CN106370862B - 一种稳定、灵敏的纤维结合蛋白检测试剂 - Google Patents
一种稳定、灵敏的纤维结合蛋白检测试剂 Download PDFInfo
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Abstract
本发明涉及血清纤维结合蛋白检测技术领域，特别涉及一种纤维结合蛋白检测试剂，试剂R1中含有缓冲液，γ‑Fe2O3/聚噻吩(γ‑Fe2O3‑PTP) 复合纳米颗粒，氟碳表面活性剂FC‑4430，防腐剂；试剂R2中含有缓冲液，羊抗人纤维结合蛋白抗体，明胶，黄原胶，氯化钠，海藻糖，PEG‑6000，氟碳表面活性剂FC‑4430，防腐剂。通过优化反应体系，采用HEPES（4‑羟乙基哌嗪乙磺酸）缓冲液，并添加NaCl、海藻糖、PEG6000、明胶、黄原胶等多种稳定剂，可以显著改善试剂的稳定性；试剂中添加γ‑Fe2O3/聚噻吩(γ‑Fe2O3‑PTP) 复合纳米颗粒，在检测时可以通过静电吸附抗原抗体复合物，可以显著增强试剂的灵敏度和准确度。此外，优选的新型氟碳表面活性剂FC‑4430，可以促进并维持抗体稳定，防止体系浑浊，进一步增强试剂的稳定性。
Description
技术领域
本发明涉及纤维结合蛋白（Fn）检测技术领域，特别涉及一种纤维结合蛋白（Fn）检测试剂，还涉及使用此检测试剂的检测方法。
背景技术
纤维结合蛋白（又称纤维连接蛋白，英文缩写Fn），广泛存在于动物组织和组织体液中，是由两个几乎相同的亚单位以二硫键连接而成的具有非免疫性调理素作用且具有多种生物活性的一种高分子糖蛋白。血清或血浆纤维结合蛋白升高常见于脑梗死、妊娠高血压等，降低常见于肝硬化、营养不良、中风、烧伤、中毒呼吸衰竭、肺部感染等。此外，纤维结合蛋白（Fn）测定有助于脑血栓与脑出血的鉴别诊断，动态观察脑血管疾病患者血浆Fn含量有助于检测病情恢复和疗效。
目前，纤维结合蛋白的检测方法有火箭电泳法、酶联免疫检测法( ELISA) 、免疫比浊法等。其中火箭电泳法、酶联免疫检测法( ELISA)需要特定设备，费时费力，不易推广使用；而免疫比浊法使用简单方便，便于自动化操作，但存在灵敏度低、稳定性差等缺点。
鉴于此，本发明在免疫比浊法基础上，通过优化反应体系，采用HEPES（4-羟乙基哌嗪乙磺酸）缓冲液，并添加NaCl、海藻糖、PEG6000、明胶、黄原胶等多种稳定剂，显著改善了试剂的稳定性；并且试剂中添加γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒，在检测时可以通过静电吸附抗原抗体复合物，可以显著增强试剂的灵敏度和准确度。此外，优选的新型氟碳表面活性剂FC-4430，可以促进并维持抗体稳定，防止体系浑浊，进一步增强试剂的稳定性。该试剂操作简便快速，适用于自动化分析，是一种更加稳定、灵敏的纤维结合蛋白（Fn）试剂。
发明内容
本发明旨在提供一种用于检测血清或血浆中纤维结合蛋白（Fn）的试剂及使用该试剂检测血清纤维结合蛋白（Fn）的方法。该试剂盒采用免疫比浊法，可以有效检测纤维结合蛋白的含量，具有灵敏度高，稳定性好等优点。
基本原理：
样本中的抗原（纤维结合蛋白，Fn）与试剂中特异性抗体（羊抗人纤维结合蛋白抗体）反应，形成不溶性免疫复合物，在波长340nm处检测其浊度变化，其变化程度与样本中纤维结合蛋白浓度成正比。
本发明是通过以下步骤得到的：
一种纤维结合蛋白检测试剂，包括试剂R1和试剂R2，所述试剂R1和试剂R2的组成如下：
试剂R1的组分为：
缓冲液················································50mmol/L，
γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒·············1.0%~2.0%（W/V），
氟碳表面活性剂FC-4430·································2g/L，
防腐剂················································0.1g/L；
试剂R2的组分为：
缓冲液················································80mmol/L，
羊抗人纤维结合蛋白抗体································15%~35%（W/V），
明胶··················································0.2%~0.8%（W/V），
黄原胶················································0.2%~1%（W/V），
氯化钠················································2g/L，
海藻糖················································25g/L，
PEG-6000··············································5g/L，
氟碳表面活性剂FC-4430·································2g/L，
防腐剂················································0.1g/L。
所述的纤维结合蛋白检测试剂，所述复合纳米颗粒为γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP)，粒径约30-50nm。
本发明所使用的γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒的制备方法如下：
取适量γ-Fe2O3纳米颗粒加入到聚乙二醇200（PEG-200）中，搅拌均匀后，使γ-Fe2O3纳米颗粒表面包裹一层PEG200。然后，加入氯仿（CHCl3），利用超声分散，接着倒入烧瓶。此时，向烧瓶内加入噻吩，冰浴，机械搅拌一段时期。然后再加人一定量无水FeCl3，搅拌使反应完全，最后加入1mol/L的HCl，除去过量的无水FeCl3，抽滤、洗涤、真空干燥，即可得到γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP)纳米材料，粒径约30-50nm。通过该法得到的γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP)复合纳米材料，具有很好的稳定性、水溶性和分散性。
所述的纤维结合蛋白检测试剂来检测纤维结合蛋白含量的检测方法，使用全自动生化分析仪利用终点法进行测定，检测主波长为340nm。
所述的检测方法，R1试剂和R2试剂的比例为3:1。
本发明的有益效果：
优化反应体系，试剂R2采用HEPES（1,4-哌嗪二乙磺酸）缓冲液，并添加NaCl、海藻糖、PEG6000、明胶、黄原胶等多种稳定剂，可以显著改善试剂的稳定性。
2）试剂中添加γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒，在检测时可以通过静电吸附抗原抗体复合物，可以显著增强试剂的灵敏度和准确度。
优选的新型氟碳表面活性剂FC-4430，可以促进并维持抗体稳定，防止体系浑浊，进一步增强试剂的稳定性。
试剂的准确度和稳定性良好，抗干扰性强，使用方便，完全可以满足临床需要。
附图说明
图1为本发明试剂与对照组试剂效期稳定性曲线图；
图2为实施例1试剂检测操作方法；
图3 为本发明试剂与对照组试剂批内精密度检测结果对比；
图4为本发明试剂与对照组试剂准确度检测结果对比；
图5为本发明试剂与对照组试剂灵敏度检测结果对比。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例对本发明进行进一步说明：
实施例1
本实施例所描述的纤维结合蛋白的检测试剂，包括试剂R1和试剂R2：
试剂R1的组分为：
HEPES（4-羟乙基哌嗪乙磺酸）缓冲液(pH=7.6，25℃) ········50mmol/L，
γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒············1.0%（W/V），
氟碳表面活性剂FC-4430································2g/L，
甲基异噻唑啉酮（MIT）··································0.1g/L；
试剂R2的组分为：
HEPES（4-羟乙基哌嗪乙磺酸）缓冲液(pH=7.6，25℃) ········80mmol/L，
羊抗人纤维结合蛋白抗体·······························15%（W/V），
明胶·················································0.2%（W/V），
黄原胶···············································0.2%（W/V），
氯化钠···············································2g/L，
海藻糖···············································25g/L，
PEG-6000·············································5g/L，
氟碳表面活性剂FC-4430································2g/L，
甲基异噻唑啉酮（MIT）··································0.1g/L。
本实施例试剂的使用方法：
本实施例描述的纤维结合蛋白检测试剂，在使用时采用具有双试剂功能的全自动生化分析仪，如日立7180全自动分析仪等，利用终点法进行测定。将R1和R2按照3:1的比例放置到对应的试剂位上，在样品盘的对应位置放置好蒸馏水、标准品和样本，操作如图2。
实施例2
本实施例所描述的纤维结合蛋白的检测试剂，包试剂R1和试剂R2（检测方法同实施例1）：
试剂R1的组分为：
HEPES（4-羟乙基哌嗪乙磺酸）缓冲液(pH=7.6，25℃) ········50mmol/L，
γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒············1.5%（W/V），
氟碳表面活性剂FC-4430································2g/L，
甲基异噻唑啉酮（MIT）··································0.1g/L；
试剂R2的组分为：
HEPES（4-羟乙基哌嗪乙磺酸）缓冲液(pH=7.6，25℃) ········80mmol/L，
羊抗人纤维结合蛋白抗体·······························20%（W/V），
明胶·················································0.5%（W/V），
黄原胶···············································0.5%（W/V），
氯化钠···············································2g/L，
海藻糖···············································25g/L，
PEG-6000·············································5g/L，
氟碳表面活性剂FC-4430································2g/L，
甲基异噻唑啉酮（MIT）··································0.1g/L。
实施例3
本实施例所描述的纤维结合蛋白的检测试剂，包试剂R1和试剂R2（检测方法同实施例1）：
试剂R1的组分为：
HEPES（4-羟乙基哌嗪乙磺酸）缓冲液(pH=7.6，25℃) ········50mmol/L，
γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒············2.0%（W/V），
氟碳表面活性剂FC-4430································2g/L，
甲基异噻唑啉酮（MIT）··································0.1g/L；
试剂R2的组分为：
HEPES（4-羟乙基哌嗪乙磺酸）缓冲液(pH=7.6，25℃) ········80mmol/L，
羊抗人纤维结合蛋白抗体······························35%（W/V），
明胶·················································0.8%（W/V），
黄原胶···············································1%（W/V），
氯化钠···············································2g/L，
海藻糖···············································25g/L，
PEG-6000·············································5g/L，
氟碳表面活性剂FC-4430································2g/L，
甲基异噻唑啉酮（MIT）··································0.1g/L。
实施例4
试剂批内精密度试验：取具有溯源性的高值质控物、低值质控物各一份，分别用实施例1、实施例2、实施例3配方配制试剂，与市场常见的国家食品药品监督管理局认可的某公司的纤维结合蛋白（Fn）检测试剂盒进行对照检测，对每份质控物进行 20 次检测，将共20次检测结果计算平均值、标准差和变异系数。结果见图3。
由图3中变异系数可知，与对照试剂相比，实施例1、实施例2、实施例3配方配制试剂具有更高的批内精密度，这说明通过优化反应体系，添加γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒，极大的提高了试剂的批内精密度。
实施例5 试剂准确度对比试验：取具有溯源性的血清高值质控物、血清低值质控物各一份，分别用实施例1、实施例2、实施例3配方配制试剂，与市场常见的国家食品药品监督管理局认可的某公司的纤维结合蛋白（Fn）检测试剂盒进行对照检测，各检测 5 次，计算平均值，与质控物靶值进行对照。结果见图4。
由图4中检测结果可知，与对照试剂相比，实施例1、实施例2、实施例3配方配制试剂具有更高的准确度，这说明通过优化反应体系，添加γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒和新型表面活性剂氟碳表面活性剂FC-4430，极大的提高了试剂的准确度。
实施例6
试剂灵敏度的对比试验 ：取具有朔源性的校准品由低到高稀释出6个浓度样本，分别用实施例1、实施例2、实施例3配方配制试剂，与市场常见的国家食品药品监督管理局认可的某公司的纤维结合蛋白（Fn）检测试剂对照检测，将检测结果与理论浓度相比较。结果见图5。
由图5中检测结果可知，在样本浓度低至5mg/L时，对照试剂测试值为0，而实施例1、实施例2实施例3配方配制试剂仍可以检测出样本的准确值；并且与对照试剂相比，实施例1、实施例2实施例3配方配制试剂检测接近线性下限的低值样本时的准确度更高。这表明实施例1、实施例2实施例3配方配制试剂拥有更高的分析灵敏度和准确度。因此可以说明通过添加γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒和新型氟碳表面活性剂FC-4430，极大的提高了试剂的分析灵敏度。
实施例7
试剂的稳定性对比试验：对实施例1、实施例2、实施例3中的试剂，分别均匀分装13组，每组的试剂量为R1为18mL，R2为6mL；并且取13组市场常见的国家食品药品监督管理局认可的某公司的纤维结合蛋白试剂盒作对照。放置到2-8℃冰箱中，每月的同一天取出一组试剂检测Fn质控品（靶值为324mg/L），检测结果如图1所示，实施例1、实施例2、实施例3试剂在2-8℃储存条件下比市场常见的纤维结合蛋白测定试剂盒更加稳定。
通过验证，本试剂与同类检测试剂对比灵敏度高、重复性好，能够达到市场对产品的应用要求，并且准确度高，是一种更加稳定、良好的纤维结合蛋白检测试剂。
Claims (4)
1.一种纤维结合蛋白（Fn）检测试剂，其特征在于包括试剂R1和试剂R2，其中试剂R1组成为： 50mmol/L的缓冲液、1.0%~2.0%（W/V）的γ-Fe2O3/聚噻吩(γ-Fe2O3-PTP) 复合纳米颗粒、2g/L的氟碳表面活性剂FC-4430、0.1g/L的防腐剂；试剂R2组成为80mmol/L的缓冲液、15%~35%（W/V）的羊抗人纤维结合蛋白抗体、0.2%~0.8%（W/V）的明胶、0.2%~1%（W/V）的黄原胶、2g/L的氯化钠、25g/L的海藻糖、5g/L的PEG-6000、2g/L的氟碳表面活性剂FC-4430、0.1g/L的防腐剂。
2.根据权利要求1所述的纤维结合蛋白检测试剂，其特征在于试剂R1中缓冲液为25℃，pH为7.6的HEPES缓冲液。
3.根据权利要求1所述的纤维结合蛋白检测试剂，其特征在于试剂R2中缓冲液为25℃，pH为7.4的HEPES缓冲液。
4.根据权利要求1所述的纤维结合蛋白检测试剂，其特征在于所述防腐剂为甲基异噻唑啉酮（MIT）。
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