CN1206538C - 用于消除含有脂蛋白颗粒的脂血样品浑浊现象的试剂 - Google Patents

用于消除含有脂蛋白颗粒的脂血样品浑浊现象的试剂 Download PDF

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CN1206538C
CN1206538C CNB021017824A CN02101782A CN1206538C CN 1206538 C CN1206538 C CN 1206538C CN B021017824 A CNB021017824 A CN B021017824A CN 02101782 A CN02101782 A CN 02101782A CN 1206538 C CN1206538 C CN 1206538C
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G·冈泽
T·拉金
A·普福茨纳
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Beckman Coulter Biomedical Ltd
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    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
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Abstract

用于消除生物样品浑浊现象的试剂，它包含0.5-10mM的苯酚，0.5-15%的聚氧乙基化甘油三酯和至少一种能溶解聚氧乙基化甘油三酯的0.5-15%范围内的非离子型表面活性剂。

Description

用于消除含有脂蛋白颗粒的脂血样品浑浊现象的试剂
技术领域
本发明涉及用于消除生物样品、尤其是血清或血浆样品浑浊现象的试剂。
背景技术
当血浆或血清样品(脂血样品)中富含甘油三酯的脂蛋白颗粒如乳糜微粒的含量增加时通常会发生浑浊。
对于化学或临床诊断中脂血样品的光度分析来说尤其需要能消除浑浊的已知试剂。如果要加以分析的组分在与引起浑浊的甘油三酯的吸收波长一致的波长下吸收光线的话，对于所研究组分的正确的光度分析要么不可能，即使可能也将是困难的。当要加以测定的组分的浓度非常低、例如在被分析物为细胞活性蛋白(CRP)的情况下尤其是如此。
因此，必须要排除浑浊产生的干扰的一个光度测定操作的典型实例是CRP的测定。该测定使用与样品中的CRP特异性反应形成不溶性聚集体的抗-人CRP抗体。该测定通过加入抗体开始，并在340nm下用光度计测量CRP-抗体-聚集体的增值。脂血样品中的甘油三酯也在这一波长下吸收，于是就有覆盖消光信号的危险。
为了解决脂血样品的这个问题，在现有技术中已知可加入若干种试剂来消除浑浊。在与此有关的美国专利4708939中公开了一种试剂，它在水溶液中包含聚乙氧基化甘油三酯和二级正链烷磺酸酯。该已知试剂需要相当高的洗涤剂浓度，这会影响某些测定，尤其是免疫比浊测定。另外，正链烷磺酸酯是一种积极的(agressive)洗涤剂，它还可抑制所研究的反应。因此该已知试剂不能使样品完全澄清和得到最佳的抗体-抗原反应。
发明内容
本发明的目的是提供一种用于消除浑浊的试剂，它尤其适用于CRP测定，但也可用于其他测定。
本发明的试剂包含0.5-10mM的苯酚、0.5-15％的聚氧乙基化甘油三酯和0.5-15％的至少一种另外的非离子型表面活性剂(tenside)。优选包括两种另外的非离子型表面活性剂的结合物。
在本发明的试剂中，聚乙氧基化甘油三酯(Triglyceridethoxylate)是得到完全澄清的样品所必需的。合适的聚乙氧基化甘油三酯可具有4-16范围内的HLB(亲水亲油平衡)值，优选为6。优选的浓度范围是0.5-2％。优选的聚氧乙基化甘油三酯可以商品名Mulsifan RT163(Zschimmer & Schwarz GmbH & Co.)或TagatCH-40(Goldschmidt AG)购得。
单独的聚氧乙基化甘油三酯不能溶于混合物而导致沉淀。溶解作用需要至少一种另外的有助于聚乙氧基化甘油三酯在样品中的溶解的非离子型表面活性剂。
可以使用的非离子型表面活性剂的典型实例包括：Thesit、Tergitol、Triton、Brij、Nonidet(诺乃清洁剂)P-40、吐温20(Sigma-Aldrich Ltd.)。
所需的非离子型表面活性剂是10-18个碳原子的乙氧基化程度低(每分子2-10个氧化乙烯单元)的直链或支链聚氧乙烯醚。
优选的另外的非离子型表面活性剂是聚氧乙烯(8)异十三烷基醚，它可以商品名Genapol X 080购得。这种非离子型表面活性剂可溶于反应混合物，但单独的它不具有澄清作用。使聚乙氧基化甘油三酯溶解是必需的(如上所述)。可以使用的非离子型表面活性剂的优选的浓度范围是0.5-2％。
另一种优选的非离子型表面活性剂是可从Sigma购得的聚氧乙烯-10-十三烷基醚。这种表面活性剂也可溶于反应混合物并帮助聚乙氧基化甘油三酯溶解。优选的浓度范围是0.5-2％。
在一个优选的实施方案中提供聚氧乙烯(8)异十三烷基醚和聚氧乙烯-10-十三烷基醚的结合物。这种结合物可使聚氧乙基化甘油三酯非常有效地溶解并由此提高本发明试剂的澄清作用。
最后，苯酚没有溶解脂质的作用，但可与表面活性剂协同起作用来增加它们的澄清作用和消除浑浊。苯酚的存在有助于减少所需表面活性剂的有效浓度。不存在苯酚时将需要较高浓度的表面活性剂，这会导致样品具有太高的粘度。
本发明试剂中的组分都是已知物质，它们已经在现有技术中用在已知用于消除浑浊的试剂中。
从EP 0004857可知苯酚作为协同作用组分的用途。从EP0004857还可知使用Genapol作为非离子型表面活性剂。从上文中提及的美国专利4708939可知另外的非离子型表面活性剂的应用。不过，现有技术只公开了单一组分或其亚结合物的应用。这些文献中没有一篇显示象本发明的试剂那样将所有这些组分结合。
结果令人惊奇地，包括在本发明试剂中的所有组分都是有效地消除浑浊同时又对可能的抗体-抗原反应作用最小所必需的。理论上，Genapol和聚氧乙烯-10-十三烷基醚在一起可起到澄清作用。但是，澄清作用所需的浓度如此之高，以致任何抗体-抗原反应都被完全抑制了。如果按照本发明加入Mulsifan和苯酚，Genapol和聚氧乙烯-10-十三烷基醚的浓度就可以减小，从而达到澄清效果而没有影响免疫反应。
原则上，本发明试剂可以用于由于脂血引起的相互作用可能性的所有免疫测定形式。
附图说明
图1表示用实施例1的配方制备的试剂对脂血样品中浑浊现象的消除。
具体实施方式
下面给出的实施例采用免疫比浊形式。本领域技术人员将意识到对其他形式的相关益处，其他形式包括(但不限于)乳汁增强测定、磁性颗粒化学发光免疫测定、免疫荧光测定(偏光和非偏光的)、ELISA测定、免疫色谱测定、或需要减少脂血和/或伴随的其他非特异性结合问题的任何测定形式，此时抗体结合特性的有益的维持是通过本发明中反应物的结合介导的。
以下描述了将要用于检测CRP的测定的本发明澄清剂的典型配方。另外还给出了一个显示本发明试剂消除脂血样品中浑浊现象作用的例子。
用于CRP-测定的澄清缓冲液(R1)的配方：
试剂                               范围       功能
净化水
Tris(100mM)/升                     50-300mM   缓冲剂
NaCl(100mM)/升                     50-150mM   帮助澄清和反应
PEG6000(2％)(w/w)                  1-3％      反应率最佳化需要的
苯酚(2mM)(升)                      0.5-10mM   脂质澄清组分
聚氧乙烯10-十三烷基醚(1.0％)(v/v)  0.5-2％    脂质澄清组分
Genapol X 80(1.0％)(v/v)           0.5-2％    脂质澄清组分
Mulsifan RT 163(1.1％)(v/v)        0.5-2％    脂质澄清组分
NaN3                                         抗微生物剂
4M HCl                                        调pH7.5的pH试剂
硫酸庆大霉素溶液                              稳定剂
将缓冲液R1调节至pH7.5(可能的范围是pH3-9)并优选在37℃(可能的范围是15-40℃)下使用。
对于CRP-测定，将250μl R1与18μl样品混合，然后加入30μl抗血清溶液(R2；包括抗CRP抗体)。样品与R1缓冲液和R2抗血清溶液混合后，样品中的CRP特异性地与R2中的抗人CRP抗体反应得到不溶性聚集体。这些聚集体的吸光度与样品中的CRP浓度成比例。
R1缓冲液的目的是将脂血样品中的脂质溶解和为免疫反应提供最佳条件。
脂血样品中浑浊现象的消除
在该试验中使用具有以下成分的澄清缓冲液(R3)：
用HCl调节至pH7.5的100mM Tris缓冲剂，100mM NaCl，2％聚乙二醇6000，2mM苯酚，1.0％聚氧乙烯10十三烷基醚，1.0％Genapol X 80和1.0％ Mulsifan RT 163。
将18μl强脂血血清在37℃下在比色杯中与250μl R3混合。在340nm下测定吸光度相对于时间的变化。结果图解表示在图1中。
图1中显示的吸光度的变化过程表明，在样品/试剂比为18μl∶250μl的情况下，与空白试剂比较，约为1.5的浊度在约1.5分钟内降低到吸光度约为0.1的澄清水平。因此，1.5分钟后已完全消除了浑浊。

Claims (3)

1、用于消除含有脂蛋白颗粒的脂血样品浑浊现象的试剂，它包含
0.5-10mM的苯酚，
0.5-2％的聚氧乙基化甘油三酯和
两种非离子型表面活性剂，每种浓度0.5-2％，其特征在于所述非离子型表面活性剂是每分子具有2-10个氧化乙烯单位且含有10-18个碳原子的聚氧乙烯醚。
2、按照权利要求1的试剂，其特征在于非离子型表面活性剂是聚氧乙烯-10-十三烷基醚和聚氧乙烯-8-异十三烷基醚。
3、按照权利要求1或2所述的试剂用于检测细胞活性蛋白的用途。
CNB021017824A 2001-02-19 2002-01-18 用于消除含有脂蛋白颗粒的脂血样品浑浊现象的试剂 Expired - Fee Related CN1206538C (zh)

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EP01103991A EP1233269B1 (en) 2001-02-19 2001-02-19 Agent for the removal of turbidity in biological samples

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CN105067815A (zh) * 2015-09-16 2015-11-18 浙江凯成生物科技有限公司 一种测定人血清中胃蛋白酶原i/ii含量的试剂盒
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CN112834758B (zh) * 2021-01-07 2023-06-16 北京九强生物技术股份有限公司 B因子的检测试剂盒

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EP1233269A1 (en) 2002-08-21
EP1233269B1 (en) 2003-11-19
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