CN103728453B - 一种tafi含量的体外检测试剂盒及其检测方法 - Google Patents
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Abstract
本发明涉及一种TAFI含量的体外检测试剂盒及其检测方法。所述该TAFI含量的体外检测试剂盒组成包括：含有与TAFI特异性结合的单克隆抗体纳米金标记物R试剂；用于制作计算待检样品中的TAFI抗原含量的标准曲线的TAFI标准品和TAFI质控品；体外检测试剂盒的体外检测方法，利用纳米金与抗体结合的生物特性，与传统生化检测方法相结合，将其用于TAFI含量的体外检测，开辟了TAFI新的体外诊断学方法，提高了TAFI体外检测的灵敏度。它解决了现有技术存在的设备昂贵，提高相关疾病的检出率和准确率，减少被检者不必要的痛苦和医疗费用支出，提高被检者的生存质量。
Description
技术领域
本发明涉及一种用于临床预测心脑血管疾病等发生可能性的检测凝血酶激活的纤溶抑制物（thrombin-activatablefibrinolysisinhibitor，简称TAFI）含量的新方法，特别是一种TAFI含量的体外检测试剂盒及其检测方法。
背景技术
众所周知，血栓性疾病是一类严重影响健康的疾病，尤其是心脑血管栓塞性疾病已成为我国人口死亡的首位原因。由于血栓性疾病的临床表现千差万别，诊断和治疗往往非常复杂，因此，早期发现、早期治疗尤为重要。目前，血栓性疾病的检查方法主要有心电图、超声心动图、胸部X射线、血压监测、头颅X射线、脑CT扫描、脑MRI检查、DSA、MRA、经颅多普勒超声检查等。射线类检查会对人体产生一定程度的电离辐射，组织血流灌注的功能性成像检查方法较多，这些检查设备通常比较昂贵，增加了成本，有的还需特殊装备，成像时间较长，无法短期获得结果。在医学检验中，尚缺乏一种简单、快捷、准确的用于临床预测心脑血管疾病等发生可能性的检测方法，不利于对疾病的早期发现，早期治疗。
据相关文献报导，TAFI具有被凝血酶激活后下调纤溶活性的功能，是一种存在于血浆中的含金属Zn的羧肽酶前体，又被称为血浆羧肽酶原B、血浆羧肽酶原U、血浆羧肽酶原R。TAFI前体由肝脏合成，是一条包含423个氨基酸的单链糖蛋白，在循环至血液中的过程中，切掉N端22个氨基酸的信号肽，形成401个氨基酸的56kDa的TAFI；TAFI也存在于血小板内，血小板激活时释放至血液。凝血酶、胰蛋白酶、纤溶酶等蛋白水解酶可以作用于TAFI的Arg92，将其切割成20kDa的激活肽和具有羧肽酶活性的36kDa的TAFIa。TAFIa具有切除部分降解的纤维蛋白C端Arg或Lys的能力，使纤溶蛋白酶原不能被激活，因而具有下调纤溶系统的功能，但其构象具有高度不稳定性。
TAFI于1988年被首次发现，活化后具有内在不稳定性，通常在37℃条件下孵育2h失去活性，并且能特异性地分解底物上精氨酸。后经多位科技工作者对其cDNA进行分离以及测定，并利用纤溶酶原-琼脂糖柱层析的方法从血浆中分离、提纯其酶原蛋白pro-pCPB，进一步分析发现TAFI通过凝血酶激活后具有羧肽酶活性，发挥纤溶抑制作用。
TAFI与心脑血管疾病有一定关系。动脉粥样硬化（atherosclerosis，简称AS）是缺血性心脑血管疾病（如心绞痛、心肌梗死、脑卒中等）共同的病理基础，也是导致血管内血栓形成的主要原因。近几年的研究表明，AS的发生发展与炎症、凝血-纤溶调节系统密切相关。目前研究表明，TAFI通过降低纤溶活性可促进静脉血栓的形成，血浆高TAFI水平能增加静脉血栓事件。
目前，用来检测TAFI水平的方法主要有两类，一类是通过测定酶活力的方法间接测定TAFI含量，即利用凝血酶和凝血酶调节蛋白激活TAFI，测定TAFIa的酶活力；另一类是免疫学方法，包括酶联免疫吸附剂测定（enzymelinkedimmunosorbentassay，ELISA）和火箭免疫电泳。其中，夹心ELISA法检测TAFI抗原是目前相对成熟的检测方法，检测灵敏度高，特异性强，准确性好，检测方法安全易行。2005年出现了两种针对TAFI的特异性单克隆抗体，成功运用ELISA方法检测TAFI、TAFIa和失活的TAFI。但这些方法因自身存在一定的缺陷，都没有成为国际公认的TAFI参照标准法。
发明内容
本发明的目的是提供一种TAFI含量的体外检测方法，它解决了现有技术存在的设备昂贵，检测时间较长等缺陷，与其它检测方法相比，具有操作简便、快捷、成本低廉，诊断敏感，检出限低等优点，作为用于临床预测心脑血管疾病的辅助诊断，可显著提高疾病的检出率和准确率，有利于疾病的早期发现，早期治疗；减少被检者不必要的痛苦和医疗费用支出，提高被检者的生存质量。
一种TAFI含量的体外检测试剂盒，所述该TAFI含量的体外检测试剂盒组成包括：含有与TAFI特异性结合的单克隆抗体纳米金标记物R试剂；用于制作计算待检样品中的TAFI抗原含量的标准曲线的TAFI标准品和TAFI质控品，用于解释试剂盒使用方法的说明书。
所述纳米金标记物R试剂是针对重组人TAFI抗原所制备的鼠抗人单克隆抗体经纳米金标记所得，鼠抗人单克隆抗体包含3B1和4E7，两种鼠抗人单克隆抗体纳米金结合物的混合比例为1:2。
所述3B1为特异性结合TAFI和失活的TAFI，所述4E7仅与TAFI结合。
所述生化仪设置为单试剂检测时，纳米金标记物R试剂的组分是偶联了3B1和4E7两种抗体的纳米金及其保存液。所述生化仪设置为双试剂检测时，纳米金标记物R试剂的组分包含R1、R2试剂，R1试剂是单4E7偶联的纳米金及其保存液，R2试剂是单3B1偶联的纳米金及其保存液。
所述保存液是由Tris0-0.5%；pvp400-1%；BSA0-1%；PEG200000-0.5%；10%蔗糖0-5%；TWEEN200-0.8%；叠氮钠0-0.02‰，NaCl0.85-1.5%.PEG60001-4%,余量为水组成。
本发明所采用的技术方案的具体操作步骤如下：
步骤一采集人个体生物样品立即于30分钟内，4℃条件下离心取上清作为待检样品，置于冰盒1小时内使用或-20℃保存备用；
步骤二利用体外检测试剂盒，对待检样品进行TAFI含量测定；
所述该TAFI含量的体外检测试剂盒组成包括：含有与TAFI特异性结合的单克隆抗体纳米金标记物R试剂；用于制作计算待检样品中的TAFI抗原含量的标准曲线的TAFI标准品和TAFI质控品；
步骤三取出试剂R放置室温下至温度平衡；
步骤四将R试剂和TAFI标准品放在生化分析仪相应的位置，制定标准曲线；
步骤五检测TAFI质控品，并根据线性回归，确定检测结果有效性；
步骤六检测TAFI样本，设置加入R试剂100-400ul，加入样本10ul，第5点作为第一点检测，第45点为第二点检测，以两点差值为检测值，通过标准曲线回归得出最终结果。
步骤一所述样本直接用于检测，无需任何处理。
步骤四所述标准品以标化TAFI作为标准品，稀释成梯度浓度为0ug/ml、10ug/ml、20ug/ml、30ug/ml、40ug/ml做标准曲线，ELISA方法测定人混合血浆浓度，根据测定结果，利用该血浆及100mMpH7.3的乙二胺四乙酸二钠溶液、100mM壳聚糖溶液将购买的TAFI抗原稀释并充分混匀，制成终浓度为含TAFI、乙二胺四乙酸二钠5mM和壳聚糖20mM的溶液，取如上所述梯度浓度的各浓度标准品溶液100μl分装至灭菌过的冻存管底部，液体或冷冻干燥后4℃保存。
所述TAFI含量的体外检测方法的应用：其技术要点是：用于检测人体生物样品中的血液、血清、血浆、尿液、粘液、粪便、脑脊液、胸腔积液、腹水、唾液、组织或细胞。
所述TAFI含量的体外检测方法的应用：其技术要点是：将对人个体样品中TAFI含量的测定，根据TAFI与相关疾病的关系，应用于辅助心脑血管疾病、急性早幼粒细胞白血病、内源性凝血系统缺陷疾病、炎症等的与TAFI含量相关联疾病的诊断。
本发明具有的优点及积极效果是：由于本发明所采用的体外检测试剂盒的内容物中包括针对TAFI的特异性单克隆抗体和纳米金标记测定所需试剂，与新型纳米金免疫检测方法相结合，用于检测人体生物样品的TAFI含量的体外检测，开辟了TAFI新的体外诊断学方法，所以它解决了现有技术存在的操作繁琐，检测时间较长等缺陷。将这种体外诊断学方法用于临床预测心脑血管疾病的可能性，其操作简便、快捷、成本低廉，诊断敏感，检出限低。样品中的TAFI抗原与标记了纳米金的特异抗体进行免疫学反应，通过纳米金颜色变化的放大作用，在700nm波长下检测吸光值，吸光值与抗原浓度呈正比关系，从而达到定量测定抗原含量的目的。将TAFI检测和纳米金技术相结合可以明显提高检测的灵敏度和特异性，这种体外诊断学方法优于现有技术中采用的其它普通方法。因此，本发明的方法可应用于大量样品中TAFI含量的体外测定，根据TAFI与相关疾病的关系，作为辅助诊断，提高相关疾病的检出率和准确率，同时检测时间很短，达到了早期发现，早期治疗的目的；减少被检者不必要的痛苦和医疗费用支出，提高被检者的生存质量。
本发明所用的体外检测试剂盒与人纤溶抑制因子/凝血酶激活的纤溶抑制物（TAFI）酶联免疫分析试剂盒进行比较实验。实验材料为已确诊的心脑梗死患者血浆60例，使用TAFI酶联免疫分析试剂盒和本发明所用的生化检测试剂盒同时进行酶联免疫测定，本发明所用的生化仪检测试剂盒与TAFI酶联免疫分析试剂盒测定的阳性率分别为40.2%和20.6%，同时，本发明检测TAFI用生化试剂盒，检测时间仅为10-15min，节约大量的时间，该结果显示，本发明方法对TAFI抗原的检测特异性更高，更准确，速度更快。
附图说明
图1是TAFI生化检测产品标准曲线；
图2是TAFI生化检测示意图。
具体实施方式
本发明是采用体外检测试剂盒的内容物中的针对TAFI的特异性单克隆抗体和纳米金法测定所需试剂，与现有的纳米金法检测试剂相结合，用于检测人体生物样品的TAFI含量的体外检测。该TAFI含量的体外检测试剂盒组成包括：含有与TAFI特异性结合的单克隆体纳米金标记物R试剂；用于制作计算待检样品中的TAFI抗原含量的标准曲线的TAFI标准品，1套（共5只，0、10、20、30、40μg/ml）；质控品一支（25μg/ml）。检测操作时，按照正常生化试剂使用，根据不同的生化仪，设置杯空白、水空白及试剂空白等，仪器准备好后，直接将生化仪设置为单试剂检测，设置检测点（4,45），波长为700nm，R试剂使用量200μl,样本使用量为10μl，定标，标准曲线拟合为直线回归，拟和结果为R2≥0.9801，检测质控品，利用标准曲线进行结果回归，测定值为30±3μg/ml，质控品检测合格后，即可以正常检测样本。
本发明所采用的TAFI含量的体外检测试剂盒的内容物的制备方法如下：
1、纳米金的制备
本发明使用的纳米金，采用柠檬酸钠和氯金酸制备，首先将氯金酸溶液加入微量纳米金保护剂，再用电炉加热至沸腾，按照4:5质量比加入柠檬酸三钠溶液，继续加热10min后既得纳米金溶液，计算理论制备纳米金溶液的体积，定容后，利用核酸蛋白分析仪或紫外分光光度计对其进行全波长扫描，确定其峰值，本发明所使用的纳米金溶液全波长扫描，其波峰在515-530nm之间。
2、单克隆抗体和金标抗体的制备
单克隆抗体和金标抗体是针对重组人TAFI抗原所制备的鼠抗人单克隆抗体，鼠抗人单克隆抗体包含3B1和4E7。
杂交瘤制备：以纯化的人血浆TAFI为免疫原，按常规方法免疫BALB/c小鼠；取其脾细胞与SP2/0骨髓瘤细胞融合；对杂交瘤细胞进行筛选，阳性孔经3次有限稀释法克隆，最终获得2株持续且稳定分泌抗人TAFI单克隆抗体的杂交瘤细胞株，分别命名为3B1和4E7。
抗体鉴定：按照罗氏公司抗体亚类试剂盒说明书测定这两种单克隆抗体的类型均为IgG1，κ；以模拟血浆作为对照，免疫印迹和ELISA分析显示，3B1和4E7这两种单克隆抗体均与人源TAFI抗原特异性结合，其中3B1可以特异性结合TAFI和失活的TAFI，如图1所示，3B1能够特异性识别血浆中的TAFI。图2单克隆抗体4E7与TAFI特异性结合ELISA曲线。以15μg/ml4E7包被酶标板，以TAFI、TAFI和4E7混合物、TAFI和小鼠IgG混合物分别作为抗原，3B1作为酶联抗体，进行ELISA反应。从曲线图可以看出，游离4E7竞争性抑制TAFI与固相4E7结合，说明4E7仅与TAFI特异性结合。
抗体制备：杂交瘤细胞体外培养法。
抗体纯化：中空纤维滤柱（美国通用公司）浓缩杂交瘤培养上清，蛋白A-琼脂糖亲和层析柱（美国通用公司）分离纯化浓缩液中的单克隆抗体。
抗体效价：经ELISA法测定纯化后的3B1和4E7单克隆抗体，效价分别105和106。
3、纳米金标记物R试剂的制备：
当R为单试剂：具体制备方法为：每1ml纳米金使用0-20μl的0.2MK2CO3调节PH值后，按照1-25μgTAFI抗体，反应2-15min后，加入BSA、PEG、Casein等进行封闭，封闭时间为2-15min，离心纯化，离心时间为3-20min，转速为5000-12000r/min,离心后去上清，沉淀使用含TRIS0-2%、Casein-Na0-2%、PEG200000-4%、BSA0-4%、TWEEN200-4%、PVP400-5%、蔗糖0-10%、叠氮钠0-0.5‰的纳米金复溶液进行复溶，纳米金复溶后，加入含PEG60000-20%、Casein-Na0-5%、NaCl0-25%的纳米金稀释液，比例为1:1。两种抗体标记后按照1:2混合后的溶液即为R试剂。
当R为双试剂：具体制备方法为：取1ml纳米金加0-6ｕl的0.2MK2CO3，混合后再加入10-20ug的4E7或3B1抗体，混匀静置5-20min，加5-20ｕlBSA、PEG或者Casein-Na封闭，再次混匀静置5-20min，离心5-10min（9000r/min），弃上清，沉淀用1-2ml复溶液复溶，复溶后补充NaCl,PEG6000,使得NaCl终浓度为0.85-1.5%，PEG6000终浓度为；1-4%。最后使用0.45um的滤膜过滤后，密封保存既得R1或R2试剂，R1和R2按照1/4至4/1比例混合后既得R试剂，所述复溶液为Tris0-0.5%；pvp400-1%；BSA0-1%；PEG200000-0.5%；10%蔗糖0-5%；TWEEN200-0.8%；10%叠氮钠2/万，余量为水。
TAFI标准品的制备
从德国Merck公司购买人血浆纯化的TAFI，相对分子质量60000，作为标化浓度的TAFI，离心机为转速为9000r/min，离心机为珠海黑马医学仪器有限公司的16K型，。利用人混合血浆与标化浓度的TAFI进行比对，稀释适当倍数，获得含预期浓度TAFI的血浆，作为实验用标准品。该方法制备TAFI标准品无需纯化，操作简单，成本低廉，适合大批量制备。具体操作步骤如下：
以标化TAFI作为标准品，倍比稀释做标准曲线，ELISA方法测定人混合血浆浓度。根据测定结果，利用该血浆及100mMpH7.3的乙二胺四乙酸二钠溶液、100mM壳聚糖溶液将购买的TAFI抗原稀释并充分混匀，制成终浓度为含TAFI（0、10、20、30、40μg/ml）、乙二胺四乙酸二钠5mM、壳聚糖20mM的溶液，取各浓度标准品溶液100μl分装至灭菌过的冻存管底部，液体或冷冻干燥后4℃保存。
4、100mMpH7.3的乙二胺四乙酸二钠溶液的配制
取37.22g乙二胺四乙酸二钠溶解于800ml蒸馏水中，1mol/lNaOH调节pH至7.3，定容至1L。
5、封闭液的配制
取0-20gBSA、0-10Gpeg20000、0-2gCasein-Na溶于80ml蒸馏水中，，室温定容至100mL。
实施例一：
现以人血浆为例，用本发明方法对60例确诊为心脑梗死的患者血浆样品及300例健康血浆样品进行TAFI含量测定。具体操作步骤如下：
步骤一采集人个体生物样品立即于30分钟内，4℃条件下离心取上清作为待检样品，置于冰盒1小时内使用或-20℃保存备用；
步骤二利用体外检测试剂盒，对待检样品进行TAFI含量测定；
所述体外检测试剂盒的内容物包括：TAFI生化检测试剂R、TAFI标准品、TAFI质控品，及使用说明。
步骤三取出试剂R放置室温下至温度平衡；
步骤四将R试剂和TAFI标准品放在生化分析仪相应的位置，制定标准曲线；
步骤五检测TAFI质控品，并根据线性回归，确定检测结果有效性；
步骤六检测TAFI样本，设置加入R试剂200ul，加入样本10ul，第5点作为第一点检测，第45点为第二点检测，以两点差值为检测值，通过标准曲线回归得出最终结果；
经TAFI检验，心脑梗死组血浆TAFI含量与健康组TAFI含量的P<0.01（P=0.0031），两组数据具有显著差异。以健康组含量平均值与2倍标准偏差的和为参考上限，心脑梗死的检出率可以达到40.2%。因此该方法作为辅助诊断，对心脑血管疾病的预测具有意义。
实施例二：
本发明方法还可以测定人尿液中的TAFI含量。采集人晨尿，于30分钟内，4℃条件下，1500rpm离心5分钟，取上清作为待检样品。后续步骤同实施例一。
实施例三：
现以人血浆为例，用本发明方法对195例确诊为心脑血管疾病的患者血浆样品及30例健康血浆样品进行TAFI含量测定。操作步骤同实施例一。经T检验，心脑血管疾病组血浆TAFI含量与健康组TAFI含量的P<0.01（P=0.0068），两组数据具有显著差异。以健康组含量平均值与2倍标准偏差的和为参考上限，心脑血管疾病的检出率可以达到23.4%。因此该方法作为辅助诊断，对心脑血管疾病的预测具有意义。
Claims (3)
1.一种TAFI含量的体外检测试剂盒，其特征在于：所述TAFI含量的体外检测试剂盒组成包括：含有与TAFI特异性结合的单克隆抗体纳米金标记物R试剂；用于制作计算待检样品中的TAFI抗原含量的标准曲线的TAFI标准品和TAFI质控品；
所述纳米金标记物R试剂是针对重组人TAFI抗原所制备的鼠抗人单克隆抗体经纳米金标记所得，鼠抗人单克隆抗体包含3B1和4E7，3B1纳米金结合物：4E7纳米金结合物为1:2；
生化仪设置为单试剂检测时，纳米金标记物R试剂的组分是分别偶联了3B1和4E7两种抗体的纳米金的混合液及其保存液；生化仪设置为双试剂检测时，纳米金标记物R试剂的组分包含R1、R2试剂，R1试剂是单4E7偶联的纳米金及其保存液，R2试剂是单3B1偶联的纳米金及其保存液；
当R为单试剂：具体制备方法为：每1ml纳米金使用0-20μl的0.2MK2CO3调节pH值后，按照每毫升1-25μgTAFI抗体，反应2-15min后，加入BSA、PEG或Casein-Na进行封闭，封闭时间为2-15min，离心纯化，离心时间为3-20min，转速为5000-12000r/min,离心后去上清，沉淀使用含TRIS0-2％、Casein-Na0-2％、PEG200000-4％、BSA0-4％、TWEEN200-4％、PVP400-5％、蔗糖0-10％、叠氮钠0-0.5‰的纳米金复溶液进行复溶，纳米金复溶后，加入含PEG60000-20％、Casein-Na0-5％、NaCl0-25％的纳米金稀释液，比例为1:1,两种抗体标记后按照3B1纳米金结合物：4E7纳米金结合物为1:2混合后的溶液即为R试剂；
当R为双试剂：具体制备方法为：取1ml纳米金加0-6μl的0.2MK2CO3，混合后再加入10-20μg的4E7或3B1抗体，混匀静置5-20min，加5-20μlBSA、PEG或者Casein-Na封闭，再次混匀静置5-20min，离心5-10min，转速为9000r/min，弃上清，沉淀用1-2ml复溶液复溶，复溶后补充NaCl,PEG6000,使得NaCl终浓度为0.85-1.5％，PEG6000终浓度为1-4％，最后使用0.45μm的滤膜过滤后，密封保存即得R1或R2试剂，双试剂复溶液为Tris0-0.5％；pvp400-1％；BSA0-1％；PEG200000-0.5％；10％蔗糖0-5％；TWEEN200-0.8％；叠氮钠0.02‰，余量为水。
2.根据权利要求1所述的TAFI含量的体外检测试剂盒，其特征在于：所述3B1特异性结合TAFI和失活的TAFI，所述4E7仅与TAFI结合。
3.根据权利要求1所述的TAFI含量的体外检测试剂盒，其特征在于：所述保存液是由Tris0-0.5％；pvp400-1％；BSA0-1％；PEG200000-0.5％；10％蔗糖0-5％；TWEEN200-0.8％；叠氮钠0-0.02‰，NaCl0.85-1.5％，PEG60001-4％,余量为水组成。
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