki Kato and Junichi Goto: "Preparation of antiserum for use in the immunoassay of unconjugated and conjugated 4beta-hydroxycholic acids" Anal.Sci.10. 349 (1994)

Toshifumi Niwa、Chiaki Yoshida、Toyoaki Kato 和 Junichi Goto：“用于非缀合和缀合 4β-羟基胆酸免疫测定的抗血清的制备”Anal.Sci.10。

Takashi Iida: "Potential bile acid metabolites. 23. Synthesis of 3-glucosides of nonamidated and glycine-and taurine-amidated bile acids." J. Lipid Res.36. 628-638 (1995)

Takashi Iida：“潜在的胆汁酸代谢物。23。非酰胺化胆汁酸、甘氨酸酰胺化胆汁酸和牛磺酸酰胺化胆汁酸的 3-葡萄糖苷的合成。”

Shigeo Ikegawa: "Stereoisomeric inversion of (25R)-and (25S)-3α, 7α, 12α-trihydroxy-5β-cholestanoic acids in rat liver peroxisome." Biol. Pharm. Bull.18. 1027-1029 (1995)

Shigeo Ikekawa：“大鼠肝脏过氧化物酶体中 (25R)-和 (25S)-3α、7α、12α-三羟基-5β-胆甾烷酸的立体异构反转”，Pharm.1027-1029。

Norio Awata, Fumio Toba, Masayuki Ando, Hitoshi Shimada, Shinichi Miyairi, Toyoaki Kato, Junichi Goto, and Toshio Nambara: "Immunoaffinity extraction of 4-hydroxy-2- (4-for determination by gaschromatography-mass spectrometry" Biol.Pharm.Bull.17. 843 (199

Norio Awata、Fumio Toba、Masayuki Ando、Hitoshi Shimada、Shinichi Miyairi、Toyoaki Kato、Junichi Goto 和 Toshio Nambara：“4-羟基-2-（4-的免疫亲和提取，用于通过气相色谱-质谱法测定”Biol.Pharm。