CN100360938C - 甾体类激素-碱性磷酸酶结合物制备方法 - Google Patents
甾体类激素-碱性磷酸酶结合物制备方法 Download PDFInfo
- Publication number
- CN100360938C CN100360938C CNB031100988A CN03110098A CN100360938C CN 100360938 C CN100360938 C CN 100360938C CN B031100988 A CNB031100988 A CN B031100988A CN 03110098 A CN03110098 A CN 03110098A CN 100360938 C CN100360938 C CN 100360938C
- Authority
- CN
- China
- Prior art keywords
- steroid hormone
- liquid
- polylysine
- alkaline phosphatase
- place
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000003270 steroid hormone Substances 0.000 title claims abstract description 15
- 102000002260 Alkaline Phosphatase Human genes 0.000 title claims abstract description 11
- 108020004774 Alkaline Phosphatase Proteins 0.000 title claims abstract description 11
- 239000003960 organic solvent Substances 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 36
- 229920000656 polylysine Polymers 0.000 claims description 22
- 150000003431 steroids Chemical class 0.000 claims description 21
- 238000002360 preparation method Methods 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 12
- 239000004472 Lysine Substances 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 12
- 150000002923 oximes Chemical class 0.000 claims description 12
- 239000000376 reactant Substances 0.000 claims description 12
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 11
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 11
- 108010039918 Polylysine Proteins 0.000 claims description 10
- 238000004108 freeze drying Methods 0.000 claims description 9
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 claims description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000003760 magnetic stirring Methods 0.000 claims description 6
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 5
- 230000021615 conjugation Effects 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- PROQIPRRNZUXQM-UHFFFAOYSA-N (16alpha,17betaOH)-Estra-1,3,5(10)-triene-3,16,17-triol Natural products OC1=CC=C2C3CCC(C)(C(C(O)C4)O)C4C3CCC2=C1 PROQIPRRNZUXQM-UHFFFAOYSA-N 0.000 claims description 3
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 3
- 230000001174 ascending effect Effects 0.000 claims description 3
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 3
- 239000000539 dimer Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 229960005309 estradiol Drugs 0.000 claims description 3
- 229930182833 estradiol Natural products 0.000 claims description 3
- PROQIPRRNZUXQM-ZXXIGWHRSA-N estriol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H]([C@H](O)C4)O)[C@@H]4[C@@H]3CCC2=C1 PROQIPRRNZUXQM-ZXXIGWHRSA-N 0.000 claims description 3
- 229960001348 estriol Drugs 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 235000011187 glycerol Nutrition 0.000 claims description 3
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims description 3
- 239000008108 microcrystalline cellulose Substances 0.000 claims description 3
- 229940016286 microcrystalline cellulose Drugs 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000000178 monomer Substances 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 239000000186 progesterone Substances 0.000 claims description 3
- 229960003387 progesterone Drugs 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 229960003604 testosterone Drugs 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 4
- 238000004020 luminiscence type Methods 0.000 abstract description 2
- 230000007246 mechanism Effects 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 abstract 2
- 206010020751 Hypersensitivity Diseases 0.000 abstract 1
- 208000026935 allergic disease Diseases 0.000 abstract 1
- 238000003556 assay Methods 0.000 abstract 1
- 239000003124 biologic agent Substances 0.000 abstract 1
- 238000003745 diagnosis Methods 0.000 abstract 1
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- 239000003814 drug Substances 0.000 abstract 1
- 230000009610 hypersensitivity Effects 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 description 2
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
Abstract
化学发光免疫分析技术是当代最流行的一类超灵敏度检测手段，广泛应用于生物医学基础研究和临床疾病的诊疗。对于甾体类激素的检测其标记物是碱性磷酸酶和甾体类激素的结合物。甾体类激素是一类脂溶性较强的物质，而碱性磷酸酶在一些低浓度的有机溶剂中即可变性失活，故制备这种标记物均为国外某些高科技生物制剂机构所垄断。本研究工艺简便易行、成本低廉，为开发此类试剂盒替代国外商品创造了条件。
Description
技术领域
本发明涉及一种甾体类激素-碱性磷酸酶结合物的制备方法，适合于以碱性磷酸酶标记的甾体类激素小分子物质在化学发光免疫分析技术中的应用。
背景技术
甾体类激素是一组具有环戊烷多氢菲基本结构的化合物，脂溶性很强，在水中溶解性极微，而碱性磷酸酶是一种分子量100kDa的蛋白质，在有机溶剂中易失活。甾体类激素-碱性磷酸酶结合物是研发化学发光免疫分析试剂盒中的重要组分。目前制备这种标记物均为国外某些高科技生物制剂机构所垄断，关于其制造工艺国内外尚未有报道。
发明内容
本发明的目的在于提供一种甾体类激素-碱性磷酸酶结合物制备方法，该方法简便易行。
本发明提供的甾体类激素-碱性磷酸酶结合物的制备方法，是将脂溶性的甾体类激素经过聚赖氨酸联接降低其脂溶性，在微量的有机溶剂中完全溶解，形成甾体类激素-聚赖氨酸，当甾体类激素-聚赖氨酸和碱性磷酸酶联结时不降低其活性，采用戊二醛法联结，形成甾体类激素-碱性磷酸酶结合物。
所述的甾体类激素经过与聚赖氨酸联接形成甾体类激素-聚赖氨酸是采用如下方法：
(1)取甾体类激素羧甲基肟(睾酮、雌二醇、雌三醇或孕酮)300ug溶于100μl 50％吡啶中，另取EDC 2mg溶于100μl双蒸水中，两液混合，室温反应1.5小时，为A液，取聚赖氨酸600ug溶于100μl双蒸水中，为B液。
(2)A液置磁力搅拌器上，将B液滴加至A液中，反应4-6h，4℃冰箱过夜。
(3)将反应液冷冻干燥，加40％的乙醇100μl溶解，充分振荡后离心去沉淀，上清液用划线法点在硅胶板GF254 10×20cm上，展开液为氯仿-甲醇-水系统，在254nm反射紫外光检测仪显示两条区带，样品参照位经茚三酮呈色显出两条斑点，将相应的经鉴定为具有茚三酮显色又在254nm紫外光下显出区带处刮下，洗脱并干燥后即为产物。
所述的甾体类激素-聚赖氨酸和碱性磷酸酶联结是采用如下方法：
(1)取甾体类激素-聚赖氨酸150ug溶于40％乙醇100μl中，为A液，另取经透析的碱性磷酸酶1mg(SIGMA产品，活性3870U/mg)，补加生理盐水至400μl，为B液。
(2)将A液置于磁力搅拌器上，取B液缓缓滴加至A液中，此时应完全透明，即刻加入1％戊二醛30μl，放室温暗处反应3h。
(3)将反应液装入8mm直径透析袋，4℃搅拌对PBS透析48h，再移至50mmol/l pH7.6 Tris-HCL缓冲液透析24h。离心10000r/min 20分钟，上清液加等量AR级丙三醇及适量优级BSA，分装50μl/支，4℃保存。
前面所述的聚赖氨酸采用如下方法制备：
(1)取赖氨酸3mg溶于500μl生理盐水(pH6.5)中，另取EDC 5mg(300μl)滴加至赖氨酸溶液中，磁力搅拌下室温反应5-6h，移至4℃冰箱内过夜。
(2)将反应液冷冻干燥，加150μl双蒸水溶解，离心5000r/min 20分钟。取上清液在10×20cm微晶纤维素薄板上(自制厚度1～1.2mm)，在板的左1/4处点10μl反应液，采用上行层析在层析筒中展开，展开剂为正丁醇-冰乙酸-水系统。待展层液展至上端2cm处，取出放室温下干燥。
(3)用相同大小的玻板将点样处覆盖，向左1/4处喷1mg/ml茚三酮液，放50℃干燥箱30分钟，可见3-4个相同色的斑点，根据预先测得赖氨酸的Rf值，刮去赖氨酸单体，将其余斑点刮下至离心管中，用适量双蒸水洗3次，洗脱液合并，冷冻干燥，产物为二聚体和三聚体，产率40％。
采用上述本发明方法制备的甾体类激素-碱性磷酸酶结合物简便易行。
具体实施方式
实施例1 聚赖氨酸的制备
(1)取赖氨酸3mg溶于500μl生理盐水(pH6.5)中，另取EDC 5mg(300μl)滴加至赖氨酸溶液中，磁力搅拌下室温反应5-6h，移至4℃冰箱内过夜。
(2)将反应液冷冻干燥，加150μl双蒸水溶解，离心5000r/min 20分钟。取上清液在10×20cm微晶纤维素薄板上(自制厚度1～1.2mm)，在板的左1/4处点10μl反应液，采用上行层析在层析筒中展开，展开剂为正丁醇-冰乙酸-水系统。待展层液展至上端2cm处，取出放室温下干燥。
(3)用相同大小的玻板将点样处覆盖，向左1/4处喷1mg/ml茚三酮液，放50℃干燥箱30分钟，可见3-4个相同色的斑点，根据预先测得赖氨酸的Rf值，刮去赖氨酸单体，将其余斑点刮下至离心管中，用适量双蒸水洗3次，洗脱液合并，冷冻干燥，产物为二聚体和三聚体，产率40％。
实施例2-5 甾体类激素-碱性磷酸酶结合物制备
(1)分别取不同的甾体类激素羧甲基肟(睾酮羧甲基肟、雌二醇羧甲基肟、雌三醇羧甲基肟、孕酮羧甲基肟)300ug溶于100μl 50％吡啶中，另取EDC 2mg溶于100μl双蒸水中，两液混合，室温反应1.5小时，为A液，取聚赖氨酸600ug溶于100μl双蒸水中，为B液。
(2)A液置磁力搅拌器上，将B液滴加至A液中，反应4-6h，4℃冰箱过夜。
(3)将反应液冷冻干燥，加40％的乙醇100μl溶解，充分振荡后离心去沉淀，上清液用划线法点在硅胶板GF254 10×20cm上，展开液为氯仿-甲醇-水系统，在254nm反射紫外光检测仪显示两条区带，样品参照位经茚三酮呈色显出两条斑点，将相应的经鉴定为具有茚三酮显色又在254nm紫外光下显出区带处刮下，洗脱并干燥后即为甾体类激素-聚赖氨酸。
(4)取制备的甾体类激素-聚赖氨酸约150ug溶于40％乙醇100μl中，为A液，另取经透析的碱性磷酸酶1mg(SIGMA产品，活性3870U/mg)，补加生理盐水至400μl，为B液。
(5)将A液置于磁力搅拌器上，取B液缓缓滴加至A液中，此时应完全透明，即刻加入1％戊二醛30μl，放室温暗处反应3h。
(6)将反应液装入8mm直径透析袋，4℃搅拌对PBS透析48h，再移至50mmol/l pH7.6 Tris-HCL缓冲液透析24h。离心10000r/min 20分钟，取上清液加等量AR级丙三醇及适量优级BSA，分装50μl/支，4℃保存。
Claims (4)
1、一种甾体类激素-碱性磷酸酶结合物的制备方法，其特征在于脂溶性的甾体类激素经过聚赖氨酸联接降低其脂溶性，在微量的有机溶剂中完全溶解，形成甾体类激素-聚赖氨酸，当甾体类激素-聚赖氨酸和碱性磷酸酶联结时不降低其活性，采用戊二醛法联结，形成甾体类激素-碱性磷酸酶结合物。
2、根据权利要求1所述的制备方法，其特征在于所述的甾体类激素经过聚赖氨酸联接形成甾体类激素-聚赖氨酸是采用如下方法：
(1)取甾体类激素羧甲基肟300ug溶于100μl 50％吡啶中，另取EDC 2mg溶于100μl双蒸水中，两液混合，室温反应1.5小时，为A液，取聚赖氨酸600ug溶于100μl双蒸水中，为B液，其中所述甾体类激素羧甲基肟选自睾酮羧甲基肟、雌二醇羧甲基肟、雌三醇羧甲基肟、孕酮羧甲基肟；(2)A液置磁力搅拌器上，将B液滴加至A液中，反应4-6h，4℃冰箱过夜；
(3)将反应液冷冻干燥，加40％的乙醇100μl溶解，充分振荡后离心去沉淀，上清液用划线法点在硅胶板GF254 10×20cm板上，展开液为氯仿-甲醇-水系统，在254nm反射紫外光检测仪显示两条区带，样品参照位经茚三酮呈色显出两条斑点，将相应的经鉴定为具有茚三酮显色又在254nm紫外光下显出区带处刮下，洗脱并干燥后即为产物。
3、根据权利要求1或2所述的制备方法，其特征在于所述的甾体类激素-聚赖氨酸和碱性磷酸酶联结是采用如下方法：
(1)取甾体类激素-聚赖氨酸150ug溶于40％乙醇100μl中，为A液，另取经透析的SIGMA公司生产，活性为3870U/mg的碱性磷酸酶1mg，补加生理盐水至400μl，为B液；
(2)将A液置于磁力搅拌器上，取B液缓缓滴加至A液中，此时应完全透明，即刻加入1％戊二醛30μl，放室温暗处反应3h；
(3)将反应液装入8mm直径透析袋，4℃搅拌对PBS透析48h，再移至50mmol/l pH7.6 Tris-HCl缓冲液透析24h，离心10000r/min 20分钟，上清液加等量AR级丙三醇及适量优级BSA，分装50μl/支，4℃保存。
4、根据权利要求1或2所述的制备方法，其特征在于所述的聚赖氨酸由如下方法制备：
(1)取赖氨酸3mg溶于500μl生理盐水pH6.5中，另取EDC 5mg滴加至赖氨酸溶液中，磁力搅拌下室温反应5-6h，移至4℃冰箱内过夜；
(2)将反应液冷冻干燥，加150μl双蒸水溶解，离心5000r/min20分钟，取上清液在10×20cm微晶纤维素薄板上，自制厚度1～1.2mm，在板的左1/4处点10μl反应液，采用上行层析在层析筒中展开，展开剂为正丁醇-冰乙酸-水系统，待展层液展至上端2cm处，取出放室温下干燥；
(3)用相同大小的玻板将点样处覆盖，向左1/4处喷1mg/ml茚三酮液，放50℃干燥箱30分钟，可见3-4个相同色的斑点，根据预先测得赖氨酸的Rf值，刮去赖氨酸单体，将其余斑点刮下至离心管中，用适量双蒸水洗3次，洗脱液合并，冷冻干燥，产物为二聚体和三聚体，产率40％。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031100988A CN100360938C (zh) | 2003-04-23 | 2003-04-23 | 甾体类激素-碱性磷酸酶结合物制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031100988A CN100360938C (zh) | 2003-04-23 | 2003-04-23 | 甾体类激素-碱性磷酸酶结合物制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1540347A CN1540347A (zh) | 2004-10-27 |
| CN100360938C true CN100360938C (zh) | 2008-01-09 |
Family
ID=34319624
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031100988A Expired - Lifetime CN100360938C (zh) | 2003-04-23 | 2003-04-23 | 甾体类激素-碱性磷酸酶结合物制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100360938C (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102331503A (zh) * | 2011-08-31 | 2012-01-25 | 内蒙古科慧生物科技有限责任公司 | 睾酮(t)定量测定试剂盒及其检测方法 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0114615A2 (en) * | 1983-01-25 | 1984-08-01 | Abbott Laboratories | Alkaline phosphatase labeled steroid hormone glucoronides |
| EP0114614A2 (en) * | 1983-01-25 | 1984-08-01 | Abbott Laboratories | Determination of steroid hormone glucuronides |
| US5028535A (en) * | 1989-01-10 | 1991-07-02 | Biosite Diagnostics, Inc. | Threshold ligand-receptor assay |
| US5939272A (en) * | 1989-01-10 | 1999-08-17 | Biosite Diagnostics Incorporated | Non-competitive threshold ligand-receptor assays |
-
2003
- 2003-04-23 CN CNB031100988A patent/CN100360938C/zh not_active Expired - Lifetime
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0114615A2 (en) * | 1983-01-25 | 1984-08-01 | Abbott Laboratories | Alkaline phosphatase labeled steroid hormone glucoronides |
| EP0114614A2 (en) * | 1983-01-25 | 1984-08-01 | Abbott Laboratories | Determination of steroid hormone glucuronides |
| US5028535A (en) * | 1989-01-10 | 1991-07-02 | Biosite Diagnostics, Inc. | Threshold ligand-receptor assay |
| US5939272A (en) * | 1989-01-10 | 1999-08-17 | Biosite Diagnostics Incorporated | Non-competitive threshold ligand-receptor assays |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1540347A (zh) | 2004-10-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fevery et al. | Bilirubin conjugates in bile of man and rat in the normal state and in liver disease | |
| McManus et al. | Polyphenol interactions. Part 1. Introduction; some observations on the reversible complexation of polyphenols with proteins and polysaccharides | |
| Carlson et al. | Polyuronic acids produced by Pseudomonas aeruginosa | |
| Spivak et al. | Reverse-phase hplc separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on hplc of their ethyl anthranilate azo derivatives | |
| US4238473A (en) | Artificial oligosaccharide antigenic determinants | |
| Alpert et al. | Multiplicity of hepatic excretory mechanisms for organic anions | |
| Heirwegh et al. | Recent advances in the separation and analysis of diazo-positive bile pigments | |
| Low-Beer et al. | Effects of sulfation of taurolithocholic and glycolithocholic acids on their intestinal transport | |
| CN102994604A (zh) | 两步酶促法制备结合态熊去氧胆酸的方法 | |
| Weiner et al. | Renal excretion of bile acids: taurocholic, glycocholic, and cholic acids | |
| Roux et al. | Condensed tannins. 13. Interrelationships of flavonoid components from the heartwood of Robinia pseudacacia | |
| JPH0323528B2 (zh) | ||
| CN105277424A (zh) | 一种莱克多巴胺免疫磁珠分离富集试剂盒及其应用 | |
| Spivak et al. | Non-enzymic hydrolysis of bilirubin mono-and diglucuronide to unconjugated bilirubin in model and native bile systems. Potential role in the formation of gallstones | |
| JPS63196596A (ja) | 末端にイノシト−ル残基を結合したグルコオリゴ糖およびその製造方法 | |
| CN100360938C (zh) | 甾体类激素-碱性磷酸酶结合物制备方法 | |
| CN102590520B (zh) | Eddp胶体金检测试剂盒及其制备方法 | |
| CN111196827A (zh) | 一种人乳中的功能组分及其制备和应用 | |
| CN102539412A (zh) | 检测克伦特罗的磁颗粒化学发光试剂盒及其应用 | |
| CN101315376A (zh) | 喹诺酮类抗生素多残留酶联免疫检测方法 | |
| US8129363B2 (en) | Method for obtaining conjugated estrogen mixtures from pregnant mare urine and use of a macroporous resin in the method | |
| Kandrac et al. | Isolation and determination of bile acids | |
| CN107652369A (zh) | 一种抗凝血女贞花多糖及其提取分离方法和应用 | |
| Anderson et al. | Studies on carbohydrate-metabolizing enzymes. Trans-β-glucosylation by barley enzymes | |
| Baker et al. | Lesions of potato sprout and extracted potato sprout alkaloid toxicity in Syrian hamsters |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: BEIJING CHEMCLIN BIOTECH CO., LTD. BEIKEYUAN Free format text: FORMER OWNER: BEIMIAN-DONGYA INST. OF BIOTECHNOLOGY, BEIJING Effective date: 20060428 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20060428 Address after: 100094 Beijing city Haidian District industrial base in Yongfeng Feng Xian Road No. 7 North Park Applicant after: Beijing Chemclin Biotech Co.,Ltd. Address before: 100012 Beijing Chaoyang District Andingmen Dayangfang No. 6 Applicant before: Beimian-Dongya Inst. of Biotechnology, Beijing |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: BEIJING KEMEI BIOLOGICAL TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: KEMEI DONGYA BIOLOGICAL TECHNOLOGY CO., LTD., BEIJING Effective date: 20110519 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 100094 USTB SCIENCE PARK, NO. 7, FENGXIAN MIDDLE ROAD, YONGFENG INDUSTRIAL BASE, HAIDIAN DISTRICT, BEIJING TO: 100094 USTB SCIENCE PARK, NO. 7, FENGXIAN MIDDLE ROAD, YONGFENG BASE, HAIDIAN DISTRICT, BEIJING |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20110519 Address after: 100094 Beijing city Haidian District Yongfeng base Feng Xian Road No. 7 North Park Patentee after: BEIJING CHEMCLIN BIOTECH Co.,Ltd. Address before: 100094 Beijing city Haidian District industrial base in Yongfeng Feng Xian Road No. 7 North Park Patentee before: Beijing Chemclin Biotech Co.,Ltd. |
|
| CP03 | Change of name, title or address |
Address after: 100094 Beijing Haidian District Yongfeng Base Fengxianzhong Road 7 Beike Modern Manufacturing Park Incubation Building Patentee after: Kemei Diagnostic Technology Co.,Ltd. Address before: 100094 Beijing city Haidian District Yongfeng base Feng Xian Road No. 7 North Park Patentee before: BEIJING CHEMCLIN BIOTECH Co.,Ltd. |
|
| CP03 | Change of name, title or address | ||
| CX01 | Expiry of patent term |
Granted publication date: 20080109 |
|
| CX01 | Expiry of patent term |