CN111381040A - 一种性能高效的检测结合珠蛋白检测试剂 - Google Patents
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Abstract
本发明公开了一种性能高效的结合珠蛋白检测试剂，属于临床体外检测技术领域。该试剂包括试剂R1和试剂R2，其中试剂R1中含有缓冲液、表面活性剂、掩蔽剂、稳定剂、封闭剂、防腐剂，试剂R2中含有缓冲液、防腐剂、表面活性剂、保护剂、羊抗人结合珠蛋白(Hp)抗体。本试剂具有良好的特异性和灵敏性，显著改善了当前该方法在临床中假阳性和假阴性等问题，为临床中的推广和应用提供了良好的保障。
Description
技术领域
本发明涉及结合珠蛋白检测技术领域，特别涉及一种性能高效的检测血清或血浆中结合珠蛋白检测试剂。
背景技术
结合珠蛋白简称Hp，Hp的主要功能是能与红细胞中释出的自由形式存在的血红蛋白(Hb)结合，每分子Hp可以结合两分子的Hb。结合是不可逆的，一旦结合后，复合物在几分钟之内转运到肝，肝细胞上有特异受体，可十分有效地结合Hp-Hb复合物进入肝细胞而被降解，氨基酸和铁可被机体再利用，因此Hp可以防止Hb从肾丢失而为机体有效地保留铁。在一次急性血管内溶血时血循环中的Hp可以结合3g以上的Hb。Hp在溶血后含量急剧降低，Hp与Hb结合后不能重新被利用，但急性溶血后其在血浆中的浓度一般在一周内即可由再生而恢复。
结合珠蛋白又是一种急性期时相反应蛋白。当机体处在应激状态时，血液中的结合珠蛋白明显增多，如心肌梗塞、肿瘤、炎症、创伤、感染等病理状态时，以及应用某些激素，如皮质激素和雄性激素后，其血清含量常有显著升高，并与严重程度和预后有关。研究表明，血清结合珠蛋白和铁蛋白含量的变化，对急性肺栓塞和深部静脉血栓形成的病人具有一定的诊断价值。另外在进行糖尿病合并血管病变患者的基础研究中，也发现血清结合珠蛋白含量与疾病进展发生相关性变化，Hp基因型有可能是糖尿病冠状动脉病变的一个独立的风险因素，因此认为，对结合珠蛋白含量的动态监测，有益于糖尿病血管病变患者的治疗，尤其是在建立糖尿病患者血管病变预防策略时，具有十分重要的意义。由于结合珠蛋白的合成与降解均在肝脏，并且在结合珠蛋白与血红蛋白的复合物形成与降解的过程中，结合珠蛋白不能重复利用，因此当肝脏功能出现问题时，体内的结合珠蛋白数量常发生明显的变化，合成不足则其含量减少，降解不足则其含量增多，此时化验检查血液中的结合珠蛋白含量是否减少或增加，对诊断肝脏疾病，判断疾病的预后，很有帮助。
目前检测结合珠蛋白的方法主要是基于免疫学方法的有ELISA方法、免疫比浊法等。ELISA方法检测血清中结合珠蛋白该检测方法灵敏度高，准确可靠，但是成本较高、效率低、操作复杂，主要应用于科研使用而不利于临床的推广使用。而后期推出的免疫比浊法检测结合珠蛋白方法，该方法的测试结果稳定，操作简单，在临床上非常实用，但是随着临床中需求进一步提高和应用发现，目前该方法存在特异性差、灵敏度低等缺点，容易造成临床中假阳性和假阴性等问题，严重影响和制约着临床中的应用和结果的判断，对此本发明研制了一种性能高效的结合珠蛋白检测试剂，具有良好的特异性和灵敏性，解决了该方法中存在的缺陷和问题，为临床中的推广和应用提供了良好的保障。
发明内容
为解决上述问题，提供一种性能高效的检测血清或血浆中结合珠蛋白(Hp)的试剂，本发明的检测试剂采用免疫比浊法检测血清或血浆中的结合珠蛋白的含量。将血清或血浆中存在干扰的物质进行封闭，而Hp被有效的解离出来，可与其相应抗体(羊抗人Hp抗体)产生抗原抗体的反应，形成抗原抗体复合物，并产生浊度变化，通过测定特定波长下浊度的变化，计算出Hp的含量。
本发明提供了一种性能高效的结合珠蛋白检测试剂，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中含有：
试剂R2中含有：
优选的，本发明试剂R1中缓冲液为25℃，PH＝8.0的DIPSP(3-双(2-羟乙基)氨基-2-羟基丙磺酸)缓冲液；试剂R2中缓冲液为25℃，pH为6.0的N-(2-乙酰氨基)亚氨基二乙酸(ADA)缓冲液；试剂R1中表面活性剂为m PEG5000；试剂R1中掩蔽剂为铁氰化钾；试剂R1中稳定剂为甲基脲；试剂R1中封闭剂为小牛血清白蛋白；试剂R1和试剂R2中防腐剂为PC-300；试剂R2中表面活性剂为聚氧乙烯硬脂基醚(EMULGEN 306P)；所述试剂R2中保护剂为丙氨酸。
本发明一种性能高效的结合珠蛋白检测试剂的检测方法，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，R1试剂和R2试剂的比例为4:1。
本发明的有益效果是：
1)在试剂R1和R2中分别使用了DIPSP(3-双(2-羟乙基)氨基-2-羟基丙磺酸)生物缓冲液和ADA缓冲液，对在保证试剂具备良好的缓冲能力的同时，有利于促进生物反应环境的构建；
2)在试剂R1中优选选用了活化后的mPEG5000聚乙二醇作为表面活性剂，能够大大提高Hp与加入的抗体的结合能力，提高反应的灵敏度；
3)在R1中加入掩蔽剂铁氰化钾和封闭剂小牛血清白蛋白来去除血清中残存的血红蛋白物质的干扰，有利于降低血红蛋白对Hp检测的干扰性，同时在R1中加入甲基脲，来提高反应的稳定性以及检测结果的重复性；
4)在试剂R2中添加了氨基酸丙氨酸和表面活性剂来保证液体试剂中抗体的稳定，以及提高抗体的反应效价。
具体实施方式
下面结合具体实施例对本发明进行进一步说明：
实施例1
一种性能高效的结合珠蛋白检测试剂，包括试剂R1和试剂R2，所述试剂R1和试剂R2的组成如下：
1)试剂R1中含有：
2)试剂R2中含有：
实施例2
本实施例描述的是一种重要组成部分含量增加的性能高效的结合珠蛋白检测试剂，包括试剂R1和试剂R2，所述试剂R1和试剂R2的组成如下：
1)试剂R1中含有：
2)试剂R2中含有：
检测使用方法：
上述2实施例描述的结合珠蛋白检测试剂，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。分别将实施例1和实施例2的R1和R2按照4:1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，操作如表1：
表1实施例试剂检测方法
计算：结合珠蛋白含量(g/L)＝(ΔA测定÷ΔA标准)×C标准。
干扰性试验：
取新鲜混合血清，分成2等份，然后将每等份再分成5等份，加入不同的干扰物质，使其在血清中的浓度达到表2的要求。然后分别用实施例1和实施例2所得试剂，同时对比测定血清中结合珠蛋白的含量，加入不同干扰物质后各组的测定结果见表2。相对偏差(％)＝(干扰样本的测定均值－无干扰物质的样本的测定均值)/无干扰物质的测定均值×100％。
由表2可以看出，实施例1试剂在抗坏血酸≤2000μmol/L、胆红素≤986μmol/L、血红蛋白≤10.5g/L、甘油三酯≤20.6mmol/L、类风湿因子≤120IU/mL对测试结果没有明显干扰。
表2实施例试剂抗干扰性能比较
灵敏度实验：
将已知浓度为2g/L的Hp血清样本进行1/2、1/4、1/8、1/16、1/32利用实施例1和实施例2配方配制试剂以及ELISA方法进行同步比对检测，观察在这些浓度梯度最先能准确测量的浓度，来判断试剂的灵敏度情况，检测结果如表3。
表3实施例试剂灵敏度实验
通过表3中的实验数据可知，实施例1和实施例2方法配置的检测试剂与经典的ELISA方法进行灵敏度比较，实施例1和实施例2方法在进行32倍样本稀释后，检测结果要优于ELISA方法，从而可见，实施例1和实施例2方法具有良好的灵敏性，从而保证了检测结果的灵敏可靠。
Claims (6)
1.一种性能高效的结合珠蛋白检测试剂，其特征在于，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中含有：
试剂R2中含有：
2.根据权利要求1所述的一种性能高效的结合珠蛋白检测试剂，试剂R1中缓冲液为25℃，PH＝8.0的DIPSP(3-双(2-羟乙基)氨基-2-羟基丙磺酸)缓冲液，试剂R2中缓冲液为25℃，PH＝6.0的N-(2-乙酰氨基)亚氨基二乙酸(ADA)缓冲液。
3.根据权利要求1所述的一种性能高效的结合珠蛋白检测试剂，所述试剂R1中表面活性剂为m PEG5000；所述试剂R1中掩蔽剂为铁氰化钾，所述试剂R1中稳定剂为甲基脲，所述试剂R1中封闭剂为小牛血清白蛋白。
4.根据权利要求1所述的一种性能高效的结合珠蛋白检测试剂，所述试剂R1和试剂R2中防腐剂为PC-300。
5.根据权利要求1所述的一种性能高效的结合珠蛋白检测试剂，所述试剂R2中表面活性剂为聚氧乙烯硬脂基醚(EMULGEN 306P)，所述试剂R2中保护剂为丙氨酸；
6.根据权利要求1-5中任一项所述的一种性能高效的结合珠蛋白检测试剂，其特征在于，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，R1试剂和R2试剂的比例为4:1。
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