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1. 发明公开

EP0300682A1 A homogeneous, lipsome-based signal amplification method for assays involving enzymes 失效
标题翻译： Ein匀浆，au f。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。
公开(公告)号：EP0300682A1
公开(公告)日：1989-01-25
申请号：EP88306427.1
申请日：1988-07-13
申请人： THE UNIVERSITY OF TENNESSEE RESEARCH CORPORATION
发明人： Huang, Leaf
IPC分类号： G01N33/58 , C12Q1/68 , G01N33/74 , G01N33/53
CPC分类号： G01N33/581 , A61K9/1271 , G01N33/586
摘要： The present invention is directed to a procedure for enhancing the enzyme-dependent readout of diagnostic assays using specially prepared liposomes.
Microgenic's CEDIA assay for digoxin was employed as an illustrative example of the present invention. The magnitude of signal was only 25 mA/min/ng digoxin/ml for the unamplified assay, while the signal magnitude for the amplified (i.e., liposome-enhanced) assay was 1,000 mA/min/ng digoxin/mL, i.e., a 40-fold amplification.
Stable liposomes were prepared with unsaturated PE stabilized with 5 mole percent of ganglioside GM₁. Glucose-6-phosphate dehydrogenase (G6PHD) was entrapped in these unilamellar liposomes in high concentrations. Addition of beta-galactosidase (B-gal) caused rapid (3-5 min.) lysis of liposomes revealing the latent G6PDH activity, due to the enzymatic degalactosylation of MG₁.
This simple, rapid and homogenous signal amplification procedure will be useful in many enzyme dependent assays, such as ELISA, CEDIA, Gene-probe assays and Immunoliposome assays.
摘要翻译：本发明涉及使用特别制备的脂质体来增强诊断测定酶的酶依赖性读出的方法。作为本发明的说明性实施例，使用了用于地高辛的微生物CEDIA测定。对于未扩增测定，信号的幅度仅为25mA / min / ng地高辛/ ml，而扩增（即，脂质体增强）测定的信号幅度为1,000mA / min / ng地高辛/ mL，即40 倍放大。用5摩尔％的神经节苷脂GM1稳定的不饱和PE制备稳定的脂质体。葡萄糖-6-磷酸脱氢酶（G6PHD）以高浓度包埋在这些单层脂质体中。加入β-半乳糖苷酶（B-gal）引起脂质体的快速（3-5分钟）裂解，显示由于MG1的酶脱羧基化而引起潜在的G6PDH活性。这种简单，快速和均匀的信号扩增程序将在许多酶依赖性测定中有用，例如ELISA，CEDIA，基因探针测定和免疫脂质体测定。

2. 发明公开

EP0277776A2 Solid core liposomes 失效
标题翻译： Liposome mit harten Kernen。
公开(公告)号：EP0277776A2
公开(公告)日：1988-08-10
申请号：EP88300750.2
申请日：1988-01-28
申请人： THE UNIVERSITY OF TENNESSEE RESEARCH CORPORATION
发明人： Huang, Leaf
IPC分类号： A61K9/50
CPC分类号： B82Y30/00 , A61K9/1277 , Y10S436/829 , Y10S514/963 , Y10T428/2984
摘要： Preferred solid core liposomes are prepared in four major steps:

(1) Preparation of prevesicles with encapsulated solid cores of agarose-gelatin by emulsification of agarose-gelatin sol in organic solvent containing emulsifiers followed by cooling;
(2) Extraction of lipophilic components from prevesicles to obtain microspherules of agarose - gelatin;
(3) In an optional step, colloidal gold particles are introduced into the microspherules, which are then coated with a protein or peptide molecule layer;
(4) Encapsulation of the microspherules is conducted using a modified organic solvent spherule evaporation method for the formation of the liposomes.

Electron micrographs indicate that if liposomes are prepared by using a lipid mixture containing dioleoyl phosphatidyl choline, cholesterol, dioleoylphosphatidylglycerol, and triolein (molar ratio 4.5:4.5:1:1), there is only a single continuous bilayer membrane for each solid core liposome.
摘要翻译：优选的固体核脂质体以四个主要步骤制备：（1）通过在含有乳化剂的有机溶剂中乳化琼脂糖凝胶溶胶随后冷却来制备具有封装的琼脂糖凝胶固体核心的前囊泡; （2）从前囊中提取亲脂性成分，得到琼脂糖凝胶微珠; （3）在任选的步骤中，将胶体金颗粒引入微球体中，然后用蛋白质或肽分子层包被; （4）使用用于形成脂质体的改性有机溶剂微球蒸发法进行微球的封装。电子显微照片表明，如果通过使用含有二油酰磷脂酰胆碱，胆固醇，二油酰磷脂酰甘油和三油酸甘油酯（摩尔比为4.5:4.5:1:1）的脂质混合物制备脂质体，则每个固体核脂质体仅存在单个连续双层膜。

3. 发明公开

EP0277776A3 Solid core liposomes 失效
标题翻译：固体核脂质体
公开(公告)号：EP0277776A3
公开(公告)日：1989-09-27
申请号：EP88300750.2
申请日：1988-01-28
申请人： THE UNIVERSITY OF TENNESSEE RESEARCH CORPORATION
发明人： Huang, Leaf
IPC分类号： A61K9/50
CPC分类号： B82Y30/00 , A61K9/1277 , Y10S436/829 , Y10S514/963 , Y10T428/2984
摘要： Preferred solid core liposomes are prepared in four major steps:
(1) Preparation of prevesicles with encapsulated solid cores of agarose-gelatin by emulsification of agarose-gelatin sol in organic solvent containing emulsifiers followed by cooling; (2) Extraction of lipophilic components from prevesicles to obtain microspherules of agarose - gelatin; (3) In an optional step, colloidal gold particles are introduced into the microspherules, which are then coated with a protein or peptide molecule layer; (4) Encapsulation of the microspherules is conducted using a modified organic solvent spherule evaporation method for the formation of the liposomes. Electron micrographs indicate that if liposomes are prepared by using a lipid mixture containing dioleoyl phosphatidyl choline, cholesterol, dioleoylphosphatidylglycerol, and triolein (molar ratio 4.5:4.5:1:1), there is only a single continuous bilayer membrane for each solid core liposome.

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