WO2023225776A1 - 一种制备高灵敏性稳定化学发光底物液方法及其在免疫检测中应用 - Google Patents

一种制备高灵敏性稳定化学发光底物液方法及其在免疫检测中应用 Download PDF

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WO2023225776A1
WO2023225776A1 PCT/CN2022/094382 CN2022094382W WO2023225776A1 WO 2023225776 A1 WO2023225776 A1 WO 2023225776A1 CN 2022094382 W CN2022094382 W CN 2022094382W WO 2023225776 A1 WO2023225776 A1 WO 2023225776A1
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liquid
add
weigh
sodium
tris
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孙嘉怡
高雪
袁扬尘
祝长俊
李礼
韩加如
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孙嘉怡
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence

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  • the invention relates to the technical field of highly sensitive and stable chemiluminescence, and specifically relates to a method for preparing a highly sensitive and stable chemiluminescence substrate solution and its application in immune detection.
  • Chemiluminescence technology is one of the immunodiagnostic technologies.
  • the difference between immunodiagnostic technologies is generally in the two aspects of antigen coating and signal detection.
  • the chemiluminescent HRP substrate solution on the market is used in scientific research or When used in medical immunodiagnosis, most of them rely on imports and are expensive.
  • most of the domestically produced luminescent fluids have low sensitivity, unstable luminescence, or damage to the polyethylene fiber membrane used in Western blot experiments.
  • the present invention provides a method for preparing a highly sensitive and stable chemiluminescence substrate liquid, which adopts the most advanced international sensitization technology and secondary sensitization, so that the substrate liquid has high sensitivity, stable luminescence, and a clearer background. Low, can reach low fg level.
  • the liquid A is composed of Tris, sodium 3-(10-phenothiazine)-propionate, sodium 5-oxyphenothiazine-10-propylpropionate, and 4-methylpropionate. It is compounded of linylpyridine, luminol, NaOH solution, pure water and hydrochloric acid; the B liquid is compounded of peroxyurea, glacial acetic acid, pure water and NaoH solution.
  • the invention also provides a method for preparing the above-mentioned highly sensitive and stable chemiluminescence substrate liquid, which includes the following steps:
  • Tris buffer weigh 24.23g Tris and add 900mL of ultrapure water to dissolve it to obtain Tris buffer;
  • the primary catalyst contains 3-(10-phenothiazine)-sodium propionate and 5-oxyphenothiazine-10-propylpropionate sodium ;
  • the secondary catalyst is 4-morpholinopyridine.
  • the substrate solution has high sensitivity, which is higher than similar products from foreign Thermo Pierce manufacturers. It has stable luminescence, lower background, and can reach low fg level, which greatly saves money.
  • the amount of antibody and sample is particularly suitable for detecting low-abundance protein content.
  • Figure 1 shows the changes in fluorescence intensity at different times after substrate solutions from different manufacturers react with the same concentration of HRP antibody protein.
  • Figure 2 shows the effect of luminescent liquid on fluorescence stability at different temperatures.
  • Figure 3 shows the application of substrate solutions from different manufacturers in Bcl-2 protein detection (femtogram level).
  • Tris buffer weigh 24.23g Tris and add 900mL of ultrapure water to dissolve it to obtain Tris buffer;
  • Figure 1 shows the changes in fluorescence intensity at different times after the interaction between substrate solutions from different manufacturers and the same concentration of HRP antibody protein. It can be seen that under the same conditions, the fluorescence value of the self-developed product is significantly better than that of Thermo's SuperSignal West Dura product, while the blank The background value is equivalent to that of Thermo products of the same level, indicating that the self-developed product is highly sensitive and stable, with almost no change in fluorescence within 1 hour.
  • Figure 2 shows the changes in signal-to-noise ratio (S/N) of the luminescent liquid in the kit when stored at 4°C or 37°C at different time periods. In comparison, temperature has less impact on the self-developed chemiluminescent liquid. Fluorescence is relatively stable.
  • Figure 3 shows Western blot detection using substrate solutions from different manufacturers. It can be seen that in the self-developed product for Bcl-2 protein detection, the protein band display is relatively clear and the band is thick, indicating that the sensitivity is higher than that of thermo products of the same grade.
  • the background is basically the same;

Abstract

一种制备高灵敏性稳定化学发光底物液方法及其在免疫检测中应用，底物液由A液和B液等体积配制而成，A液为1000mL，pH=9.0，其中，含有Tris 24.23g、3-(10-吩噻嗪)-丙酸钠及5-氧化吩噻嗪-10-丙基丙酸钠混合物 2.123 g、4-吗啉基吡啶0.7g、鲁米诺0.44g、NaOH 0.04g、余量为盐酸和纯水；B液为1000 mL，pH=3.0~5.5，其中，含有过氧尿素 1.175g、冰乙酸 2.875m L、余量为纯水与5mol/L 的NaoH溶液。通过二次增敏，使得底物液灵敏度高，发光平稳，背景更低，可达到低 fg级，节省了抗体与样品的用量，特别适用于检测低丰度蛋白质含量。

Description

一种制备高灵敏性稳定化学发光底物液方法及其在免疫检测中应用 技术领域
本发明涉及高灵敏性稳定化学发光技术领域，具体涉及一种制备高灵敏性稳定化学发光底物液方法及其在免疫检测中应用
背景技术
化学发光技术是免疫诊断技术中的一种，而免疫诊断技术的差别，大体上在抗原体包被和信号检测这两个环节，目前市面上化学发光HRP底物液无论应用于科研中或是应用于医学免疫诊断，大部分都依赖于进口，且价格昂贵，而国产的大部分发光液灵敏度不高，发光不稳定或是对Western blot实验操作的聚乙烯纤维膜存在损伤性。
发明内容
为解决上述问题，本发明提供了一种高灵敏稳定化学发光底物液的制备方法，采用目前国际最先进的增敏技术，二次增敏，使得底物液灵敏度高，发光平稳，背景更低,可达到低fg级。
为实现上述目的，本发明采取的技术方案为：
一种高灵敏稳定化学发光底物液，所述A液由Tris、3-(10-吩噻嗪)-丙酸钠、5-氧化吩噻嗪-10-丙基丙酸钠、4-吗啉基吡啶、鲁米诺、NaOH溶液、纯水、盐酸复配而成；所述B液由过氧尿素、冰乙酸、纯水、NaoH溶液复配而成。所述A液为1000mL,pH＝9.0，其中，含有Tris 24.23g；3-(10-吩噻嗪)-丙酸钠及5-氧化吩噻嗪-10-丙基丙酸钠2.123g；4-吗啉基吡啶0.7g；鲁米诺0.44g；NaOH 0.04g；余量为盐酸和纯水；
所述B液为1000mL,pH＝3.0～5.5，其中，含有过氧尿素1.175g；冰乙 酸2.875m L；余量为5mol/L的NaoH溶液和纯水。
本发明还提供了上述一种高灵敏稳定化学发光底物液的制备方法，包括如下步骤：
S1、A液的配置
S11、配置Tris缓冲液：称取24.23g Tris加900mL超纯水溶解，得Tris缓冲液；
S12、称取3-(10-吩噻嗪)-丙酸钠及5-氧化吩噻嗪-10-丙基丙酸钠2.123g，加入到所得的Tris缓冲液中，搅拌至溶解；
S13、称取4-吗啉基吡啶0.7g，加入到上述的Tris缓冲液中，搅拌至溶解；
S14、称取鲁米诺0.44g，加入1mL 1mol/L的NaOH溶液，充分溶解后，再加入到缓冲液中，搅拌至溶解；
S15、向所得的缓冲液中加入适量盐酸调pH至9.0，定容为1000mL，配置完后避光保存；
S2、B液配置方法
S21、取2.875mL冰乙酸加入到950mL超纯水中，用5mol/L的NaOH溶液调pH至3.0～5.5；
S22、称取过氧尿素1.175g加入到上述液体中，搅拌至溶解，定容为1000mL，得B液；
S3、将A液与B液等体积混合后，即得，现用现配。
本发明具有以下有益效果：
采用目前国际最先进的增敏技术，采用两种类型增敏催化剂：一级催化剂含3-(10-吩噻嗪)-丙酸钠、5-氧化吩噻嗪-10-丙基丙酸钠；二级催化剂为4-吗啉基吡啶，通过二次增敏，使得底物液灵敏度高，高于国外Thermo Pierce厂家同类产品，发光平稳，背景更低,可达到低fg级，大大节省了抗体与样品的用量，特别适用于检测低丰度的蛋白质含量。
附图说明
图1为不同厂家的底物液与相同浓度的HRP抗体蛋白作用后不同时间荧光强度变化情况。
图2为发光液在不同的温度下对荧光稳定性影响
图3为不同厂家底物液在Bcl-2蛋白检测中应用(飞克级)。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
本发明实施例的一种高灵敏稳定化学发光底物液，通过以下步骤配置所得：
S1、A液的配置
S11、配置Tris缓冲液：称取24.23g Tris加900mL超纯水溶解，得Tris缓冲液；
S12、称取3-(10-吩噻嗪)-丙酸钠及5-氧化吩噻嗪-10-丙基丙酸钠2.123g，加入到所得的Tris缓冲液中,搅拌至溶解；
S13、称取4-吗啉基吡啶0.6g，加入到上述的Tris缓冲液中,搅拌至溶解；
S14、称取鲁米诺0.44g，加入1mL 1mol/L的NaOH溶液，充分溶解后，再加入到缓冲液中,搅拌至溶解；
S15、向所得的缓冲液中加入适量盐酸调pH至9.0，定容为1000mL，配置完后避光保存；
S2、B液配置方法
S21、取2.875mL冰乙酸加入到950mL超纯水中，用5mol/L的NaOH溶液调pH至3.0～5.5；
S22、称取过氧尿素1.175g加入到上述液体中，搅拌至溶解,定容为1000mL，得B液；
S3、将A液与B液等体积混合后，即得，现用现配。
图1为不同厂家的底物液与相同浓度的HRP抗体蛋白作用后不同时间荧光强度变化情况，可见，同等条件下，自研产品的荧光值都显著优于Thermo的SuperSignal West Dura产品,而空白背景值与Thermo同级别产品相当，说明自研产品灵敏度较高，且稳定，1h之内荧光几乎没有变化。
图2为试剂盒中发光液存储在4℃或37℃，于不同的时间段，检测信噪比(S/N)变化情况，相比较而言温度对自主研发的化学发光液影响较小，荧光相对稳定。
图3为用不同的厂家底物液进行Western blot检测，可知，在Bcl-2蛋白检测中自研产品，蛋白条带显示比较清晰，条带较粗，说明灵敏度比thermo同等级产品灵敏度高，背景基本相同；
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变化或修改，这并不影响本发明的实质内容。在不冲突的情况下，本申请的实施例和实施例中的特征可以任意相互组合。

Claims (3)

  1. 一种制备高灵敏稳定化学发光底物液方法，该底物液由A液和B液等体积复配而成，其特征在于：所述A液由Tris、3-(10-吩噻嗪)-丙酸钠及5-氧化吩噻嗪-10-丙基丙酸钠混合物、4-吗啉基吡啶、鲁米诺、NaOH溶液、纯水、盐酸复配而成；所述B液由过氧尿素、冰乙酸、纯水、NaoH溶液复配而成。
  2. 如权利要求1所述的一种制备灵敏稳定化学发光底物液方法，其特征在于：所述A液为1000mL,pH＝9.0，其中，含有Tris 24.23g；；3-(10-吩噻嗪)-丙酸钠及5-氧化吩噻嗪-10-丙基丙酸钠混合物2.123g；4-吗啉基吡啶0.7g；鲁米诺0.44g；NaOH 0.04g；余量为盐酸和纯水；
    所述B液为1000mL,pH＝3.0～5.5，其中，含有过氧尿素1.175g；冰乙酸2.875mL；余量为5mol/L的NaoH溶液和纯水。
  3. 如权利要求1所述的一种制备灵敏稳定化学发光底物液方法，其特征在于：通过以下步骤配置：
    S1、A液的配置
    S11、配置Tris缓冲液：称取24.23g Tris加900mL超纯水溶解，得Tris缓冲液；
    S12、称取；3-(10-吩噻嗪)-丙酸钠及5-氧化吩噻嗪-10-丙基丙酸钠混合物2.123g，加入到所得的Tris缓冲液中，搅拌至溶解；
    S13、称取4-吗啉基吡啶0.7g，加入到上述的Tris缓冲液中，搅拌至溶解；
    S14、称取鲁米诺0.44g，加入1mL 1mol/L的NaOH溶液，充分溶解后，再加入到缓冲液中，搅拌至溶解；
    S15、向所得的缓冲液中加入适量盐酸调pH至9.0，定容为1000mL，配置 完后避光保存；
    S2、B液配置方法
    S21、取2.875mL冰乙酸加入到950mL超纯水中，用5mol/L的NaOH溶液调pH至3.0～5.5；
    S22、称取过氧尿素1.175g加入到上述液体中，搅拌至溶解，定容为1000mL，得B液；
    S3、将A液与B液等体积混合后，即得，现用现配。
PCT/CN2022/094382 2022-05-23 2022-05-23 一种制备高灵敏性稳定化学发光底物液方法及其在免疫检测中应用 WO2023225776A1 (zh)

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