WO2022252241A1 - 一种稳定、灵敏的髓过氧化物酶检测试剂盒 - Google Patents

一种稳定、灵敏的髓过氧化物酶检测试剂盒 Download PDF

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WO2022252241A1
WO2022252241A1 PCT/CN2021/098510 CN2021098510W WO2022252241A1 WO 2022252241 A1 WO2022252241 A1 WO 2022252241A1 CN 2021098510 W CN2021098510 W CN 2021098510W WO 2022252241 A1 WO2022252241 A1 WO 2022252241A1
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myeloperoxidase
reagent
detection kit
add
latex particles
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PCT/CN2021/098510
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French (fr)
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甘宜梧
谢清华
李志明
胡晓飞
董雯
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山东博科生物产业有限公司
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)

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  • the invention belongs to the technical field of biochemical detection, in particular to a stable and sensitive myeloperoxidase detection kit.
  • Myeloperoxidase (myeloperoxidase, MPO) is a heme protease of the heme prosthetic group, a member of the heme peroxidase superfamily, and exists in myeloid cells (mainly neutrophils and monocytes) In the azurophilic blue granules, it is a specific marker of myeloid cells.
  • MPO gene polymorphisms lead to individual differences in susceptibility to some diseases, which are closely related to the occurrence and development of various human diseases, so more and more attention has been paid by domestic and foreign scholars.
  • MPO exists in three subtypes in myeloid cells, namely MPOI,II,III.
  • the three subtypes mainly have differences in the heavy chain, and the difference in the light chain is small, which leads to their differences in relative molecular mass and hydrophobicity.
  • the functional differences of the three subtypes are still unclear and need to be further studied.
  • the present invention provides a stable and sensitive myeloperoxidase detection kit.
  • the kit can effectively detect the content of myeloperoxidase, and has the advantages of high sensitivity, good stability, wide linear range and the like.
  • a stable and sensitive myeloperoxidase detection kit comprising reagent R1 and reagent R2, the composition and content of the reagent R1 are:
  • composition and content of the reagent R2 are:
  • the buffers of the reagents R1 and R2 are DIPSO buffers at 25°C and pH 6-8.
  • the preservative is o-o-cymene-5-ol.
  • preparation steps of the rabbit anti-human myeloperoxidase antibody-coated latex particles are as follows:
  • Step 1 Take 1 mg of carboxyl latex particles with a particle size of 83nm and carboxyl latex particles with a particle size of 185nm, add MOPS-HCL buffer respectively, oscillate and mix well, then centrifuge, discard the supernatant, then add MOPS-HCL buffer and ultrasonically weigh Suspended to obtain 83nm carboxyl latex particle buffer system and 185nm carboxyl latex particle buffer system;
  • Step 2 Add EDC and NHS to the 83nm carboxyl latex particle buffer system and the 185nm carboxyl latex particle buffer system obtained in step 1, respectively, and supplement MOPS-HCL buffer to 5mL, shake and react at a certain temperature and rotation speed for a period of time, and carry out activation;
  • Step 3 After the activation, centrifuge, remove the supernatant, add MOPS-HCL buffer for ultrasonic resuspension, repeat once, then add PIPES for ultrasonic resuspension, and set aside;
  • Step 4 Add myeloperoxidase antibody to the liquid obtained in step 3, mix well, and react in a constant temperature shaking box;
  • Step 5 After the reaction is over, add BSA and block at 2-8°C for a period of time;
  • Step 6 Centrifuge, discard the supernatant, and the resulting precipitates are 83nm rabbit anti-human myeloperoxidase antibody-coated latex particles and 185nm rabbit anti-human myeloperoxidase antibody-coated latex particles.
  • centrifugation speed corresponding to the carboxyl latex particles with a particle diameter of 83nm is 20000rpm and the time is 45min; the centrifugation speed corresponding to the carboxyl latex particles with a particle diameter of 185nm is 12000rpm and the time is 45min.
  • the kit uses an automatic biochemical analyzer for determination by the endpoint method, the main detection wavelength is 570nm, and the ratio of the R1 reagent to the R2 reagent is 4:1.
  • the basic principle of the detection of the present invention is: the antigenic myeloperoxidase in the sample forms an insoluble immune complex with the hypersensitized rabbit anti-human myeloperoxidase antibody latex particles in the reagent, and the turbidity change is detected at a wavelength of 570nm.
  • the degree of change is proportional to the concentration of myeloperoxidase in the sample.
  • the present invention adopts latex-enhanced immune turbidimetry, by optimizing the reaction system, reagent R1 and reagent R2 add stabilizers such as D-idose and erythritol in the buffer solution, optimize the proportioning of each stabilizer, Significantly improved reagent stability.
  • the preferred new surfactant triisopropanolamine polyoxypropylene polyoxyethylene ether can promote and maintain antibody stability, prevent system turbidity, and significantly enhance the stability and anti-interference ability of the reagent.
  • the reagent is easy and quick to operate, and is suitable for automatic analysis. It is a more stable, sensitive, and wide linear range myeloperoxidase reagent.
  • the reagent has good accuracy and stability, strong anti-interference, and is easy to use. It can fully meet the clinical needs.
  • Fig. 1 is a reagent calibration curve diagram of the stable and sensitive myeloperoxidase detection kit of the present invention.
  • Fig. 2 is a reagent stability comparison curve of the stable and sensitive myeloperoxidase detection kit of the present invention.
  • the detection kit for myeloperoxidase provided in this example includes reagent R1 and reagent R2:
  • the reagent R1 includes the following components:
  • the reagent R2 includes the following components:
  • the DIPSO buffer of the reagent R1 is a buffer with a pH of 6.35 at 25°C
  • the DIPSO buffer of the reagent R2 is a buffer with a pH of 7.88 at 25°C.
  • Step 2 Add 0.6 mg EDC and 0.4 mg NHS to each 1 mg of carboxyl latex particles in the 83 nm carboxyl latex particle buffer system and 185 nm carboxy latex particle buffer system obtained in step 1, and then add MOPS-HCL buffer to 5 mL, at 32 °C, rotating speed 110rpm shaking reaction 25min, to activate;
  • Step 4 Add 3 mg of myeloperoxidase antibody to the solution obtained in Step 3, mix well, and react in a constant temperature shaking box at 40°C for 3 hours;
  • Step 5 After the reaction, add 200 ⁇ L 50mg/mL BSA, and block for 12 hours at 2-8°C;
  • Step 6 Centrifuge (carboxyl latex particles with a particle size of 83nm at a centrifugal speed of 20,000rpm for 45 minutes; carboxyl latex particles with a particle size of 185nm at a centrifugal speed of 12000rpm for 45 minutes), discard the supernatant, and the resulting precipitate is the 83nm rabbit anti-human Myeloperoxidase antibody-coated latex particles and 185nm rabbit anti-human myeloperoxidase antibody-coated latex particles.
  • the myeloperoxidase detection kit described in this example uses an automatic biochemical analyzer with dual reagent functions, such as Hitachi 7180 automatic analyzer, etc., and uses the endpoint method for determination. Place R1 and R2 on the corresponding reagent positions according to the ratio of 4:1, and place distilled water, standards and samples on the corresponding positions of the sample plate. The operation is shown in Table 1.
  • the myeloperoxidase detection kit described in this example includes reagent R1 and reagent R2:
  • reagent R1 The components of reagent R1 include:
  • the reagent R1 comprises the following components:
  • the reagent R2 includes the following components:
  • MPO myeloperoxidase
  • Example 1 Comparative example 1 Average (X) 319.7 291.7 287.4 Standard Deviation (S) 2.621 2.246 10.318 Coefficient of Variation CV 0.82% 0.77% 3.59%
  • Example 1 Comparative example 1 Average (X) 79.6 79.2 78.1 Standard Deviation (S) 1.425 1.433 3.836 Coefficient of Variation CV 1.79% 1.81% 4.91%
  • Example 1 Comparative example 1 maximum value 321.0 320.8 321.6 minimum value 319.0 319.4 310.6 average value 320.3 320.6 313.6
  • Example 1 Example 2 Comparative example 1 maximum value 80.0 79.2 79.8 minimum value 77.0 76.3 74.6 average value 79.1 79.6 76.3
  • the detection values of the formula preparation kits in Example 1 and Example 2 are closer to the target value and have higher accuracy, which shows that this
  • the invention uses latex-enhanced immune turbidimetry (combination of two kinds of carboxyl nano-latex particles with different particle sizes), and at the same time uses new surfactants triisopropanolamine polyoxypropylene polyoxyethylene ether, carboxylated ferric oxide magnetic nano Particles and carboxylated ferric oxide magnetic nanoparticles, scientifically proportioned D-idose, erythritol and other stabilizers, optimize the reaction system, and greatly improve the accuracy of the reagent.
  • Example 1 Take the traceable calibrator and dilute 7 concentration samples from low to high, use the formulas of Example 1, Example 2 and Comparative Example 1 to prepare reagents respectively, and prepare a myeloperoxidase detection kit for control detection. The test results were compared with the theoretical concentrations. The results are shown in Table 4.
  • Example 2 Comparative example 1 2.5ng/mL 2.8 2.3 0.5 12.5ng/mL 12.8 12.3 9.8 25ng/mL 25.5 26.3 23.5 400ng/mL 401.0 401.4 400.4 600ng/mL 602.4 602.6 601.4 800ng/mL 800.4 800.5 751.5 1300ng/mL 1304.8 1302.6 1108.2
  • embodiment 1, embodiment 2 formula preparation reagents have higher analytical sensitivity, accuracy and wider linear range.
  • the present invention enhances immune turbidimetry by adopting latex (two kinds of carboxyl nano-latex particles with different particle sizes are used in combination), and adopts novel surfactant triisopropanolamine polyoxypropylene polyoxyethylene ether, and carboxylated trioxide Diferromagnetic nanoparticles, scientifically proportioned D-idose, erythritol and other stabilizers optimize the reaction system and greatly improve the analytical sensitivity of the reagent.
  • Reagents were prepared with the formulations of Example 1, Example 2 and Comparative Example 1 to prepare a myeloperoxidase detection kit for calibration detection.
  • the absorbance results of each concentration point are shown in Table 5, and the calibration curve is shown in Figure 1.
  • Example 2 Comparative example 1 0ng/mL 0.12 0.54 1.14 300ng/mL 852.21 785.13 421.02 600ng/mL 1822.15 1752.32 1225.21 900ng/mL 3200.33 3001.23 2450.1 1300ng/mL 5200.25 5012.45 3785.36
  • the present invention enhances immune turbidimetry by adopting latex (2 kinds of carboxyl nano-latex particles with different particle sizes are used in combination), and adopts novel surfactant triisopropanolamine polyoxypropylene polyoxyethylene ether, and carboxylated three Ferric oxide magnetic nanoparticles, scientifically proportioned D-idose, erythritol and other stabilizers, optimize the reaction system, and greatly improve the analytical sensitivity and linear range of the reagent.
  • Example 2 and Comparative Example 1 13 groups were evenly distributed, and the reagent volume of each group was 20 mL for R1 and 5 mL for R2. Place it in the refrigerator at 2-8°C, take out a group of reagents to detect the myeloperoxidase quality control product (target value is 320ng/mL) on the same day every month, the test results are as shown in Figure 2, the reagents in Example 1-2 It is more stable than the comparative example under the storage condition of 2-8°C.
  • the present invention enhances immune turbidimetry by adopting latex (2 kinds of carboxyl nano-latex particles with different particle diameters are combined), and adopts novel surfactant triisopropanolamine polyoxypropylene polyoxyethylene ether, scientific proportioning D- Stabilizers such as idose and erythritol optimize the reaction system and greatly improve the stability of the reagent.
  • the kit provided by the present invention has higher sensitivity, better repeatability, and wider linear range than the kit of Comparative Example 1, and can meet the application requirements of the market for products, and has high accuracy.
  • Stable and sensitive myeloperoxidase detection kit is provided by the present invention.

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Abstract

提供了一种髓过氧化物酶检测试剂盒。所述检测试剂盒包括试剂R1、R2，试剂R1中含有缓冲液、氯化锂、D-艾杜糖、赤藓糖醇、PEG-10000、三异丙醇胺聚氧丙烯聚氧乙烯醚、防腐剂；试剂R2中含有缓冲液、D-艾杜糖、赤藓糖醇、三异丙醇胺聚氧丙烯聚氧乙烯醚、羧基化三氧化二铁磁性纳米颗粒、83nm的兔抗人髓过氧化物酶抗体包被胶乳颗粒、185nm的兔抗人髓过氧化物酶抗体包被胶乳颗粒、防腐剂。该试剂盒可有效检测髓过氧化物酶的含量，灵敏度高、稳定性好、线性范围宽。

Description

一种稳定、灵敏的髓过氧化物酶检测试剂盒 技术领域
本发明属于生化检测技术领域，具体涉及一种稳定、灵敏的髓过氧化物酶检测试剂盒。
背景技术
髓过氧化物酶(myeloperoxidase，MPO)是血红素辅基的血红素蛋白酶，是血红素过氧化物酶超家族成员之一，存在于髓系细胞(主要是中性粒细胞和单核细胞)的嗜苯胺蓝颗粒中，是髓细胞的特异性标志。随着对MPO研究的深入，人们发现MPO基因多态性导致个体对一些疾病易感性的差异，与人类多种疾病的发生、发展密切相关，因此越来越受到国内外学者的重视。
MPO以3种亚型存在于髓系细胞中，分别为MPOⅠ、Ⅱ、Ⅲ。3种亚型主要是重链有差异，轻链的差异较小，导致它们在相对分子质量及疏水性等方面不同，3种亚型在功能上的差异还不明确，有待进一步研究。
目前，测定髓过氧化物酶常用的方法有流式细胞仪测定法、ELISA法、连续监测法等。其中流式细胞仪测定法的技术程序比较复杂，需使用特殊仪器；ELISA法，操作繁琐，灵敏度低，检测结果多为定性；连续监测法，操作简单、使用方便，但灵敏度和稳定性较差，线性范围比较窄。
发明内容
为解决上述问题，本发明提供一种稳定、灵敏的髓过氧化物酶检测试剂盒。该试剂盒可以有效检测髓过氧化物酶的含量，具有灵敏度高、稳定性好、线性范围宽等优点。
为解决上述技术问题，本发明提供技术方案如下：
一种稳定、灵敏的髓过氧化物酶检测试剂盒，包括试剂R1和试剂R2，所述试剂R1的成分和含量为：
Figure PCTCN2021098510-appb-000001
所述试剂R2的成分和含量为：
Figure PCTCN2021098510-appb-000002
进一步的，所述试剂R1、R2的缓冲液为25℃、pH为6-8的DIPSO缓冲液。
进一步的，所述防腐剂为o-邻伞花烃-5-醇。
进一步的，所述兔抗人髓过氧化物酶抗体包被胶乳颗粒的制备步骤如下：
步骤1：各取1mg粒径为83nm的羧基胶乳颗粒和185nm的羧基胶乳颗粒，分别加入MOPS-HCL缓冲液，振荡混匀，然后离心，弃上清，再分别加入 MOPS-HCL缓冲液超声重悬，得到83nm羧基胶乳颗粒缓冲体系和185nm羧基胶乳颗粒缓冲体系；
步骤2：于步骤1得到的83nm羧基胶乳颗粒缓冲体系和185nm羧基胶乳颗粒缓冲体系中分别加入EDC和NHS，并补充MOPS-HCL缓冲液至5mL，于一定温度及转速下振荡反应一段时间，进行活化；
步骤3：活化结束后，离心，去上清，分别加入MOPS-HCL缓冲液进行超声重悬，重复一次，之后加入PIPES超声重悬，待用；
步骤4：在步骤3所得液体中分别加入髓过氧化物酶抗体，混匀，于恒温振荡箱反应；
步骤5：反应结束后，加入BSA，于2-8℃封闭一段时间；
步骤6：离心，弃上清，所得沉淀即为83nm兔抗人髓过氧化物酶抗体包被胶乳颗粒和185nm兔抗人髓过氧化物酶抗体包被胶乳颗粒。
进一步的，粒径为83nm的羧基胶乳颗粒对应的离心转速为20000rpm，时间45min；粒径为185nm的羧基胶乳颗粒对应的离心转速为12000rpm，时间45min。
进一步的，该试剂盒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570nm，R1试剂和R2试剂的比例为4:1。
本发明检测的基本原理为：样本中的抗原髓过氧化物酶与试剂中超敏化的兔抗人髓过氧化物酶抗体胶乳颗粒形成不溶性免疫复合物，在波长570nm处检测其浊度变化，其变化程度与样本中髓过氧化物酶浓度成正比。
本发明的有益效果在于：
(1)本发明采用胶乳增强免疫比浊法，通过优化反应体系，试剂R1和试剂R2在缓冲液中添加D-艾杜糖、赤藓糖醇等稳定剂，优化各稳定剂的配比， 显著改善了试剂的稳定性。
(2)优选的新型表面活性剂三异丙醇胺聚氧丙烯聚氧乙烯醚，可以促进并维持抗体稳定，防止体系浑浊，显著增强了试剂的稳定性和抗干扰能力。
(3)添加羧基化三氧化二铁磁性纳米颗粒，通过科学配比，大大增强了试剂的反应灵敏度和线性范围，试剂的重复性和抗干扰能力更强。
(4)采用胶乳增强免疫比浊法，且83nm和185nm两种不同粒径的羧基胶乳微球联用，科学配比、大大增强了试剂的反应灵敏度和线性范围，使试剂的重复性和抗干扰能力更强。
(5)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、线性范围宽的髓过氧化物酶试剂，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为本发明的稳定、灵敏的髓过氧化物酶检测试剂盒的试剂校准曲线图。
图2为本发明的稳定、灵敏的髓过氧化物酶检测试剂盒的试剂稳定性对比曲线图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合具体对比例与实施例来进一步详细说明。
实施例1
本实施例提供的髓过氧化物酶的检测试剂盒，包括试剂R1和试剂R2：
(1)所述试剂R1包括如下含量的组分：
Figure PCTCN2021098510-appb-000003
Figure PCTCN2021098510-appb-000004
(2)所述试剂R2包括如下含量的组分：
Figure PCTCN2021098510-appb-000005
(3)所述试剂R1的DIPSO缓冲液为25℃、pH为6.35的缓冲液，所述试剂R2的DIPSO缓冲液为25℃、pH为7.88的缓冲液。
(4)所述83nm和185nm兔抗人髓过氧化物酶抗体包被胶乳颗粒的制备步骤如下：
步骤1：各取1mg粒径为83nm的羧基胶乳颗粒和185nm的羧基胶乳颗粒，分别加入5mL pH＝5.8的MOPS-HCL缓冲液，振荡混匀，然后离心(粒径为83nm的羧基胶乳颗粒，离心速度20000rpm，时间45min；粒径为185nm的羧基胶乳颗粒，离心速度12000rpm，时间45min)，弃上清，加入3mL pH＝5.8的MOPS-HCL缓冲液超声重悬，得到83nm羧基胶乳颗粒缓冲体系和185nm羧基胶乳颗粒缓 冲体系；
步骤2：于步骤1得到的83nm羧基胶乳颗粒缓冲体系和185nm羧基胶乳颗粒缓冲体系中，分别每1mg羧基胶乳颗粒加入0.6mg EDC和0.4mg NHS，然后补充MOPS-HCL缓冲液至5mL，于32℃、转速110rpm振荡反应25min，进行活化；
步骤3：活化结束后，离心(粒径为83nm的羧基胶乳颗粒，离心速度20000rpm，时间45min；粒径为185nm的羧基胶乳颗粒，离心速度12000rpm，时间45min)，去上清，加入3mL MOPS-HCL缓冲液超声重悬，重复一次，加入5mL pH＝7.15的PIPES超声重悬，待用；
步骤4：在步骤3所得溶液中加入3mg髓过氧化物酶抗体，混匀，于恒温振荡箱40℃反应3h；
步骤5：反应结束后，加入200μL 50mg/mL BSA，2-8℃封闭12小时；
步骤6：离心(粒径为83nm的羧基胶乳颗粒，离心速度20000rpm，时间45min；粒径为185nm的羧基胶乳颗粒，离心速度12000rpm，时间45min)，弃上清，所得沉淀即为83nm兔抗人髓过氧化物酶抗体包被胶乳颗粒和185nm兔抗人髓过氧化物酶抗体包被胶乳颗粒。
(5)本实施例试剂盒的使用方法：
本实施例描述的髓过氧化物酶检测试剂盒，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试剂检测方法表
Figure PCTCN2021098510-appb-000006
Figure PCTCN2021098510-appb-000007
计算：髓过氧化物酶含量(ng/mL)＝(ΔA测定÷ΔA标准)×C标准
实施例2
本实施例所描述的髓过氧化物酶的检测试剂盒，包试剂R1和试剂R2：
(1)试剂R1的组分包括：
所述试剂R1包括如下含量的组分：
Figure PCTCN2021098510-appb-000008
(2)所述试剂R2包括如下含量的组分：
Figure PCTCN2021098510-appb-000009
Figure PCTCN2021098510-appb-000010
具体制备过程及试剂使用方法同实施例1。
对比例1
本对比例采用市场常见的国家食品药品监督管理局认可的髓过氧化物酶(MPO)检测试剂盒。该试剂盒未采用2种胶乳颗粒，也未添加新型表面活性剂三异丙醇胺聚氧丙烯聚氧乙烯醚和羧基化三氧化二铁磁性纳米颗粒(DMSA-Fe2O3)。
下面将对实施例1、实施例2及对比例1进行对比试验。
试验一 精密度试验
取具有溯源性的高值质控物(靶值320ng/mL)、低值质控物(靶值80ng/mL)各1份，分别用实施例1、实施例2和对比例1配方配制试剂，制得髓过氧化物酶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。结果见表2-1及表2-2。
表2-1高值质控物精密度试验数据表
  实施例1 实施例2 对比例1
平均值(X) 319.7 291.7 287.4
标准偏差(S) 2.621 2.246 10.318
变异系数CV 0.82％ 0.77％ 3.59％
表2-2低值质控物精密度试验数据表
  实施例1 实施例2 对比例1
平均值(X) 79.6 79.2 78.1
标准偏差(S) 1.425 1.433 3.836
变异系数CV 1.79％ 1.81％ 4.91％
由表2-1及表2-2中变异系数可知，与对比例1相比，实施例1、实施例2配方配制试剂盒检测数值更接近靶值，标准偏差和变异系数均较小，具有更高的批内精密度，这说明本发明通过采用胶乳增强免疫比浊法(2种不同粒径羧基纳米胶乳颗粒联用)，并且采用新型表面活性剂三异丙醇胺聚氧丙烯聚氧乙烯醚，以及羧基化三氧化二铁磁性纳米颗粒，科学配比，优化了反应体系，极大的提高了试剂的批内精密度。
试验二 准确度对比试验
取具有溯源性的高值质控物(靶值320ng/mL)、低值质控物(靶值80ng/mL)各1份，分别用实施例1、实施例2和对比例1配方配制试剂，制得髓过氧化物酶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。结果见表3-1及表3-2。
表3-1高值质控物准确度试验数据表
  实施例1 实施例2 对比例1
最大值 321.0 320.8 321.6
最小值 319.0 319.4 310.6
平均值 320.3 320.6 313.6
表3-2低值质控物准确度试验数据表
  实施例1 实施例2 对比例1
最大值 80.0 79.2 79.8
最小值 77.0 76.3 74.6
平均值 79.1 79.6 76.3
由表3-1及表3-2中检测结果可知，与对比例1相比，实施例1、实施例2配方配制试剂盒检测数值更接近靶值，具有更高的准确度，这说明本发明通过采用胶乳增强免疫比浊法(2种不同粒径羧基纳米胶乳颗粒联用)，同时采用新型表面活性剂三异丙醇胺聚氧丙烯聚氧乙烯醚、羧基化三氧化二铁磁性纳米颗 粒以及羧基化三氧化二铁磁性纳米颗粒，科学配比D-艾杜糖、赤藓糖醇等稳定剂，优化了反应体系，极大地提高了试剂的准确度。
试验三 试剂灵敏度的对比试验
取具有溯源性的校准品由低到高稀释出7个浓度样本，分别用实施例1、实施例2和对比例1配方配制试剂，制得髓过氧化物酶检测试剂盒进行对照检测，将检测结果与理论浓度相比较。结果见表4。
表4灵敏度对比试验数据表
理论浓度 实施例1 实施例2 对比例1
2.5ng/mL 2.8 2.3 0.5
12.5ng/mL 12.8 12.3 9.8
25ng/mL 25.5 26.3 23.5
400ng/mL 401.0 401.4 400.4
600ng/mL 602.4 602.6 601.4
800ng/mL 800.4 800.5 751.5
1300ng/mL 1304.8 1302.6 1108.2
由表4中检测结果可知，在样本浓度低至2.5ng/mL时，对比例1检测值为0.5，而实施例1、实施例2配方配制试剂仍可以检测出样本的准确值；并且与对比例1相比，实施例1、实施例2配方配制试剂检测接近线性下限的低值样本(2.5-25ng/mL)时的准确度更高。另外，在样本浓度在线性上限1300ng/mL时，对比例检测值为1108.2ng/mL，测试值明显偏低，而实施例1、实施例2配方配制试剂仍可以检测出样本的准确值，这表明实施例1、实施例2配方配制试剂拥有更高的分析灵敏度、准确度及更宽的线性范围。这说明本发明通过采用胶乳增强免疫比浊法(2种不同粒径羧基纳米胶乳颗粒联用)，并且采用新型表面活性剂三异丙醇胺聚氧丙烯聚氧乙烯醚，以及羧基化三氧化二铁磁性纳米颗粒，科学配比D-艾杜糖、赤藓糖醇等稳定剂，优化了反应体系，极大地提高了试剂的分析灵敏度。
试验四 试剂校准曲线对比试验
用实施例1、实施例2和对比例1配方配制试剂，制得髓过氧化物酶检测试剂盒进行校准检测。各浓度点的吸光度结果见表5，校准曲线见附图1。
表5各试剂盒校准对比试验数据表
理论浓度 实施例1 实施例2 对比例1
0ng/mL 0.12 0.54 1.14
300ng/mL 852.21 785.13 421.02
600ng/mL 1822.15 1752.32 1225.21
900ng/mL 3200.33 3001.23 2450.1
1300ng/mL 5200.25 5012.45 3785.36
由表5中检测结果可知，校准品浓度为300ng/mL时，对比例1的吸光度值为421.02，而实施例1、实施例2配方配制试剂的吸光度为852.21和785.13；另外，在样本浓度在线性上限1300ng/mL时，对比例1的吸光度值为3785.36，远远低于实施例1-2配方配制试剂，这表明实施例1、实施例2配方配制试剂盒拥有更高的分析灵敏度、更宽的线性范围。因此可以说明本发明通过采用胶乳增强免疫比浊法(2种不同粒径羧基纳米胶乳颗粒联用)，并且采用新型表面活性剂三异丙醇胺聚氧丙烯聚氧乙烯醚，以及羧基化三氧化二铁磁性纳米颗粒，科学配比D-艾杜糖、赤藓糖醇等稳定剂，优化了反应体系，极大的提高了试剂的分析灵敏度和线性范围。
试验五 试剂的稳定性对比试验
对实施例1、实施例2和对比例1中的试剂，分别均匀分装13组，每组的试剂量为R1为20mL，R2为5mL。放置到2-8℃冰箱中，每月的同一天取出一组试剂检测髓过氧化物酶质控品(靶值为320ng/mL)，检测结果如图2所示，实施例1-2试剂在2-8℃储存条件下比对比例更加稳定。
由检测结果可知，在保存的13个月时间内，对比例1检测数值为257.7-326.5ng/mL，与靶值相差6.5-62.3ng/mL，并且检测值呈现明显的随储存时间延长而降低趋势；而实施例1、实施例2的试剂检测数值为316.4-330.9ng/mL，与 靶值相差3.6-10.9；这表明实施例1、实施例2的试剂拥有更高的稳定性。这说明本发明通过采用胶乳增强免疫比浊法(2种不同粒径羧基纳米胶乳颗粒联用)，并且采用新型表面活性剂三异丙醇胺聚氧丙烯聚氧乙烯醚，科学配比D-艾杜糖、赤藓糖醇等稳定剂，优化了反应体系，极大的提高了试剂的稳定性。
综上所述，通过验证，本发明提供的试剂盒比对比例1的试剂盒灵敏度高、重复性好、线性范围宽，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、灵敏的髓过氧化物酶检测试剂盒。

Claims (6)

  1. 一种稳定、灵敏的髓过氧化物酶检测试剂盒，其特征在于，包括试剂R1和试剂R2，所述试剂R1的成分和含量为：
    Figure PCTCN2021098510-appb-100001
    所述试剂R2的成分和含量为：
    Figure PCTCN2021098510-appb-100002
  2. 根据权利要求1所述的髓过氧化物酶检测试剂盒，其特征在于，所述试剂R1、R2的缓冲液为25℃、pH为6-8的DIPSO缓冲液。
  3. 根据权利要求1所述的髓过氧化物酶检测试剂盒，其特征在于，所述防腐剂为o-邻伞花烃-5-醇。
  4. 根据权利要求1所述的髓过氧化物酶检测试剂盒，其特征在于，所述兔 抗人髓过氧化物酶抗体包被胶乳颗粒的制备步骤如下：
    步骤1：各取1mg粒径为83nm的羧基胶乳颗粒和185nm的羧基胶乳颗粒，分别加入MOPS-HCL缓冲液，振荡混匀，然后离心，弃上清，再分别加入MOPS-HCL缓冲液超声重悬，得到83nm羧基胶乳颗粒缓冲体系和185nm羧基胶乳颗粒缓冲体系；
    步骤2：于步骤1得到的83nm羧基胶乳颗粒缓冲体系和185nm羧基胶乳颗粒缓冲体系中分别加入EDC和NHS，并补充MOPS-HCL缓冲液至5mL，于一定温度及转速下振荡反应一段时间，进行活化；
    步骤3：活化结束后，离心，去上清，分别加入MOPS-HCL缓冲液进行超声重悬，重复一次，之后加入PIPES超声重悬，待用；
    步骤4：在步骤3所得液体中分别加入髓过氧化物酶抗体，混匀，于恒温振荡箱反应；
    步骤5：反应结束后，加入BSA，于2-8℃封闭一段时间；
    步骤6：离心，弃上清，所得沉淀即为83nm兔抗人髓过氧化物酶抗体包被胶乳颗粒和185nm兔抗人髓过氧化物酶抗体包被胶乳颗粒。
  5. 根据权利要求4所述的髓过氧化物酶检测试剂盒，其特征在于，粒径为83nm的羧基胶乳颗粒对应的离心转速为20000rpm，时间45min；粒径为185nm的羧基胶乳颗粒对应的离心转速为12000rpm，时间45min。
  6. 根据权利要求1所述的髓过氧化物酶检测试剂盒，其特征在于，该试剂盒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570nm，R1试剂和R2试剂的比例为4:1。
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