Preparation of affinity column using MAb and its application for isolation and optical resolution.

MAb 亲和柱的制备及其在分离和光学分离中的应用。

• 批准号: 08457586
• 负责人: SHOYAMA Yukihiro
• 金额: $ 4.99万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457586/
• 关键词: MAb; forskolin; ELISA; affinity column; pulitication; optical resolution; 小型化抗体

Forskol in, a labdane diterpenoid, was isolated from the tuberous roots of Coleus foreskohlii Briq. (Lamiaceae). C.forskohlii has been used as an important folk medicine in India, and has been found to be a potent activator of adenylate cyclase, leading to an increase of c-AMP.The full measuring range of the assay extends from 5ng/ml to 5mug/ml of forskolin.The concentration of forskolin in the stem base (4.04mug/mg dwt) exceeded that found in both the upper stem (1.58mug/mg dwt) and near the shoot tip (0.34mug/mg). This tendency to accumulate forskolin near the base stem was observed in individual branches, except for the lowest branch base which contained the highest amount of forskolin (5.97mug/mg dwt). In order to confirm the purity of the IgG.the MALDI mass spectrum was measured.The molecular weight was 148600, which is in good agreement with that of human IgG being detaemined as 146000.Since forskolin was almost insoluble in water, various buffer solutions were tested for the dis … More solution of forskolin. It became evident that 6% of CH_3OH in PBS was necessary to dissolve forskolin. The forskolin concentrations eluted increased rapidly from 20% CH_3OH to the maximum at 45%. The crude diethyl ether extracts od tuberous root of C.forskohlii were loaded on the immunoaffinity column, washed 5 times with PBS containing 6% of CH_3OH,and eluted with PBS containing 45% CH_3OH.Fractions 2 to 8 conteined 45 mug of forskolin, which is greater than the column capacity, together with the related compounds, 1-deoxyforskolin, 1.9-dideoxyforskolin, 7-deacety forskolin and 6-acetyl-7-deacetyl forskolin, and other unknown compounds which were deteced by TLC.The peak-of fractions 26 to 30 shows the elution of forskolin (21mug) eluted with the PBS containing 45% CH_3OH.Forskolin eluted by the washing solution (fraction 2 to 8) was repeatedly loaded and finally isolated. However, forskoklin purified in this way by the immunoaffinity column was still contaminated by a small amount of 7-deacetyl forskoklin because this compound has 5.5% of cross-reactivity against MAb as previously indicated. Therefore, the mixture was treated with pyridine and acetic anhydride at 4ﾟC for 2h to give forskolin. Less

毛喉素为唇形科二萜类化合物，从鞘蕊花（Coleus foreskohlii Briq.）（唇形科）。毛喉梭菌在印度已被用作重要的民间药物，并且已被发现是腺苷酸环化酶的有效激活剂，该测定的整个测量范围从5 ng/ml延伸到5 μ g/ml毛喉素。（4.04 μ g/mg dwt）超过茎上部（1.58 μ g/mg dwt）和茎尖附近（0.34 μ g/mg dwt）。在单个分枝中观察到毛喉素在基部茎附近积累的趋势，除了最低的分支基部含有最高量的毛喉素（5.97 mug/mg dwt）。为确定IgG的纯度，测定了其MALDI质谱，其分子量为148600，与人IgG的分子量146000相符。由于毛喉素几乎不溶于水，因此对各种缓冲液进行了检测，结果表明，毛喉素的分子量为148600，与人IgG的分子量146000相符。 ...更多信息 Forskolin溶液。结果表明，溶解佛司可林需要6%的甲醇溶液。从20%CH_3OH洗脱到45%时，毛喉素浓度迅速增加。将毛喉黄连块根乙醚粗提物上免疫亲和柱，用含6%CH_3OH的PBS洗涤5次，用含45%CH_3OH的PBS洗脱，洗脱级分2 - 8含有45 μ g毛喉素，大于柱容量，以及相关化合物1-脱氧毛喉素，1.9-双脱氧毛喉素，经薄层色谱检测，发现有7-去乙酰基毛喉素和6-乙酰基-7-去乙酰基毛喉素等未知化合物，其中26 ~ 30级分峰为用含45%CH_3OH的PBS洗脱的毛喉素洗脱液（21 μ g），洗脱液（2 ~ 8级分）经反复上样分离。然而，通过免疫亲和柱以这种方式纯化的毛喉素仍然被少量的7-脱乙酰基毛喉素污染，因为该化合物对MAb具有5.5%的交叉反应性，如前所述。因此，将混合物用吡啶和乙酸酐在4 ° C下处理2小时以得到毛喉素。少

项目成果

H.Tanaka, R.Takahashi, S.Morimoto, Y.Shoyama: "A new cannabinoid, DELTA9-tetrahydrocannabinol 2-0-beta-D-glucopyranoside, Biotransformed by plant tissue." J.Nat.Prod.60. 168-170 (1997)

H.Tanaka、R.Takahashi、S.Morimoto、Y.Shoyama：“一种新的大麻素，DELTA9-四氢大麻酚 2-0-β-D-吡喃葡萄糖苷，由植物组织生物转化。”

H.Tanaka et al: "A simple determination of steroidal alkaloid glycosides by thinlayer chromotography immunostoining using monoclonal antibody agoinst solamargine" FEBS Letters. 404. 279-282 (1997)

H.Tanaka 等人：“使用单克隆抗体 solamargine 通过薄层色谱免疫染色法简单测定甾体生物碱糖苷”FEBS Letters。

H.Yanagihara, R.Sakata, Y.Shoyama, H.Murakami: "Rapid analysis of small samples containing forskolin using monoclonal antibodies." Planta Medica. 62. 169-172 (1996)

H.Yanagihara、R.Sakata、Y.Shoyama、H.Murakami：“使用单克隆抗体快速分析含有毛喉素的小样品。”

H.Tanaka et al: "Monoclonal antibody based enzyme immunoassay for marihuana(Cannabinoid)compounds" Jounal of Immunoassay. 17(4). 321-342 (1996)

H.Tanaka 等人：“基于单克隆抗体的大麻（大麻素）化合物酶免疫测定”《免疫测定杂志》。

H.Tanaka et al: "Cannabis 25,Biotransformation of cannabidiol and cannabidiolic acid by Pinellia ternata tissue segments." Plant Cell Rep.,. 15. 819-823 (1996)

H.Tanaka 等人：“大麻 25，半夏组织片段对大麻二酚和大麻二酚酸的生物转化。”