CN102565427A - Preparation method for prothrombin time determination reagent - Google Patents

Preparation method for prothrombin time determination reagent Download PDF

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CN102565427A
CN102565427A CN 201110446209 CN201110446209A CN102565427A CN 102565427 A CN102565427 A CN 102565427A CN 201110446209 CN201110446209 CN 201110446209 CN 201110446209 A CN201110446209 A CN 201110446209A CN 102565427 A CN102565427 A CN 102565427A
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reagent
method
pt
preparation
time
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CN 201110446209
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王小良
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苏州良辰生物医药科技有限公司
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Abstract

The invention relates to a preparation method for prothrombin time determination reagent (PT reagent), comprising the following steps. Cow or sheep tissue factor with the purity of more than 90% is prepared by the genetic engineering method, phospholipid is dispersed in phosphate buffer solution containing surfactant, and then the tissue factor and the phospholipid are mixed and sufficiently combined, and finally, C18 resin is adopted to adsorb and separate the surfactant, and the PT reagent product is obtained after freeze-drying. The preparation method is stable in technique and strong in operability, and further, the obtained PT reagent has better stability, sensitiveness and fewer impurities, and the quality can be easily ensured.

Description

ー种凝血酶原时间测定试剂的制备方法 Preparation of reagent for measuring prothrombin time ー species

技术领域 FIELD

[0001] 本发明涉及临床血液诊断领域中的凝血酶原时间试验,具体涉及ー种凝血酶原时间测定试剂的制备方法。 [0001] The present invention relates to the field of prothrombin time test for the diagnosis of clinical blood, particularly species relates ー preparing a prothrombin time measurement reagent.

背景技术 Background technique

[0002] 临床上进行凝血试验的目的有很多,如确定接受外科手术的病人的出血倾向,以及对接受抗凝血治疗的病人进行监测以防止血液凝集等。 [0002] The purpose of the clinical coagulation tests are many, such as bleeding tendency determining a patient receiving the surgery, and to patients receiving anticoagulant therapy monitoring in order to prevent blood coagulation and the like. “凝血酶原时间(PT)”试验即是凝血试验中的ー种。 "Prothrombin time (the PT)" that is the test species ー clotting test. PT试验是通过在需要进行PT试验的血样中加入凝血活酶,从而激活外源性凝血途径而进行的。 PT test is performed by the addition of thromboplastin in a blood sample is required in the PT test, which activates the extrinsic coagulation pathway. 凝血活酶也称作组织因子(TF),是ー种膜相关蛋白,它与因子V II a 形成V II a/TF复合物后,会激活一系列凝血途径中所涉及的特异性酶,导致凝血酶和纤维蛋白的形成,血小板的激活,最终形成血块。 After the thromboplastin also called tissue factor (TF), a kind of membrane-associated proteins ー, V II a / TF complexes which are formed with Factor V II a, activates a series of specific enzymes involved in the coagulation pathway, leading to thrombin and fibrin formation, platelet activation, and finally clot formation. 常规PT试验是在体外及可控条件下,利用上述一系列酶反应,对病人凝血系统的障碍或缺陷进行诊断,血块形成所用的时间即为凝血酶原时间或PT值。 Conventional PT tests under controlled conditions and in vitro, using the above-described series of enzymatic reactions, disorders of the coagulation system or defective patient diagnosis, time spent clot formation is the prothrombin time or PT value.

[0003] 通常所称的凝血酶原时间测定试剂,即PT试剂,其主要成分就是凝血活酶(即组织因子)。 [0003] commonly referred to prothrombin time assay reagent, PT reagent, which is the main component of thromboplastin (i.e. tissue factor). 传统上,PT试剂常用从兔脑、羊脑或牛脑中初歩分离纯化的脑组织制成。 Traditionally, PT reagent used is made from rabbit brain, bovine brain, sheep brain or brain tissue ho First isolated and purified. 脑中的组织因子能够和磷脂协调作用完成血液外源性凝固系统的启动。 Brain tissue factor and phospholipid capable of coordinating role of exogenous blood coagulation promoter complete system. 但是,用初歩分离纯化的脑组织制备的PT试剂具有灵敏度差、有可能存在ー些抑制剂及制品稳定性差的缺点。 However, PT reagent prepared by Initial Purification ho brain tissue has poor sensitivity, there may exist some inhibitors and disadvantages of poor product stability ー. 国外也有报道应用基因工程制备的组织因子,主要是将含有组织因子基因的分子片段提取出并在大肠杆菌、真菌或动物細胞等中表达,从而获得组织因子,这种方法比前述自然取得组织因子的方法更为方便,得到的产品更纯并可大量生产。 Abroad have reported the preparation of tissue factor gene engineering, molecular fragments mainly containing the tissue factor gene and the expression in E. coli extracts, fungi, or animal cells or the like to obtain a tissue factor, tissue factor obtaining this method than that of the natural the method is more convenient to obtain a more pure product and mass production. 目前PT试剂中用到的这种基于基因工程的方法得到的组织因子,还主要是人组织因子和兔组织因子,但这种PT试剂及其制备方法也有缺陷,或稳定性差,或敏感性不高,或制备过程复杂エ艺难控制等。 Currently this genetic engineering tissue factor obtained based PT reagent used in further mainly human tissue factor and rabbit tissue factor, and PT reagent preparation but this method has a defect or poor stability, sensitivity, or not high, or the preparation of complex Ester Arts and difficult to control and so on.

发明内容 SUMMARY

[0004] 本发明所要解决的技术问题是克服现有技术的不足,提供一种改进的凝血酶原时间测定试剂的制备方法。 [0004] The present invention solves the technical problem to overcome deficiencies of the prior art, to provide an improved method for preparing a prothrombin time measurement reagent.

[0005] 为解决以上技术问题,本发明采取如下技术方案: ー种凝血酶原时间测定试剂的制备方法,包括如下步骤: [0005] To solve the above technical problem, the present invention adopts the following technical solutions: The method of preparing a prothrombin time measurement reagent thrombin ー species, comprising the steps of:

(1)、通过基因工程的方法制备纯度在90%以上的牛或羊组织因子,并将所述组织因子溶于水或磷酸缓冲液中形成组织因子溶液; (1), prepared in a purity of more than 90% goat or bovine tissue factor by genetic engineering, and the tissue factor is soluble in water or a phosphate buffer solution, forming a tissue factor;

(2)、将适量磷脂溶于含有表面活性剂的磷酸缓冲液中,搅拌6(Γ120分钟后,获得均勻分散的磷脂溶液;所述磷酸缓冲液的PH值在7. 2〜7. 6之间,表面活性剂的质量含量为1% ; (2), the amount of phospholipid was dissolved in a phosphate buffer solution containing a surfactant. After stirring 6 (Γ120 minutes to obtain uniform dispersion of the phospholipid solution; the PH value of the phosphate buffer in the 6 7. 2~7. between the mass content of the surfactant is 1%;

(3)、将步骤(1)制备得到的组织因子溶液加入步骤(2)所得的磷脂溶液中,使磷脂与组织因子的质量比在2. 5Χ IO3^XlO3:1之间,然后继续搅拌4(Γ90分钟,得到磷脂/组织因子溶液; (3), the step (1) Preparation of tissue factor obtained was added in step (2) resulting phospholipid solution and the mass ratio of phospholipid to tissue factor 2. 5Χ IO3 ^ XlO3 between: 1, and then stirring was continued for 4 (Γ90 minutes to obtain a phospholipid / tissue factor solution;

(4)、向步骤(3)所得的磷脂/组织因子溶液中加入体积为所述磷脂/组织因子溶液体积的1/10的完全溶胀的烷基键合硅胶,然后每间隔圹12分钟,进行一次摇勻,摇勻多次后, 得到浑浊液体; (4), the step (3) the resulting phospholipid / volume of tissue factor was added to the phospholipid / tissue factor solution volume of fully swollen 1/10 alkyl bonded silica gel, and then every 12 minutes interval tomb, for a shake, after shaking several times to give the turbid liquid;

(5)、对步骤(4)所得的浑浊液体进行离心分离,滤除沉淀,取上清液冻干,即得所述的凝血酶原时间测定试剂。 (5), step (4) the resultant cloudy liquid was centrifuged, the precipitate was filtered off, the supernatant was lyophilized to obtain the prothrombin time-measuring reagent.

[0006] 优选地,所述的表面活性剂为聚乙ニ醇对异辛基苯基醚,即曲拉通。 [0006] Preferably, the surfactant is polyvinyl alcohol Ni-isooctyl phenyl ether, i.e. triton.

[0007] 优选地,步骤(1)和步骤(2)中所述的磷酸缓冲液为PBS缓冲液。 [0007] Preferably, the step (1) and step (2) in the phosphate buffer was PBS buffer.

[0008] 优选地,所述磷脂为自然磷脂或合成磷脂或两者的混合物,且合成磷脂可选用磷脂酰胆碱、磷脂酰丝氨酸和磷脂酰乙醇胺中的ー种或多种的混合。 [0008] Preferably, the phospholipid is a natural phospholipid or a synthetic phospholipid mixture, or both, and the choice of synthetic phospholipids phosphatidylcholine, phosphatidylserine ー admixture and phosphatidylethanolamine or more.

[0009] 优选地,所述烷基键合硅胶为十八烷基键合硅胶,俗称C18树脂,常用来制作层析柱,但尚未见此种树脂应用在本发明所属领域中。 [0009] Preferably, the alkyl bonded silica gel as octadecyl-bonded silica, C18 resins commonly known, used to produce a chromatography column, but this resin has not been applied in the field of the invention.

[0010] 由于以上技术方案的实施,本发明与现有技术相比具有如下优点: [0010] Due to the technical solution of the embodiments above, the prior art and the present invention has the following advantages:

本发明将通过基因工程方法制备得到的纯度> 90%的牛或羊组织因子与磷脂相结合, 能够得到稳定性和灵敏性更好的PT试剂;用C18树脂能够非常方便和彻底地除去溶解磷脂时所用的表面活性剤,使制得的PT试剂杂质更少,质量更好;另外本发明制备方法还具有エ艺稳定,可操作性强的优点。 The purity of the present invention prepared by genetic engineering methods in> 90% of the cattle or sheep combine tissue factor with phospholipids, can be obtained a better stability and sensitivity PT reagent; with C18 resin can very easily and thoroughly remove dissolved phospholipid Ji surfactant used when the PT reagent was made less impurities, better quality; further preparation of the present invention further includes a method of stabilizing Ester arts, strong operational advantages.

具体实施方式 detailed description

[0011] 下面结合具体的实施例对本发明做进ー步详细的说明,但不限于这些实施例。 [0011] The following embodiments with reference to specific embodiments of the present invention is built into ー further described in detail, but are not limited to these embodiments.

[0012] 实施例1 [0012] Example 1

本实施例的PT试剂的制备方法,包括如下步骤: PT reagent prepared according to the present embodiment, comprising the steps of:

(1)、用基因工程的方法在大肠杆菌中表达羊组织因子,制备纯度在90%以上的羊组织因子,将该羊组织因子溶于适量PBS缓冲液中形成Mug/ml的组织因子溶液; (1), the expression of tissue factor sheep, goat prepared purity more than 90% of tissue factor in E. coli by genetic engineering methods, the sheep Tissue Factor Tissue Factor was dissolved Mug / ml of the appropriate amount of PBS buffer is formed;

(2)、取磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸各0. 3g,倒入ー个2L的玻璃烧杯中, 配制曲拉通质量含量为1%、ρΗ值为7. 4、浓度为20mmol的PBS缓冲液800ml,加入上述烧杯中,在15°C〜25°C下轻轻搅拌90分钟后,获得均勻分散的磷脂溶液; (2), taking phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine each 0. 3g, poured into a 2L glass beaker ー, the formulated mass triton content of 1%, ρΗ value of 7.4, the concentration of PBS buffer solution of 20mmol of 800ml, was added to the beaker and stirred gently for 90 minutes at 15 ° C~25 ° C, to obtain a uniform dispersion of a phospholipid solution;

(3)、取步骤(1)制备得到的组织因子溶液5ml (含羊组织因子约270ug)加入步骤(2) 所得的磷脂溶液中,并再用PBS缓冲液定容至900ml,继续轻轻搅拌60分钟,使磷脂与组织因子充分结合,得到磷脂/组织因子溶液; (3), from step (1) prepared in 5ml tissue factor solution obtained (containing tissue factor sheep approximately 270ug) was added in step (2) resulting phospholipid solution and with PBS buffer volume to 900ml, with gentle stirring continued for 60 minutes to fully phospholipid binding to tissue factor, a phospholipid obtained / tissue factor solution;

(4)、向步骤(3)所得的磷脂/组织因子溶液中加入90ml完全溶胀的C18树脂,然后每间隔10分钟,进行一次摇勻操作,摇勻5次后,得到含有沉淀的浑浊液体; (4), the step (3) was added phospholipid / tissue factor solution obtained 90ml of C18 resin fully swollen, and then every 10-minute intervals, for a shake operation, shaken 5 times to obtain a cloudy liquid containing the precipitate;

(5)、对步骤(4)所得的浑浊液体进行离心分离,用尼龙布滤除沉淀,得到上清液,将该上清液分装入IOml的血清瓶中,每瓶1ml,再通过常规技术冻干,即得PT试剂产品。 (5), step (4) the resultant cloudy liquid was centrifuged, the precipitate was filtered off with a nylon cloth to obtain a supernatant, the supernatant was dispensed into the serum bottles IOml bottle 1ml, and then by a conventional freeze-drying technique, that was the PT reagent products.

[0013] 实施例2 [0013] Example 2

本实施例的PT试剂的制备方法,包括如下步骤: PT reagent prepared according to the present embodiment, comprising the steps of:

(1)、用基因工程的方法在大肠杆菌中表达羊组织因子,制备纯度在90%以上的羊组织因子,将该羊组织因子溶于适量PBS缓冲液中形成50ug/ml的组织因子溶液; (1), the expression of tissue factor sheep, goat prepared purity more than 90% of tissue factor in E. coli by genetic engineering methods, the sheep Tissue Factor Tissue Factor was dissolved 50ug / ml of the appropriate amount of PBS buffer is formed;

(2)、取磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸各0. 3g,倒入ー个2L的玻璃烧杯中, 配制曲拉通质量含量为1%、ρΗ值为7. 4、浓度为20mmol的PBS缓冲液800ml,加入上述烧杯中,在15°C〜25°C下轻轻搅拌60分钟后,获得均勻分散的磷脂溶液;(3)、取步骤(1)制备得到的组织因子溶液5ml (含羊组织因子约250ug)加入步骤(2) 所得的磷脂溶液中,并再用PBS缓冲液定容至900ml,继续轻轻搅拌40分钟,使磷脂与组织因子充分结合,得到磷脂/组织因子溶液; (2), taking phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine each 0. 3g, poured into a 2L glass beaker ー, the formulated mass triton content of 1%, ρΗ value of 7.4, the concentration of 800ml 20mmol as PBS buffer, and added to the beaker and stirred at 15 ° C~25 ° C for 60 minutes and gently to obtain a uniform dispersion of the phospholipid solution; to give preparation (3), from step (a) tissue factor 5ml solution (containing about 250 ug tissue factor sheep) added in step (2) resulting phospholipid solution and with PBS buffer volume to 900ml, gentle stirring was continued for 40 minutes to sufficiently bind tissue factor and phospholipid, a phospholipid obtained / tissue factor solution;

(4)、向步骤(3)所得的磷脂/组织因子溶液中加入90ml完全溶胀的Cw树脂,然后每间隔10分钟,进行一次摇勻操作,摇勻5次后,得到含有沉淀的浑浊液体; (4), the step (3) a phospholipid / tissue factor resulting solution was added 90ml Cw resin fully swollen, and then every 10-minute intervals, for a shake operation, after shaking 5 times to give a turbid liquid containing the precipitate;

(5)、对步骤(4)所得的浑浊液体进行离心分离,用尼龙布滤除沉淀,得到上清液,将该上清液分装入IOml的血清瓶中,每瓶1ml,再通过常规技术冻干,即得PT试剂产品。 (5), step (4) the resultant cloudy liquid was centrifuged, the precipitate was filtered off with a nylon cloth to obtain a supernatant, the supernatant was dispensed into the serum bottles IOml bottle 1ml, and then by a conventional freeze-drying technique, that was the PT reagent products.

[0014] 实施例3 [0014] Example 3

本实施例的PT试剂的制备方法,包括如下步骤: PT reagent prepared according to the present embodiment, comprising the steps of:

(1)、用基因工程的方法在大肠杆菌中表达羊组织因子,制备纯度在90%以上的羊组织因子,将该羊组织因子溶于适量PBS缓冲液中形成60ug/ml的组织因子溶液; (1), the expression of tissue factor sheep, goat prepared purity more than 90% of tissue factor in E. coli by genetic engineering methods, the sheep Tissue Factor Tissue Factor was dissolved 60ug / ml of the appropriate amount of PBS buffer is formed;

(2)、取磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸各0. 3g,倒入ー个2L的玻璃烧杯中, 配制曲拉通质量含量为1%、ρΗ值为7. 4、浓度为20mmol的PBS缓冲液800ml,加入上述烧杯中,在15°C〜25°C下轻轻搅拌120分钟后,获得均勻分散的磷脂溶液; (2), taking phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine each 0. 3g, poured into a 2L glass beaker ー, the formulated mass triton content of 1%, ρΗ value of 7.4, the concentration of PBS buffer solution of 20mmol of 800ml, was added to the beaker and gently stirred for 120 minutes at 15 ° C~25 ° C, to obtain a uniform dispersion of a phospholipid solution;

(3)、取步骤(1)制备得到的组织因子溶液5ml (含羊组织因子约300ug)加入步骤(2) 所得的磷脂溶液中,并再用PBS缓冲液定容至900ml,继续轻轻搅拌80分钟,使磷脂与组织因子充分结合,得到磷脂/组织因子溶液; (3), from step (1) prepared in 5ml tissue factor solution obtained (containing about 300 ug tissue factor sheep) added in step (2) resulting phospholipid solution and with PBS buffer volume to 900ml, with gentle stirring continued 80 minutes, sufficient phospholipid binding to tissue factor, a phospholipid obtained / tissue factor solution;

(4)、向步骤(3)所得的磷脂/组织因子溶液中加入30ml完全溶胀的C18树脂,然后每间隔10分钟,进行一次摇勻操作,摇勻5次后,得到含有沉淀的浑浊液体; (4), the step (3) was added phospholipid / tissue factor solution obtained 30ml of C18 resin fully swollen, and then every 10-minute intervals, for a shake operation, shaken 5 times to obtain a cloudy liquid containing the precipitate;

(5)、对步骤(4)所得的浑浊液体进行离心分离,用尼龙布滤除沉淀,得到上清液,将该上清液分装入IOml的血清瓶中,每瓶1ml,再通过常规技术冻干,即得PT试剂产品。 (5), step (4) the resultant cloudy liquid was centrifuged, the precipitate was filtered off with a nylon cloth to obtain a supernatant, the supernatant was dispensed into the serum bottles IOml bottle 1ml, and then by a conventional freeze-drying technique, that was the PT reagent products.

[0015] 上述实施例中通过基因工程的方法制备牛或羊组织因子,可以制备整个分子,即包括胞外区域、脂结合区域和細胞内区域,也可以是整个分子的部分片段如胞外区域等。 [0015] In the embodiment described above by preparing cattle or sheep tissue factor genetic engineering techniques, it can be prepared in the whole molecule, i.e., comprising an extracellular region, lipid binding region and an intracellular region may be a partial fragment of the whole molecule such as an extracellular region Wait. 且基因可以在大肠杆菌中表达,也可以在动物細胞或真菌中表达。 And the gene can be expressed in E. coli, can be expressed in animal cells or fungus. 因此这种方法制备牛或羊组织因子具有容易实现、操作灵活的优点。 Thus this method of preparing cattle or sheep tissue factor is easy to realize, the advantages of flexible operation. 另外,由于牛或羊组织因子的结构明了,选用牛或羊组织因子可望制备出质量更好的PT试剂产品。 Further, since the structure of bovine or sheep clear tissue factor, tissue factor selected cattle or sheep is expected to prepare a better quality product PT reagent.

[0016] 上述实施例制备而得的PT试剂与市售PT试剂相比,敏感性显著提高,ISI值为0. 85 ;稳定性也得到显著改善,冻干品37°C下热稳定时间达5周,预计制品在2°C、で的温度条件下稳定时间可达至少3年。 [0016] The preparation of the above embodiments obtained PT reagent compared to commercially available PT reagent, significantly improve the sensitivity, the ISI value of 0.85; the stability is significantly improved thermal stability at the time of the lyophilized 37 ° C 5 weeks expected product at 2 ° C, a temperature condition で stabilization time up to at least 3 years.

[0017] 以上对本发明做了详尽的描述,其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施,并不能以此限制本发明的保护范围,凡根据本发明的精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围内。 [0017] more than to do a detailed description of the present invention, the purpose is to allow familiar with skilled people who can understand the content of the present invention and implemented, and not in order to limit the scope of the present invention, where the spirit of the invention essence made equivalent variations or modifications shall fall within the scope of the present invention.

Claims (6)

  1. 1. ー种凝血酶原时间测定试剂的制备方法,其特征在于:包括如下步骤:(1)、通过基因工程的方法制备纯度在90%以上的牛或羊组织因子,并将所述组织因子溶于水或磷酸缓冲液中形成组织因子溶液;(2)、将适量磷脂溶于含有表面活性剂的磷酸缓冲液中,搅拌6(Γ120分钟后,获得均勻分散的磷脂溶液;所述磷酸缓冲液的PH值在7. 2〜7. 6之间,表面活性剂的质量含量为1% ;(3)、将步骤(1)制备得到的组织因子溶液加入步骤(2)所得的磷脂溶液中,使磷脂与组织因子的质量比在2. 5Χ IO3^XlO3:1之间,然后继续搅拌4(Γ90分钟,得到磷脂/组织因子溶液;(4)、向步骤(3)所得的磷脂/组织因子溶液中加入体积为所述磷脂/组织因子溶液体积的1/10的完全溶胀的烷基键合硅胶,然后每间隔圹12分钟,进行一次摇勻,摇勻多次后, 得到浑浊液体;(5)、对步骤(4)所得的浑浊液 1. ー species prothrombin time measurement reagent preparation process, characterized by: comprising the steps of: (1), prepared by genetic engineering purity sheep or bovine tissue factor in more than 90% and the tissue factor dissolved in water or a phosphate buffer solution formed tissue factor; (2), the amount of phospholipid was dissolved in a phosphate buffer solution containing a surfactant. after stirring 6 (Γ120 minutes to obtain uniform dispersion of the phospholipid solution; the phosphate buffered . PH value of the solution is between 6 7. 2~7, mass content of the surfactant is 1%; (3), the step (1) preparation of tissue factor obtained was added in step (2) resulting phospholipid solution , tissue factor and phospholipids in a mass ratio of 2. 5Χ IO3 ^ XlO3:, and then stirring was continued for between 1 4 (Γ90 minutes to obtain a phospholipid / tissue factor solution; (3) obtained in (4), the step of phospholipid / tissue volume factor was added to the phospholipid solution volume / tissue factor fully swollen alkyl bonded silica 1/10, then every 12 minutes interval tomb, once shaken, after shaking several times to give the turbid liquid; (5), step (4) resulting cloudy solution 进行离心分离,滤除沉淀,取上清液冻干,即得所述的凝血酶原时间测定试剂。 Centrifuged, the precipitate was filtered off, the supernatant was lyophilized to obtain the prothrombin time-measuring reagent.
  2. 2.根据权利要求1所述的制备方法,其特征在于:所述的表面活性剂为聚乙ニ醇对异辛基苯基醚。 The production method according to claim 1, wherein: the surfactant is polyvinyl alcohol Ni-isooctyl phenyl ether.
  3. 3.根据权利要求1所述的制备方法,其特征在于:步骤(1)和步骤(2)中所述的磷酸缓冲液为PBS缓冲液。 The production method according to claim 1, characterized in that: the (2) in step (1) and the step of phosphate buffer was PBS buffer.
  4. 4.根据权利要求1所述的制备方法,其特征在于:所述磷脂为自然磷脂或合成磷脂或两者的混合物。 4. The production method according to claim 1, wherein: said phospholipid is a mixture of natural or synthetic phospholipids of the phospholipid or both.
  5. 5.根据权利要求4所述的制备方法,其特征在于:所述合成磷脂为磷脂酰胆碱、磷脂酰丝氨酸和磷脂酰乙醇胺中的ー种或多种的混合。 The production method as claimed in claim 4, wherein: said synthetic phospholipids phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine in a mixture of two or more thereof ー.
  6. 6.根据权利要求1所述的制备方法,其特征在于:所述烷基键合硅胶为十八烷基键合硅胶。 6. The production method according to claim 1, wherein: the alkyl bonded silica gel as octadecyl-bonded silica.
CN 201110446209 2011-12-28 2011-12-28 Preparation method for prothrombin time determination reagent CN102565427A (en)

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CN103852362A (en) * 2014-03-13 2014-06-11 皖南医学院 Preparation method for rabbit brain tissue factor freeze-drying preparation
CN105603048A (en) * 2016-01-18 2016-05-25 青岛古高生物科技有限公司 Prothrombin time determining reagent containing myricetin and caffeic acid and preparation method of prothrombin time determining reagent

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US5314695A (en) * 1990-11-13 1994-05-24 Corvas International, Inc. Tissue factor based prothrombin time reagent
US20040086953A1 (en) * 2002-11-05 2004-05-06 Richard Jenny Method for manufacturing a tissue factor-based prothrombin time reagent
CN1496992A (en) * 2001-11-06 2004-05-19 复旦大学 Recombination soluble tissue factor and preparation method and opplication thereof
WO2011148207A1 (en) * 2010-05-28 2011-12-01 Diagon Kft. Procedure for biphasic preparation of liposomes and application thereof in manufacturing diagnostic reagents

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US5314695A (en) * 1990-11-13 1994-05-24 Corvas International, Inc. Tissue factor based prothrombin time reagent
CN1496992A (en) * 2001-11-06 2004-05-19 复旦大学 Recombination soluble tissue factor and preparation method and opplication thereof
US20040086953A1 (en) * 2002-11-05 2004-05-06 Richard Jenny Method for manufacturing a tissue factor-based prothrombin time reagent
CN1523358A (en) * 2002-11-05 2004-08-25 生命扫描有限公司 Method for the preparation of a tissue-factor based reagent for the determination of the prothrombin time
WO2011148207A1 (en) * 2010-05-28 2011-12-01 Diagon Kft. Procedure for biphasic preparation of liposomes and application thereof in manufacturing diagnostic reagents

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103852362A (en) * 2014-03-13 2014-06-11 皖南医学院 Preparation method for rabbit brain tissue factor freeze-drying preparation
CN103852362B (en) * 2014-03-13 2016-01-20 皖南医学院 Lyophilized rabbit brain tissue factor preparation
CN105603048A (en) * 2016-01-18 2016-05-25 青岛古高生物科技有限公司 Prothrombin time determining reagent containing myricetin and caffeic acid and preparation method of prothrombin time determining reagent
CN105603048B (en) * 2016-01-18 2017-05-31 青岛古高生物科技有限公司 Myricetin containing prothrombin time measurement reagent and caffeic acid and preparation method

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