CN111662956A - 一种血栓弹力图法增强型纤溶系统检测试剂盒及其制备方法 - Google Patents
一种血栓弹力图法增强型纤溶系统检测试剂盒及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种血栓弹力图法增强型纤溶系统检测试剂盒,属于检测技术领域。该试剂盒包括以下组分:pH值为7.2~7.4的4‑羟乙基哌嗪乙磺酸缓冲液、高岭土、多聚赖氨酸、甘氨酸、牛血清白蛋白、纤溶酶原激活物、海藻糖、聚蔗糖、PEG8000,脑磷脂、NaCl、KCl、纳豆激酶和Proclin300。试剂盒中包括纤溶酶原激活物,纳豆激酶等,将纤溶检测时间缩短为30min内,能快速区分原发和继发纤溶亢进、早期诊断DIC,满足临床时效性需求。
Description
技术领域
本发明涉及检测技术领域,尤其涉及一种血栓弹力图法增强型纤溶系统检测试剂盒及其制备方法。
背景技术
人体内的纤溶系统与凝血系统和血小板在正常生理情况下存在一种动态的平衡关系,以保障血液循环的稳定和畅通。纤溶系统的功能是清除沉积于血管上的纤维蛋白,溶解血凝块,维持血流通畅,在防止血栓形成和消除已形成的血栓方面发挥作用。
血液凝固过程中形成的纤维蛋白被分解液化的过程,叫纤维蛋白溶解,简称纤溶。纤溶活性异常增强,即纤溶亢进。纤溶亢进又分为原发性纤溶亢进和继发性纤溶亢进,可致出血等严重并发症。
原发性纤溶是由于纤溶酶原激活剂增多导致纤溶酶活性增强,后者降解血浆中纤维蛋白原和多种凝血因子,使它们的血浆水平和活性下降,从而引起皮肤出血如大片淤斑,黏膜内脏出血为特征的临床表现。
继发性纤溶是指继发于血管内凝血的纤溶亢进,主要见于弥散性血管内凝血(DIC)。DIC是指在某些致病因子作用下凝血因子和血小板被激活,大量可溶性促凝物质入血,从而引起一个以凝血功能失常为主要特征的病理过程。在微循环中形成大量微血栓,同时大量消耗凝血因子和血小板,继发性纤溶过程加强,导致出血、休克、器官功能障碍和贫血等临床表现的出现。在DIC已被启动的患者中引起多器官功能障碍综合征将是死亡的主要原因。DIC病死率高达31%~80%,其临床诊断相对困难,不同患者的治疗决策迥异。DIC是炎症与凝血系统互相影响相互作用的复杂病理生理过程。血栓弹力图是1948年由德国科学家Hellmut Hartert发明,1980年代开始广泛用于临床。血栓弹力图能更准确反映凝血的全过程,可以更早诊断高凝及低凝,低凝与脓毒症不良预后密切相关,对指导术中输血取得良好效果,现已成为围术期监测凝血功能最重要的指标。
血栓弹力图与传统凝血检测相比,它使用全血作为检测标本,在体外加入激活剂启动凝血机制,从内、外源凝血系统的启动、纤维蛋白的形成到血凝块溶解进行全程监测,能够更准确、更直观、更全面的反映凝血机制中除血管内皮细胞和血管壁以外的所有凝血因素的综合状态,对临床治疗具有非常明确的指导意义。
血栓弹力图的检测过程(见图1)是装载血标本的测试杯以特定角度在一固有速度下匀速往返转动,一旦血凝块形成,与检测杯中活塞相连的金属扭丝受到血凝块形成的切应力作用,随之出现左右旋动,根据金属扭丝的旋动幅度作图即为血栓弹力图。血栓弹力图能够动态监测整个凝血过程中凝血、纤溶过程及血小板的功能性检测,并指导成份输血。
血栓弹力图主要参数有R、K、α角、MA、LY30、EPL和LY60等参数,其中 LY30、EPL和LY60为测量纤溶的指标,不同的参数代表的意义如下:
LY30:测量在MA值确定后30分钟内血凝分钟块消融(或减少)的速率(%),反应MA值确定后30分钟血液的纤溶活性。LY30﹥7.5%,表示处于高纤溶状态,即纤溶亢进。LY30﹥7.5%时,若CI≤1.0提示原发纤溶亢进,使用抗纤溶药来纠正;若CI≥3.0提示继发纤溶亢进,需抗凝处理。
CI:凝血综合指数,用来描述病人的总体凝血状况,﹤-3:低凝,-3﹤正常﹤+3,﹥+3:高凝。此参数对于血栓和出血的预测具有相当的意义。
EPL:预测在MA值确定后30分钟内血凝块将要溶解的百分比。EPL=100(MA-A30)/MA。
LY60:测量在MA值确定后60分钟内血凝块幅度减少速率,LY60﹥15%,表示处于高纤溶状态,即纤溶亢进。
R值:指血样置入测试杯开始到第一块纤维蛋白凝块形成(描记图幅度达 2mm)所需的时间,反映参加凝血过程(内源性、外源性和共同途径)所有凝血因子的综合作用。R值能因抗凝剂及凝血因子缺乏而延长,因血液呈高凝状态而缩短。
K值:从R时间终点至描记图幅度达20mm所需的时间,反映纤维蛋白和血小板在血凝块开始形成时的共同作用结果,反映血凝块形成的速率。K值的长短主要受纤维蛋白原水平高低的影响,受血小板功能的影响则较小。抗凝剂可使K值延长。
α角:从血凝块形成点至描记图最大曲线弧度作切线与水平线的夹角,α角与K值密切相关,都是反映血凝块聚合的速率。当标本处于重度低凝状态时,血凝血块最大幅度达不到20mm,此时K值无法确定。因此,α角比K值更有价值。影响α角的因素与K值相同。
MA:血栓弹力图的最大振幅,即最大切应力系数(mm)。反映形成血凝块的最大强度及血凝块形成的稳定性,反应了纤维蛋白水平和血小板功能的综合作用。
目前,血栓弹力图法常规测试平均35分钟左右确定MA值,纤溶检测需要 MA值确定后继续测试30min,因此纤溶测试时间至少60min,无法满足临床时效性需求。
发明内容
为了解决现有技术中纤溶测试时间无法满足临床时效性需求的技术问题,本发明提供一种血栓弹力图法增强型纤溶系统检测试剂盒,为此,本发明还提供了该血栓弹力图法增强型纤溶系统检测试剂盒的制备方法,使用该血栓弹力图法增强型纤溶系统检测试剂盒缩短纤溶检测时间为30min内,能够快速区分原发和继发纤溶亢进、早期诊断DIC,满足临床时效性需求。
为了实现上述目的,本发明采用了如下技术方案:
本发明的一个目的是提供一种血栓弹力图法增强型纤溶系统检测试剂盒,包括如下组分:5~100mmol/L且pH值为7.2~7.4的HEPES缓冲液、0.1~0.6g/L 的高岭土、5~30g/L的多聚赖氨酸、10~60g/L的甘氨酸、5~30g/L的BSA、 5IU~1000IU/mL纤溶酶原激活物、5~30g/L的海藻糖、1~12g/L的聚蔗糖、1~ 20g/L的PEG8000,10~60mg/L的脑磷脂、6-12g/L NaCl、4~8g/L KCl、10~ 1000IU/mL纳豆激酶、0.03%~0.05%的Proclin300。
优选地,所述纤溶酶原激活物的活性为5000IU/g;所述纳豆激酶的活性为40000IU/g。
优选地,包括如下组分:10mmol/L且pH值为7.2的HEPES缓冲液、0.5g/L 的高岭土、20g/L的多聚赖氨酸、40g/L的甘氨酸、20g/L的BSA、50IU/mL的纤溶酶原激活物、20g/L的海藻糖、8g/L的聚蔗糖、10g/LPEG8000,50mg/L的脑磷脂、9g/L的NaCl、6g/L的KCl、800IU/mL的纳豆激酶、0.05%的Proclin300。
优选地,包括如下组分:50mmol/L且pH值为7.3的HEPES缓冲液、0.25g/L 的高岭土、30g/L的多聚赖氨酸、20g/L的甘氨酸、30g/L的BSA、250IU/mL的纤溶酶原激活物、10g/L的海藻糖、12g/L的聚蔗糖、20g/LPEG8000,25mg/L 的脑磷脂、6g/L的NaCl、8g/L的KCl、400IU/mL的纳豆激酶、0.04%的Proclin300。
优选地,包括如下组分:100mmol/L且pH值为7.4的HEPES缓冲液、0.1g/L 的高岭土、10g/L的多聚赖氨酸、60g/L的甘氨酸、5g/L的BSA、800IU/mL的纤溶酶原激活物、30g/L的海藻糖、2g/L的聚蔗糖、5g/L的PEG8000,10mg/L 的脑磷脂、12g/L的NaCl、4g/L的KCl、100IU/mL的纳豆激酶、0.03%的 Proclin300。
本发明的另一个目的在于提供一种上述血栓弹力图法增强型纤溶系统检测试剂盒的制备方法,包括如下步骤:
S1,将4-羟乙基哌嗪乙磺酸加入纯化水中配置成4-羟乙基哌嗪乙磺酸缓冲液,并将4-羟乙基哌嗪乙磺酸缓冲液的pH调节为7.2~7.4;
S2,取高岭土加入纯化水中,配制高岭土储备液;
S3,取S1制备的4-羟乙基哌嗪乙磺酸缓冲液加入脑凝脂中,研磨至乳状,制备成脑凝脂储备液;
S4,取纯化水加入纤溶酶原激活物冻干粉中复溶,配置成纤溶酶原激活物储备液;
S5,取多聚赖氨酸、甘氨酸、PEG8000、聚蔗糖、纳豆激酶、海藻糖、牛血清白蛋白、NaCl和KCl,加入S1制备的4-羟乙基哌嗪乙磺酸缓冲液,配制成溶液;
S6,取S2中配制的高岭土储备液、S3中配制的脑凝脂储备液、Proclin300 和S4中配制的纤溶酶原激活物储备液加入到S5配制的溶液中,加入S1中配制的4-羟乙基哌嗪乙磺酸缓冲液,混合均匀,冻干即得血栓弹力图法增强型纤溶系统检测试剂盒。
优选地,还包括分装步骤,每试剂瓶分装20ul,冻干即得血栓弹力图法增强型纤溶系统检测试剂盒。
优选地,所述S1中配制的4-羟乙基哌嗪乙磺酸缓冲液的浓度为10mmol/L, pH值为7.2;所述S2中配制的高岭土储备液的浓度为5g/L;所述S3中配制的脑凝脂储备液的浓度为1g/L;所述S4中配制的纤溶酶原激活物储备液的浓度为5000IU/mL;所述S5中配制的溶液中称取0.2g多聚赖氨酸,0.4g甘氨酸, 0.1gPEG8000,0.08g聚蔗糖,0.2g活性为40000IU/g纳豆激酶,0.2g海藻糖, 0.2gBSA,0.09gNaCl,0.06gKCl;所述S6中配制的溶液10mL中高岭土0.5g/L, 脑凝脂50mg/L,纤溶酶原激活物50IU/ml,Proclin 0.05%,多聚赖氨酸20g/L,甘氨酸40g/L,PEG800010g/L,聚蔗糖8g/L,纳豆激酶800IU/mL,海藻糖20g/L,BSA 20g/L,NaCl9g/L,KCl 6g/L。
优选地,所述S1中配制的4-羟乙基哌嗪乙磺酸缓冲液的浓度为50mmol/L, pH值为7.3;所述S2中配制的高岭土储备液的浓度为5g/L;所述S3中配制的脑凝脂储备液的浓度为1g/L;所述S4中配制的纤溶酶原激活物储备液的浓度为5000IU/mL;所述S5中配制的溶液中称取0.3g多聚赖氨酸,0.2g甘氨酸, 0.2gPEG8000,0.12g聚蔗糖,0.1g活性为40000IU/g纳豆激酶,0.1g海藻糖, 0.3g BSA,0.06gNaCl,0.08gKCl;所述S6中配制的溶液10mL中高岭土0.25g/L, 脑凝脂25mg/L,纤溶酶原激活物250IU/ml,Proclin 0.04%,多聚赖氨酸30g/L,甘氨酸20g/L,PEG800020g/L,聚蔗糖12g/L,纳豆激酶400IU/mL,海藻糖 10g/L,BSA 30g/L,NaCl 6g/L,KCl 8g/L。
优选地,所述S1中配制的4-羟乙基哌嗪乙磺酸缓冲液的浓度为100mmol/L, pH值为7.4;所述S2中配制的高岭土储备液的浓度为5g/L;所述S3中配制的脑凝脂储备液的浓度为1g/L;所述S4中配制的纤溶酶原激活物储备液的浓度为5000IU/mL;所述S5中配制的溶液中称取0.1g多聚赖氨酸,0.6g甘氨酸, 0.05gPEG8000,0.02g聚蔗糖,0.025g活性为40000IU/g纳豆激酶,0.3g海藻糖,0.05g BSA,0.12gNaCl,0.04gKCl;所述S6中配制的溶液10mL中高岭土 0.1g/L,脑凝脂10mg/L,纤溶酶原激活物800IU/ml,Proclin 0.03%,多聚赖氨酸10g/L,甘氨酸60g/L,PEG80005g/L,聚蔗糖2g/L,纳豆激酶100IU/mL,海藻糖 30g/L,BSA 5g/L,NaCl 12g/L,KCl 4g/L。
本发明提供的一种血栓弹力图法增强型纤溶系统检测试剂盒,试剂盒中包括纤溶酶原激活物,纳豆激酶等,缩短纤溶检测时间为30min内,能够快速区分原发和继发纤溶亢进、早期诊断DIC,满足临床时效性需求。
本发明提供的一种血栓弹力图法增强型纤溶系统检测试剂盒的制备方法中,加入了纤溶酶原激活物和纳豆激酶,纤溶过程包括两部分,即纤溶酶原的激活及纤维蛋白或纤维蛋白原的降解。纤溶酶原有内源性及外源性两条激活途径。纤溶酶原激活物属于内源性激活物,纳豆激酶属于内源性和外源性激活物,二者相互协同作用,共同缩短纤溶检测时间为30min内。
附图说明
图1.血栓弹力图示意图(左:检测原理;右:结果图谱解析)
图2.曲线1为传统纤溶检测,曲线2为本发明的纤溶检测
图3.实施例1-3级对比例1-3的检测效果图。
具体实施方式
下面结合具体实施例,对本发明提供的技术方案做进一步详细说明;
需要说明的是,以下实施例所涉及的高岭土、多聚赖氨酸、甘氨酸、牛血清白蛋白、纤溶酶原激活物、海藻糖聚蔗糖、PEG8000,脑磷脂、NaCl、KCl、纳豆激酶、Proclin300及4-羟乙基哌嗪乙磺酸以及其他试剂皆市售可得。
HEPES为4-羟乙基哌嗪乙磺酸;BSA为牛血清白蛋白;Proclin300为市售的防腐剂;PEG8000为聚乙二醇;Kaolin为高岭土。
实施例1
S1,称取0.239g 4-羟乙基哌嗪乙磺酸,0.040g氢氧化钠加入纯化水中,配置10mmol/L的HEPES缓冲液100mL,测定pH值为7.2;
S2,称取0.05g高岭土加入10ml纯化水中,配制高岭土储备液5g/L;
S3,称取0.001g脑凝脂加入1ml HEPES溶液,研磨至乳状,肉眼观察无明显颗粒,储备液浓度为1g/L;
S4,活性为5000IU/g的纤溶酶原激活物冻干粉中加1mL纯化水复溶,储备液浓度为5000IU/mL;
S5,称取0.2g多聚赖氨酸,0.4g甘氨酸,0.1gPEG8000,0.08g聚蔗糖, 0.2g活性为40000IU/g的纳豆激酶,0.2g海藻糖,0.2g BSA,0.09gNaCl, 0.06gKCl,加入适量HEPES缓冲液;
S6,量取1mLS2中配制的Kaolin储备液,0.5mLS3中配制的脑磷脂储备液, 0.005mLProclin300,0.1mL S4中配制的纤溶酶原激活物储备液加入S5配制的溶液中,加入适量HEPES缓冲液,配制10mL溶液,混合均匀;
S7,每试剂瓶20ul分装,冻干即得。
实施例2
S1,称取1.197g 4-羟乙基哌嗪乙磺酸,0.20g氢氧化钠加入纯化水中,配置50mmol/L的HEPES缓冲液100mL,测定pH值为7.3;
S2,称取0.05g高岭土加入10ml纯化水中,配制高岭土储备液5g/L;
S3,称取0.001g脑凝脂加入1ml HEPES溶液,研磨至乳状,肉眼观察无明显颗粒,储备液浓度为1g/L;
S4,活性为5000IU/g的纤溶酶原激活物冻干粉中加1mL纯化水复溶,储备液浓度为5000IU/mL;
S5,称取0.3g多聚赖氨酸,0.2g甘氨酸,0.2gPEG8000,0.12g聚蔗糖, 0.1g活性为40000IU/g的纳豆激酶,0.1g海藻糖,0.3g BSA,0.06gNaCl,0.08gKCl,加入适量HEPES缓冲液;
S6,量取0.5mLS2中配制的Kaolin储备液,0.25mLS3中配制的脑磷脂储备液,0.004mL Proclin300,0.5mL S4中配制的纤溶酶原激活物储备液加入S5 配制的溶液中,加入适量HEPES缓冲液,配制10mL溶液,混合均匀;
S7,每试剂瓶20ul分装,冻干即得。
实施例3
S1,称取2.395g 4-羟乙基哌嗪乙磺酸(HEPES),0.40g氢氧化钠加入纯化水中,配置100mmol/LHEPES缓冲液100mL,测定pH值为7.4;
S2,称取0.05g高岭土加入10ml纯化水中,配制高岭土储备液5g/L;
S3,称取0.001g脑凝脂加入1ml HEPES溶液,研磨至乳状,肉眼观察无明显颗粒,储备液浓度为1g/L;
S4,活性为5000IU/g的纤溶酶原激活物冻干粉中各加1mL纯化水复溶,储备液浓度为5000IU/mL;
S5,称取0.1g多聚赖氨酸,0.6g甘氨酸,0.05gPEG8000,0.02g聚蔗糖, 0.025g活性为40000IU/g的纳豆激酶,0.3g海藻糖,0.05g BSA,0.12gNaCl, 0.04gKCl,加入适量HEPES缓冲液;
S6,量取0.2mLS2中配制的Kaolin储备液,0.1mLS3中配制的脑磷脂储备液,0.003mL Proclin300,1.6mL S4中配制的纤溶酶原激活物储备液加入S5 配制的溶液中,加入适量HEPES缓冲液,配制10mL溶液,混合均匀;
S7,每试剂瓶20ul分装,冻干即得。
对比例1
与实施例1的相同,不同之处在于,对比例1中不加入纤溶酶原激活物和纳豆激酶。
S1,称取0.239g 4-羟乙基哌嗪乙磺酸,0.040g氢氧化钠加入纯化水中,配置10mmol/L的HEPES缓冲液100mL,测定pH值为7.2;
S2,称取0.05g高岭土加入10ml纯化水中,配制高岭土储备液5g/L;
S3,称取0.001g脑凝脂加入1ml HEPES溶液,研磨至乳状,肉眼观察无明显颗粒,储备液浓度为1g/L;
S4,称取0.2g多聚赖氨酸,0.4g甘氨酸,0.1gPEG8000,0.08g聚蔗糖, 0.2g海藻糖,0.2g BSA,0.09gNaCl,0.06gKCl,加入适量HEPES缓冲液;
S5,量取1mLS2中配制的Kaolin储备液,0.5mLS3中配制的脑磷脂储备液, 0.005mLProclin300,加入适量HEPES缓冲液,配制10mL溶液,混合均匀;
S6,每试剂瓶20ul分装,冻干即得。
对比例2
与实施例1的相同,不同之处在于,对比例2中不加入活性为40000IU/g 的纳豆激酶。
S1,称取0.239g 4-羟乙基哌嗪乙磺酸,0.040g氢氧化钠加入纯化水中,配置10mmol/L的HEPES缓冲液100mL,测定pH值为7.2;
S2,称取0.05g高岭土加入10ml纯化水中,配制高岭土储备液5g/L;
S3,称取0.001g脑凝脂加入1ml HEPES溶液,研磨至乳状,肉眼观察无明显颗粒,储备液浓度为1g/L;
S4,活性为5000IU/g的纤溶酶原激活物冻干粉中加1mL纯化水复溶,储备液浓度为5000IU/mL;
S5,称取0.2g多聚赖氨酸,0.4g甘氨酸,0.1gPEG8000,0.08g聚蔗糖, 0.2g海藻糖,0.2g BSA,0.09gNaCl,0.06gKCl,加入适量HEPES缓冲液;
S6,量取1mLS2中配制的Kaolin储备液,0.5mLS3中配制的脑磷脂储备液,0.005mLProclin300,0.1mL S4中配制的纤溶酶原激活物储备液加入S5配制的溶液中,加入适量HEPES缓冲液,配制10mL溶液,混合均匀;
S7,每试剂瓶20ul分装,冻干即得。
对比例3
与实施例1的相同,不同之处在于,对比例3中不加入活性为5000IU/g的纤溶酶原激活物。
S1,称取0.239g 4-羟乙基哌嗪乙磺酸,0.040g氢氧化钠加入纯化水中,配置10mmol/L的HEPES缓冲液100mL,测定pH值为7.2;
S2,称取0.05g高岭土加入10ml纯化水中,配制高岭土储备液5g/L;
S3,称取0.001g脑凝脂加入1ml HEPES溶液,研磨至乳状,肉眼观察无明显颗粒,储备液浓度为1g/L;
S4,称取0.2g多聚赖氨酸,0.4g甘氨酸,0.1gPEG8000,0.08g聚蔗糖, 0.2g活性为40000IU/g的纳豆激酶,0.2g海藻糖,0.2g BSA,0.09gNaCl, 0.06gKCl,加入适量HEPES缓冲液;
S5,量取1mLS2中配制的Kaolin储备液,0.5mLS3中配制的脑磷脂储备液, 0.005mLProclin300,加入适量HEPES缓冲液,配制10mL溶液,混合均匀;
S6,每试剂瓶20ul分装,冻干即得。
本发明还提供了用于血栓弹力图法增强型纤溶系统检测试剂盒的检测方法用于对实施例1-3及对比例1-3制得的试剂盒进行检测。检测方法包括如下步骤:
S8,从2-8℃取出冻干后的纤溶试剂,室温平衡15分钟;
S9,加入20ul纯化水复溶,缓慢摇匀;
S10,打开弹力图仪,进入程序、标准、备样操作。
S11,装载空白测试杯;
S12,吸取20ul 0.2mol/LCaCl2加入测试杯中;
S13,吸取10ul复溶后试剂加入测试杯中;
S14,吸取340ul枸橼酸抗凝全血;
S15,将恒温槽推上去并将检测杆推到检测位置;
S16,单击软件工具栏上的开始按钮开始检测;
S17,完成测试后,可得到R,K,Angle,MA,LY30和EPL值;
传统的纤溶检测时间平均35分钟左右确定MA值,纤溶检测需要MA值确定后继续测试30min,因此纤溶测试时间至少60min。
如图3所示,图的横轴是时间,对比例1是传统纤溶检测方法所需时间至少需要60min;对比例2为只加入纤溶酶原激活物,对比例3是只加入纳豆激酶,因此对比例2和对比例3检测纤溶所需时间比传统检测时间稍短,但是仍需要较长时间;实施例1-3的纤溶检测时间相比于对比例1传统检测方法的检测时间缩短了一半以上,实施例1-3的纤溶检测时间相比于对比例2和对比例 3大幅度缩短了纤溶的检测时间。
Claims (10)
1.一种血栓弹力图法增强型纤溶系统检测试剂盒,其特征在于,包括如下组分:5~100mmol/L且pH值为7.2~7.4的HEPES缓冲液、0.1~0.6g/L的高岭土、5~30g/L的多聚赖氨酸、10~60g/L的甘氨酸、5~30g/L的BSA、5IU~1000IU/mL纤溶酶原激活物、5~30g/L的海藻糖、1~12g/L的聚蔗糖、1~20g/L的PEG8000,10~60mg/L的脑磷脂、6-12g/L NaCl、4~8g/L KCl、10~1000IU/mL纳豆激酶、0.03%~0.05%的Proclin300。
2.根据权利要求1所述的血栓弹力图法增强型纤溶系统检测试剂盒,其特征在于,所述纤溶酶原激活物的活性为5000IU/g;所述纳豆激酶的活性为40000IU/g。
3.根据权利要求1所述的血栓弹力图法增强型纤溶系统检测试剂盒,其特征在于,包括如下组分:10mmol/L且pH值为7.2的HEPES缓冲液、0.5g/L的高岭土、20g/L的多聚赖氨酸、40g/L的甘氨酸、20g/L的BSA、50IU/mL的纤溶酶原激活物、20g/L的海藻糖、8g/L的聚蔗糖、10g/LPEG8000,50mg/L的脑磷脂、9g/L的NaCl、6g/L的KCl、800IU/mL的纳豆激酶、0.05%的Proclin300。
4.根据权利要求1所述的血栓弹力图法增强型纤溶系统检测试剂盒,其特征在于,包括如下组分:50mmol/L且pH值为7.3的HEPES缓冲液、0.25g/L的高岭土、30g/L的多聚赖氨酸、20g/L的甘氨酸、30g/L的BSA、250IU/mL的纤溶酶原激活物、10g/L的海藻糖、12g/L的聚蔗糖、20g/LPEG8000,25mg/L的脑磷脂、6g/L的NaCl、8g/L的KCl、400IU/mL的纳豆激酶、0.04%的Proclin300。
5.根据权利要求1所述的血栓弹力图法增强型纤溶系统检测试剂盒,其特征在于,包括如下组分:100mmol/L且pH值为7.4的HEPES缓冲液、0.1g/L的高岭土、10g/L的多聚赖氨酸、60g/L的甘氨酸、5g/L的BSA、800IU/mL的纤溶酶原激活物、30g/L的海藻糖、2g/L的聚蔗糖、5g/L的PEG8000,10mg/L的脑磷脂、12g/L的NaCl、4g/L的KCl、100IU/mL的纳豆激酶、0.03%的Proclin300。
6.一种权利要求1-5中任一项所述的血栓弹力图法增强型纤溶系统检测试剂盒的制备方法,其特征在于,包括如下步骤:
S1,将4-羟乙基哌嗪乙磺酸加入纯化水中配置成4-羟乙基哌嗪乙磺酸缓冲液,并将4-羟乙基哌嗪乙磺酸缓冲液的pH调节为7.2~7.4;
S2,取高岭土加入纯化水中,配制高岭土储备液;
S3,取S1制备的4-羟乙基哌嗪乙磺酸缓冲液加入脑凝脂中,研磨至乳状,制备成脑凝脂储备液;
S4,取纯化水加入纤溶酶原激活物冻干粉中复溶,配置成纤溶酶原激活物储备液;
S5,取多聚赖氨酸、甘氨酸、PEG8000、聚蔗糖、纳豆激酶、海藻糖、牛血清白蛋白、NaCl和KCl,加入S1制备的4-羟乙基哌嗪乙磺酸缓冲液,配制成溶液;
S6,取S2中配制的高岭土储备液、S3中配制的脑凝脂储备液、Proclin300和S4中配制的纤溶酶原激活物储备液加入到S5配制的溶液中,加入S1中配制的4-羟乙基哌嗪乙磺酸缓冲液,混合均匀,冻干即得血栓弹力图法增强型纤溶系统检测试剂盒。
7.根据权利要求6所述的血栓弹力图法增强型纤溶系统检测试剂盒的制备方法,其特征在于,还包括分装步骤,每试剂瓶分装20ul,冻干即得血栓弹力图法增强型纤溶系统检测试剂盒。
8.根据权利要求6所述的血栓弹力图法增强型纤溶系统检测试剂盒的制备方法,其特征在于,所述S1中配制的4-羟乙基哌嗪乙磺酸缓冲液的浓度为10mmol/L,pH值为7.2;所述S2中配制的高岭土储备液的浓度为5g/L;所述S3中配制的脑凝脂储备液的浓度为1g/L;所述S4中配制的纤溶酶原激活物储备液的浓度为5000IU/mL;所述S5中配制的溶液中称取0.2g多聚赖氨酸,0.4g甘氨酸,0.1gPEG8000,0.08g聚蔗糖,0.2g活性为40000IU/g纳豆激酶,0.2g海藻糖,0.2g BSA,0.09gNaCl,0.06gKCl;所述S6中配制的溶液10mL中高岭土0.5g/L,脑凝脂50mg/L,纤溶酶原激活物50IU/ml,Proclin 0.05%,多聚赖氨酸20g/L,甘氨酸40g/L,PEG800010g/L,聚蔗糖8g/L,纳豆激酶800IU/mL,海藻糖20g/L,BSA 20g/L,NaCl 9g/L,KCl 6g/L。
9.根据权利要求7所述的血栓弹力图法增强型纤溶系统检测试剂盒的制备方法,其特征在于,所述S1中配制的4-羟乙基哌嗪乙磺酸缓冲液的浓度为50mmol/L,pH值为7.3;所述S2中配制的高岭土储备液的浓度为5g/L;所述S3中配制的脑凝脂储备液的浓度为1g/L;所述S4中配制的纤溶酶原激活物储备液的浓度为5000IU/mL;所述S5中配制的溶液中称取0.3g多聚赖氨酸,0.2g甘氨酸,0.2gPEG8000,0.12g聚蔗糖,0.1g活性为40000IU/g纳豆激酶,0.1g海藻糖,0.3g BSA,0.06gNaCl,0.08gKCl;所述S6中配制的溶液10mL中高岭土0.25g/L,脑凝脂25mg/L,纤溶酶原激活物250IU/ml,Proclin 0.04%,多聚赖氨酸30g/L,甘氨酸20g/L,PEG800020g/L,聚蔗糖12g/L,纳豆激酶400IU/mL,海藻糖10g/L,BSA 30g/L,NaCl 6g/L,KCl 8g/L。
10.根据权利要求7所述的血栓弹力图法增强型纤溶系统检测试剂盒的制备方法,其特征在于,所述S1中配制的4-羟乙基哌嗪乙磺酸缓冲液的浓度为100mmol/L,pH值为7.4;所述S2中配制的高岭土储备液的浓度为5g/L;所述S3中配制的脑凝脂储备液的浓度为1g/L;所述S4中配制的纤溶酶原激活物储备液的浓度为5000IU/mL;所述S5中配制的溶液中称取0.1g多聚赖氨酸,0.6g甘氨酸,0.05gPEG8000,0.02g聚蔗糖,0.025g活性为40000IU/g纳豆激酶,0.3g海藻糖,0.05g BSA,0.12gNaCl,0.04gKCl;所述S6中配制的溶液10mL中高岭土0.1g/L,脑凝脂10mg/L,纤溶酶原激活物800IU/ml,Proclin 0.03%,多聚赖氨酸10g/L,甘氨酸60g/L,PEG80005g/L,聚蔗糖2g/L,纳豆激酶100IU/mL,海藻糖30g/L,BSA 5g/L,NaCl12g/L,KCl 4g/L。
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