CN1172530A - 以调节化学发光为手段分析被分析物的方法 - Google Patents

以调节化学发光为手段分析被分析物的方法 Download PDF

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CN1172530A
CN1172530A CN96191746A CN96191746A CN1172530A CN 1172530 A CN1172530 A CN 1172530A CN 96191746 A CN96191746 A CN 96191746A CN 96191746 A CN96191746 A CN 96191746A CN 1172530 A CN1172530 A CN 1172530A
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逵保宏
上参乡庆一
大光男
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Abstract

一种利用化学发光物质标记的材料对被分析物进行分析的方法,包括添加猝灭剂和/或降低化学发光物质标记的探针的特定活性,从而减少化学发光的数量。

Description

以调节化学发光为手段分析被分析物的方法
技术领域
本发明涉及一种利用标记了化学发光物质的材料而进行的分析物质的方法。更具体地说，本发明涉及一种在较宽范围内，通过减少化学发光的数量而进行的分析试验方法。
背景技术
在所有的针对实验中的被分析物的分析方法中，溶液都被探针标记了化学发光物质。通过测量化学发光的数量，该方法被广泛地用于高灵敏地确定被分析物质的量。该方法用于合适数量的被分析物。然而，当存在大量的被分析物(例如通过象聚合酶链反应产生许多拷贝时)时，化学发光的数量超出了该方法测量装置的测定极限，使准确的分析变得不可能了。测量结果超过分析上限的样品，通过稀释试验溶液再次测定。这种工艺非常费事。特别地，通过基因扩增而扩增的样品，可能因稀释而导致扩增产物的污染。需要极端小心以避免污染，更进一步增加了劳动。为了扩宽分析范围而不需稀释样品，除了期待测量装置的改进外别无选择。
作为发明人，我们发现以上超过分析限制范围的样品的测定的问题可以通过减少化学发光的数量而加以解决，而不需要诸如稀释样品的费力操作，也不需要测量装置的改进。这个发现使我们完成了本发明。
本发明的公开
本发明是一种利用标记了化学发光物质的材料对物质进行分析的方法，该方法包括降低化学发光的数量。本发明进一步提供了一种分析方法，包括添加猝灭剂和/或降低化学发光物质标记的探针的特定活性。
以上表明，利用化学发光物质标记的材料进行物质的分析指的是，比如把含有被分析物的样品与标记了化学发光物质的材料反应，然后测量该共轭物质的化学发光的数量以检测或测定被分析物。
附图的简要说明
图1表示添加酚红对血清中HBV模板的定量扩增和检测的影响；
图2表示添加未标记的探针对血清中HBV模板的定量扩增和检测的影响。
本发明的最佳实施方式
(1)通过添加猝灭剂降低化学发光的数量
用于本发明的猝灭剂可以是任何能猝灭化学发光的物质。例如，它包括色料和印度油墨(印度油墨滴剂，即度油墨上清液)。色料的例子有结晶紫，溴酚蓝，胭脂红酸，氯酚红，苏木紫，溴酚紫，溴酚红，玫红酸，酚红，甲酚红，和异甲酚红。
色料用作猝灭剂时，在测量化学发光时的色料浓度可在0.01-10％，最好在0.01-1％的范围内，尽管它随着所用色料不同而不同。另一方面，印度油墨用作猝灭剂时，其在测量化学发光时的量可以在0.01-50％，最好在1-20％的范围内，依试液的量而定。猝灭剂可以在测量化学发光之前的任何时间加入。例如，可以在被分析物与标记探针反应前或反应后加入。
(2)通过降低化学发光物质标记的探针的特定活性而减少化学发光的数量。
本发明中，为了降低标记探针的特定活性，将一个未标记探针加到标记的探针上。未标记探针的加入量可以在0.1-105，最好10-103的范围，相对于标记探针的1。
(3)结合方法(1)和方法(2)
本发明中，猝灭剂的加入和化学发光物质标记的探针的特定活性的降低可以结合起来。这种结合使用两种方法的条件是遵守上述的范围。
按照本发明，分析方法的使用确保化学发光的数量能够被准确地测量，甚至当样液中被分析物的量大得超过分析的极限范围时也是如此。举例来说，从微生物或细胞的遗传信息(DNA或RNA)的基因扩增产物，可以很容易地检测或定量测定被分析物。这个办法可以从将在后面提供的实施例1-4中得到证实。
在涉及加入猝灭剂的方法中，不仅正信号而且背景(噪声)的水平也降低了。这种化学发光数量的降低使得定量地测量强的正信号成为可能，而背景(噪声)水平的降低使得分辨弱的正信号成为可能。总之，猝灭剂的加入确保了分析高强正信号的样品又不影响弱的正信号的分辨。这种作用将为以后将要提供的实施例1-3所证实。
将本发明用于减少化学发光的数量，检测或定量测定超过分析极限范围的样品或具有高强正信号水平的样品。特别地，添加猝灭剂的方法也降低了背景(噪声)水平。这样，有高强正信号的样品可以得到测量而不影响弱的正信号的样品的分析。因此，本发明的方法证明是极好的方法，它能扩宽分析范围而不用稀释样品或改进测量装置。
实施例
现在参考实施例更加详细地说明本发明，对于实施例的说明并不限定本发明。
〔实施例1〕
方法
5μl含有HBV-DNA序列的人血清(Galibert，F.，Mandavt，E.，FitioussiF.，Tiollais，P.and Charnay，P.，Nature281,646-650(1979)(每次扩增50-5,000份)与20μl碱溶液(pH13)混和，然后在97℃加热5分钟。在室温下，让混和物凉10分钟，再用缓冲液中和。加入两种引物，在室温下退火。加入DNA和RNA聚合酶后，在37℃进行基因扩增(用正式出版的日本专利公报号500759/92所述的方法)。扩增产物与吖啶酯标记的探针在60℃杂交，之后，用HPA方法检测扩增产物(Arnold JR，L.J.，Hammond，P.w.，Wiese，W.A.andNelson，N.C.，Clinical Chemistry 35，1588-1594(1988))。通过测量化学发光来检测扩增产物时，研究了添加酚红的影响。酚红以0.05％的量加入到用于化学发光测量的试液中以测量化学发光的数量。测量结果与从不含酚红的试液得到的结果比较。结果如图1所示。
讨论
如图1所示，不含酚红的样品在大约500GE(基因组当量)/AMP时，几乎达到了分析极限(饱和值)，在该值以上，化学发光的数量不再呈线性关系。对于含有酚红的样品，化学发光的数量甚至在5000GE/AMP时还保持线性关系。这意味着添加酚红使得500GE/AMP或以上的分析变为可能。与不含酚红的样品相比，含酚红的样品显著地降低了背景(噪声)水平(DNA含量＝0)，这样就使分析弱的正当信号的样品(大约50GE/AMP)成为可能。换一句话说，酚红的加入确保能分析强正信号的样品而不会影响弱的正信号的样品的测定。
〔实施例2〕
方法
在检测如实施例1中通过基因扩增得到的扩增产物时，加入不同量的酚红以研究添加酚红的影响。以0.025-0.2％的量，将酚红加入到用于化学发光测量的试液中以测量化学发光的数量。测量结果与用不含酚红的试液得到的结果比较。结果如表1所示。
表1酚红对测定化学发光数量的影响
酚红浓度(％) 正样(50GE/AMP) 负样(0GE/AMP)   正/负比
    0     237340     967     245
    0.025     73120     239     306
    0.05     41706     172     242
    0.1     19332     114     170
    0.2     7571     89     85
讨论
如表1所示，正样的化学发光的数量与负样的化学发光数量的减少依赖于所添加的酚红的量。这些发现表明，在很宽的酚红浓度范围内，本发明方法都是可行的。
〔实施例3〕
方法
以与实施例2同样的方式进行试验，除了用商业上可得到的印度油墨代替酚红之外。
向用于化学发光测量的试液中加入1.25％-10％体积的印度油墨，测量化学发光的数量。结果如表2所示，与不含印度油墨的试液的测量结果比较。
表2印度油墨对测定化学发光数量的影响
印度油墨的添加量(％体积)     正样(50GE/AMP)     负样(0GE/AMP)   正/负比值
    0     291638     1689     173
    1.25     30902     218     142
    2.5     21292     119     179
    5     6609     63     105
    10     5172     43     120
讨论
如表2所示，正样的化学发光的数量与负样的化学发光(背景)数量的减少依赖于所添加的印度油墨的数量。这些发现证明本发明方法可在很宽的印度油墨浓度范围内使用。
方法
与实施例1中同样的方式进行基因扩增。扩增产物与吖啶酯标记的探针在60℃下杂交，同时加入不同数量的未标记探针。相对于标记探针的1，未标记探针的量为10-1000。杂交后，样品中化学发光的数量用HPA方法测量。结果如图2所示，与不含未标记探针的试液的测量结果比较。
讨论
如图2所示，不含未标记探针的样品在500-5000GE/AMP时几乎达到分析极限(饱和值)，高于该值，化学发光的数值不再呈线性。相反，含有未标记探针的样品，表现出化学发光的数量的减少依赖于添加的未标记探针的数量。当添加的未标记探针的量与标记探针的量的相对值为100(未标记探针)：1(标记探针)或更高时，甚至在大约500,000GE/AMP时，分析都是可能的。这样，添加未标记探针使得分析500-500,000 GE/AMP或更高值变得可能。

Claims (6)

1.一种利用化学发光物质标记的材料对被分析物进行分析的方法，包括减少化学发光的数量。
2.权利要求1所述的分析方法，包括添加猝灭剂和/或降低化学发光物质标记的探针的特定活性。
3.权利要求1所述的分析方法，包括添加一种猝灭剂。
4.权利要求1所述的分析方法，包括降低一种化学发光物质标记的探针的特定活性。
5.权利要求3所述的分析方法，其中色料和/或印度油墨用作猝灭剂。
6.权利要求4所述的分析方法，其中把未标记的探针加到化学发光物质标记的探针上以降低其特定活性。
CN96191746A 1995-02-02 1996-02-02 以调节化学发光为手段分析被分析物的方法 Expired - Lifetime CN1100258C (zh)

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