WO2018233174A1 - Glycosylated hemoglobin on-line hemolytic enzymatic assay kit - Google Patents

Glycosylated hemoglobin on-line hemolytic enzymatic assay kit Download PDF

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WO2018233174A1
WO2018233174A1 PCT/CN2017/108541 CN2017108541W WO2018233174A1 WO 2018233174 A1 WO2018233174 A1 WO 2018233174A1 CN 2017108541 W CN2017108541 W CN 2017108541W WO 2018233174 A1 WO2018233174 A1 WO 2018233174A1
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reagent
mmol
sodium salt
acid
kit
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PCT/CN2017/108541
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Chinese (zh)
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邹炳德
邹继华
贾江花
何才郎
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美康生物科技股份有限公司
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Publication of WO2018233174A1 publication Critical patent/WO2018233174A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • 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
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
    • G01N2333/805—Haemoglobins; Myoglobins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • the invention relates to the technical field of biological preparations, in particular to a glycosylated hemoglobin online hemolysis enzyme detection kit.
  • HbA1c is a glycated Hb in which the ⁇ -amino group at the N-terminus of the ⁇ chain of Hb is glycated, and is also particularly important as a diagnostic indicator for diabetes in glycated Hb.
  • HbA1c is mainly determined by ion exchange chromatography, high performance liquid chromatography, affinity chromatography, radioimmunoassay, enzyme immunoassay, and latex immunoagglutination.
  • the above methods are expensive and require expensive equipment. Or the accuracy of detection, anti-interference, speed, etc. are not ideal, which limits its large-scale clinical application to a certain extent.
  • the newly developed glycosylated hemoglobin detection kit utilizes redox reaction, which overcomes the shortcomings of the above methods, such as low sensitivity, poor repeatability, and time-consuming and large-scale detection. It has high detection sensitivity, no pollution of reaction products and anti-interference.
  • the utility model has the advantages of strong ability and the like; the specific operation of the glycosylated hemoglobin detection test kit is: collecting EDTA anticoagulated fresh whole blood from the patient, performing natural sedimentation of the red blood cells, and then adding to the 250 uL kit hemolysis buffer, and hemolyzing for 10 minutes at room temperature; The obtained 25 ul hemolyzed sample was mixed with 112 uL of the reagent R1a and 48 uL of the reagent R1b, and the bath was incubated at 37 ° C for 3 minutes, and the absorbance A1 was read. Further, 70 uL of the reagent 2 was added to the reaction system, and the mixture was incubated at 37 ° C for 2 minutes, and the absorbance A2 was read.
  • ⁇ A A2-A1.
  • the absorbance at 751 nm and 571 nm can be selected for the measurement.
  • the Hb concentration can be obtained from the absorbance at a wavelength of 751 nm
  • the HbA1c concentration can be obtained from the absorbance at a wavelength of 571 nm.
  • glycated hemoglobin is a product of hemoglobin binding to blood sugar in red blood cells of human blood, it is necessary to dissolve red blood cells to release glycated hemoglobin, and the prior art scheme mostly uses a hemolysis reagent for hemolysis.
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a glycosylated hemoglobin online hemolysis enzyme detection kit, which is creatively constructed using a three-reagent structure, and the kit has no need for manual dissolution. Blood, short hemolysis time, small sample size, suitable for POCT detection.
  • the technical solution adopted by the present invention is: a glycated hemoglobin online hemolysis enzyme detection kit, the kit is composed of a reagent R1, a reagent R2, and a reagent R3, wherein:
  • Reagent R1 is:
  • polyoxyethylene lauryl ether dodecyl polyoxyethylene ether
  • Reagent R2 is: 0.1-10 mmol / L morpholinoethyl sulfonic acid
  • Reagent R3 is: 10-1000 mmol/L Tris-HCl
  • composition of the kit of the invention is as follows:
  • Reagent R1 is: 10-900 mmol/L morpholinopropanesulfonic acid
  • Reagent R2 is: 0.5-10 mmol / L morpholinoethyl sulfonic acid
  • Reagent R3 is: 10-1000 mmol/L Tris-HCl
  • composition of the kit of the invention is as follows:
  • Reagent R1 is: 800 mmol / L morpholinopropane sulfonic acid
  • Reagent R2 is: 5mmol / L morpholinoethyl sulfonic acid
  • Reagent R3 is: 800mmol/L Tris-HCl
  • the preparation of the above kit of the present invention is carried out according to the content of each component described above in the form of a solvent such as deionized water or the like, which is a conventional ratio of the industry.
  • the glycated hemoglobin online hemolysis enzyme detection kit of the invention is improved on the basis of the original glycosylated hemoglobin detection kit, and the four reagents of the original kit are changed into three reagents, and the process of manual hemolysis of the previous sample is removed, and hemolysis is performed.
  • the time was changed from manual hemolysis for 10 min to the instrument for 5 min.
  • the sensitivity of the kit was adjusted after the formula was adjusted, and the sample size was reduced from the original 25-30 ul to 2 ul. The expected amount of the sample was reduced.
  • it reduces the error caused by manual hemolysis which is beneficial to large-scale clinical application and is suitable for POCT detection.
  • the invention can not overcome the short reaction time caused by the original four reagents and the shortage of sample usage in the early stage of the research and development process. Later, through the creative work of the applicant, it was found that the hemolysis effect was simultaneously introduced in the reagent R1.
  • the invention adds a whole blood mixing sequence before the sample is hemolyzed, and the whole blood mixing device includes, but is not limited to, a disk-shaped automatic mixing device, and the device is rotated to achieve the purpose of automatic mixing.
  • the high-activity fructose proline oxidase of the present invention is a highly active fructose proline having the nucleotide sequence number of SEQ ID. No. 1, and the amino acid sequence number of SEQ ID. No. 2, as described in ZL200810166329.x. Oxidase.
  • the formula of the glycated hemoglobin online hemolysis enzyme detection kit was prepared by using distilled water as a solvent according to the conventional preparation reagent method, as shown in Table 1.
  • the Hb concentration can be obtained from the absorbance at a wavelength of 751 nm
  • the HbA1c concentration can be obtained from the absorbance at a wavelength of 571 nm.

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Abstract

Disclosed is a glycosylated hemoglobin on-line hemolytic enzymatic assay kit, consisting of a reagent R1, a reagent R2 and a reagent R3. The reagent R1 comprises: 10-1,000 mmol/L of morpholinopropanesulfonic acid 3-(N-morpholine) propanesulfonic acid, 10-1,000 mmol/L of N-cyclohexyl-2-aminoethanesulfonic acid, 1-200 g/L of polyoxyethylene lauryl ether (lauryl polyoxyethylene ether), 100-10,000 kU/L of metalloprotease (in a reagent R1a), and 0.1-10 mmol/L of 2-(iodophenyl)-3-(2,4-dinitrobenzene)-5-(2,4-disulfophenyl)-2H-tetrazole sodium salt. The reagent R2 comprises 0.1-10 mmol/L of morpholinoethylsulfonic acid and 0.5-50 mmol/L of CaCl2. The reagent R3 comprises 10-1,000 mmol/L of Tris-HCl, 1-150 kU/L of high-activity fructose valine oxidase, 30-600 kU/L of peroxidase, and 0.01-0.5 mmol/L of N-(carboxymethylaminocarbonyl)-4,4-diazo(dimethylamine)-diphenylamine sodium salt.

Description

糖化血红蛋白在线溶血酶法检测试剂盒Glycosylated hemoglobin online hemolysis enzyme assay kit 技术领域Technical field
本发明涉及生物制剂技术领域，具体涉及一种糖化血红蛋白在线溶血酶法检测试剂盒。The invention relates to the technical field of biological preparations, in particular to a glycosylated hemoglobin online hemolysis enzyme detection kit.
背景技术Background technique
世界卫生组织估计，全世界有1.8亿人患有糖尿病。糖化血红蛋白(糖化Hb)对于糖尿病患者来说是一个非常重要的检查指标。HbA1c为Hb的β链N末端的α氨基被糖化的糖化Hb，在糖化Hb中，也被作为糖尿病诊断等的特别重要的指标。目前，HbA1c的测定方法主要有离子交换层析法、高效液相色谱法、亲和层析法、放射免疫法、酶免疫法和胶乳免疫凝集法等，以上方法或是需专业的昂贵设备、或是检测准确度、抗干扰性、速度等尚不理想，一定程度上限制了其在临床上的大规模应用。The World Health Organization estimates that 180 million people worldwide have diabetes. Glycated hemoglobin (glycated Hb) is a very important indicator for diabetic patients. HbA1c is a glycated Hb in which the α-amino group at the N-terminus of the β chain of Hb is glycated, and is also particularly important as a diagnostic indicator for diabetes in glycated Hb. At present, HbA1c is mainly determined by ion exchange chromatography, high performance liquid chromatography, affinity chromatography, radioimmunoassay, enzyme immunoassay, and latex immunoagglutination. The above methods are expensive and require expensive equipment. Or the accuracy of detection, anti-interference, speed, etc. are not ideal, which limits its large-scale clinical application to a certain extent.
新开发的糖化血红蛋白酶法检测试剂盒利用了氧化还原反应，克服了上述几种方法的灵敏度较低、重复性差、耗时难以大量检测等缺点，具有检测灵敏度高、反应产物无污染且抗干扰能力强等优点；该糖化血红蛋白酶法检测试剂盒具体操作为：从患者采集EDTA抗凝新鲜全血，进行红细胞的自然沉降，然后加入到250uL试剂盒的溶血缓冲液中，室温溶血10分钟；将得到的25ul溶血试样与112uL试剂R1a和48uL试剂R1b混合，于37℃温浴3分钟，读取吸光度A1。再向反应体系中加入70uL试剂2，于37℃温浴2分钟，读取吸光度A2。ΔA＝A2-A1。测定时可选择751nm和571nm处的吸光值。其中，Hb浓度可由波长751nm处的吸光度求出，HbA1c浓度可由波长571nm处的吸光度求出。The newly developed glycosylated hemoglobin detection kit utilizes redox reaction, which overcomes the shortcomings of the above methods, such as low sensitivity, poor repeatability, and time-consuming and large-scale detection. It has high detection sensitivity, no pollution of reaction products and anti-interference. The utility model has the advantages of strong ability and the like; the specific operation of the glycosylated hemoglobin detection test kit is: collecting EDTA anticoagulated fresh whole blood from the patient, performing natural sedimentation of the red blood cells, and then adding to the 250 uL kit hemolysis buffer, and hemolyzing for 10 minutes at room temperature; The obtained 25 ul hemolyzed sample was mixed with 112 uL of the reagent R1a and 48 uL of the reagent R1b, and the bath was incubated at 37 ° C for 3 minutes, and the absorbance A1 was read. Further, 70 uL of the reagent 2 was added to the reaction system, and the mixture was incubated at 37 ° C for 2 minutes, and the absorbance A2 was read. ΔA = A2-A1. The absorbance at 751 nm and 571 nm can be selected for the measurement. Here, the Hb concentration can be obtained from the absorbance at a wavelength of 751 nm, and the HbA1c concentration can be obtained from the absorbance at a wavelength of 571 nm.
但是，上述的试剂盒由于糖化血红蛋白是人体血液中红细胞内的血红蛋白与血糖结合的产物,因此需要溶解红细胞以释放糖化血红蛋白，现有技术的方案多是采用的多少单独先设置一个溶血试剂进行溶血，然后再进行后续的其它检测步骤；但是这一溶血过程需要一定的时间,因此仍存在样本需要手工溶血，溶血时间长，样本量大，不适合POCT检测等一些待改进地方；此外，现有的糖化血红蛋白酶法检测试剂盒在检测过程需要将红细胞沉降回收起到一个富集的效果，而且样本需求量比较大，试剂盒结构的不合理导致的这一耗时、繁琐的过程，不适合现有POCT检测的快速、简便的检测需求。However, in the above kit, since glycated hemoglobin is a product of hemoglobin binding to blood sugar in red blood cells of human blood, it is necessary to dissolve red blood cells to release glycated hemoglobin, and the prior art scheme mostly uses a hemolysis reagent for hemolysis. And then carry out other subsequent detection steps; however, this hemolysis process requires a certain period of time, so there is still a need for manual hemolysis of the sample, long hemolysis time, large sample size, and is not suitable for some areas to be improved such as POCT detection; The glycosylated hemoglobin detection kit needs to recover the red blood cell sedimentation during the detection process to achieve an enrichment effect, and the sample demand is relatively large, and the time-consuming and cumbersome process caused by the unreasonable structure of the reagent kit is not suitable. The fast and simple detection requirements of existing POCT detection.
发明内容Summary of the invention
本发明的目的在于克服现有技术的上述不足，提供一种糖化血红蛋白在线溶血酶法检测试剂盒，该试剂盒创造性地采用三试剂组成结构，该结构的试剂盒具有无需手工溶 血，溶血时间短，样本量小，适合POCT检测的优点。The object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a glycosylated hemoglobin online hemolysis enzyme detection kit, which is creatively constructed using a three-reagent structure, and the kit has no need for manual dissolution. Blood, short hemolysis time, small sample size, suitable for POCT detection.
为了解决上述技术问题，本发明采用的技术方案为：一种糖化血红蛋白在线溶血酶法检测试剂盒，该试剂盒由试剂R1、试剂R2、试剂R3组成，其中：In order to solve the above technical problem, the technical solution adopted by the present invention is: a glycated hemoglobin online hemolysis enzyme detection kit, the kit is composed of a reagent R1, a reagent R2, and a reagent R3, wherein:
试剂R1为：Reagent R1 is:
    10-1000mmol/L吗啉代丙烷磺酸(3-(N-吗啉)丙磺酸)，10-1000 mmol/L morpholinopropanesulfonic acid (3-(N-morpholine)propanesulfonic acid),
    10-1000mmol/L N-环乙基-2氨基乙烷磺酸，10-1000 mmol/L N-cycloethyl-2-aminoethanesulfonic acid,
    1-200g/L聚氧乙烯十二烷基醚(十二烷基聚氧乙烯醚)，1-200g/L polyoxyethylene lauryl ether (dodecyl polyoxyethylene ether),
    100-10000kU/L金属蛋白酶(在试剂R1a中)，100-10000 kU/L metalloproteinase (in reagent R1a),
    0.1-10mmol/L 2-(碘苯基)-3-(2，4-二硝基苯)-5-(2，4-二磺苯基)-2H四唑钠盐；0.1-10 mmol/L 2-(iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium sodium salt;
试剂R2为：0.1-10mmol/L吗啉代乙基磺酸，Reagent R2 is: 0.1-10 mmol / L morpholinoethyl sulfonic acid,
          0.5-50mmol/L CaCl2；0.5-50mmol/L CaCl2;
试剂R3为：10-1000mmol/L Tris-HCl，Reagent R3 is: 10-1000 mmol/L Tris-HCl,
          1-150kU/L高活性果糖缬氨酸氧化酶，1-150kU/L high activity fructose proline oxidase,
          30-600kU/L过氧化物酶，30-600kU/L peroxidase,
          0.01-0.5mmol/L N-(羧甲基氨羰基)-4，4双偶(二甲胺)-二苯胺0.01-0.5 mmol/L N-(carboxymethylaminocarbonyl)-4,4 bis (dimethylamine)-diphenylamine
          钠盐。Sodium salt.
作为进一步优选，本发明试剂盒的组成如下：As a further preferred, the composition of the kit of the invention is as follows:
试剂R1为：10-900mmol/L吗啉代丙烷磺酸，Reagent R1 is: 10-900 mmol/L morpholinopropanesulfonic acid,
          10-900mmol/L N-环乙基-2氨基乙烷磺酸，10-900 mmol/L N-cycloethyl-2-aminoethanesulfonic acid,
          1-150g/L聚氧乙烯十二烷基醚，1-150g/L polyoxyethylene lauryl ether,
          100-8000kU/L金属蛋白酶，100-8000kU/L metalloproteinase,
          0.5-10mmol/L 2-(碘苯基)-3-(2，4-二硝基苯)-5-(2，4-二磺0.5-10mmol/L 2-(iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfonate
          苯基)-2H四唑钠盐；Phenyl)-2H tetrazolium sodium salt;
试剂R2为：0.5-10mmol/L吗啉代乙基磺酸，Reagent R2 is: 0.5-10 mmol / L morpholinoethyl sulfonic acid,
          0.5-40mmol/L CaCl2；0.5-40 mmol / L CaCl 2 ;
试剂R3为：10-1000mmol/L Tris-HCl，Reagent R3 is: 10-1000 mmol/L Tris-HCl,
          1-100kU/L高活性果糖缬氨酸氧化酶，1-100kU/L high activity fructose proline oxidase,
          30-500kU/L过氧化物酶，30-500kU/L peroxidase,
          0.05-0.5mmol/L N-(羧甲基氨羰基)-4，4双偶(二甲胺)-二苯胺 0.05-0.5 mmol/L N-(carboxymethylaminocarbonyl)-4,4 bis(dimethylamine)-diphenylamine
          钠盐；Sodium salt
作为最优选，本发明试剂盒的组成如下：Most preferably, the composition of the kit of the invention is as follows:
试剂R1为：800mmol/L吗啉代丙烷磺酸，Reagent R1 is: 800 mmol / L morpholinopropane sulfonic acid,
          800mmol/L N-环乙基-2氨基乙烷磺酸，800 mmol/L N-cycloethyl-2-aminoethanesulfonic acid,
          100g/L聚氧乙烯十二烷基醚，100g/L polyoxyethylene lauryl ether,
          800kU/L金属蛋白酶，800kU/L metalloproteinase,
          5mmol/L 2-(碘苯基)-3-(2，4-二硝基苯)-5-(2，4-二磺苯基)5mmol/L 2-(iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)
          -2H四唑钠盐；-2H tetrazolium sodium salt;
试剂R2为：5mmol/L吗啉代乙基磺酸，Reagent R2 is: 5mmol / L morpholinoethyl sulfonic acid,
          20mmol/L CaCl2；20mmol/L CaCl 2 ;
试剂R3为：800mmol/L Tris-HCl，Reagent R3 is: 800mmol/L Tris-HCl,
          80kU/L高活性果糖缬氨酸氧化酶，80kU/L high activity fructose proline oxidase,
          250kU/L过氧化物酶，250kU/L peroxidase,
          0.5mmol/L N-(羧甲基氨羰基)-4，4双偶(二甲胺)-二苯胺钠盐。0.5 mmol/L N-(carboxymethylaminocarbonyl)-4,4 bis (dimethylamine)-diphenylamine sodium salt.
本发明上述试剂盒的配制，就按照上述各组分的含量以水如去离子水等为溶剂进行溶解配比的，为行业常规配比方式。The preparation of the above kit of the present invention is carried out according to the content of each component described above in the form of a solvent such as deionized water or the like, which is a conventional ratio of the industry.
本发明的优点和有益效果：Advantages and benefits of the present invention:
1.本发明糖化血红蛋白在线溶血酶法检测试剂盒，在原来糖化血红蛋白酶法检测试剂盒基础上进行改进，将原来试剂盒的四试剂变成了三试剂，去除前期样本手工溶血的过程，溶血时间由手工溶血10min改为仪器直接孵育5min，试剂盒配方调整后分析灵敏度更高、创造性的实现了样本量从原来的25-30ul减少到2ul的预料不到的技术效果，该样本量的减少符合糖化血红蛋白临床检测要求自动化、快速性、大批量、经济性特点的同时降低手工溶血带来的误差，有利于临床大规模应用，并适用于POCT检测。1. The glycated hemoglobin online hemolysis enzyme detection kit of the invention is improved on the basis of the original glycosylated hemoglobin detection kit, and the four reagents of the original kit are changed into three reagents, and the process of manual hemolysis of the previous sample is removed, and hemolysis is performed. The time was changed from manual hemolysis for 10 min to the instrument for 5 min. The sensitivity of the kit was adjusted after the formula was adjusted, and the sample size was reduced from the original 25-30 ul to 2 ul. The expected amount of the sample was reduced. In line with the requirements of automated, rapid, large-volume, and economical testing of glycosylated hemoglobin, it reduces the error caused by manual hemolysis, which is beneficial to large-scale clinical application and is suitable for POCT detection.
2.同时，本发明在研发过程中前期始终不能克服原来四试剂导致的反应时间长以及样本使用量多的不足，后来通过申请人创造性的劳动，发现在试剂R1中通过同时引入的具有溶血作用的聚氧乙烯十二烷基醚以及用来水解糖化蛋白质的金属蛋白酶的结构设置，达到了在样本溶血的同时金属蛋白酶快速水解样本中的糖化蛋白质、使其分解为较小的糖化肽片段和糖化氨基酸、实现了溶血的同时水解糖化蛋白质可以缩短反应时间，然后将果糖缬氨酸氧化酶作用于该糖化蛋白质的分解物进行氧化还原反应，产生的过氧化氢的量与参与反应的糖化蛋白的量成正比，通过测定因氧化而显色的显色基质的 显色量来确定过氧化氢的量，从而间接的达到测定糖化蛋白质的目的。本发明在样本溶血前加装全血混匀时序，该全血混匀装置包括但不限于一种圆盘状的自动混匀装置，通过旋转该装置来达到自动混匀的目的。2. At the same time, the invention can not overcome the short reaction time caused by the original four reagents and the shortage of sample usage in the early stage of the research and development process. Later, through the creative work of the applicant, it was found that the hemolysis effect was simultaneously introduced in the reagent R1. The polyoxyethylene lauryl ether and the structural arrangement of the metalloprotease used to hydrolyze the glycated protein, the metalloprotease rapidly hydrolyzes the glycated protein in the sample, and decomposes it into smaller glycated peptide fragments while the sample is hemolyzed. Glycation of amino acids, hydrolysis of glycated proteins while hemolysis can shorten the reaction time, and then fructose proline oxidase acts on the decomposed products of the glycated proteins for redox reaction, the amount of hydrogen peroxide produced and the glycated proteins involved in the reaction In proportion to the amount of chromogenic substrate that is colored by oxidation The amount of coloration is used to determine the amount of hydrogen peroxide, thereby indirectly achieving the purpose of determining the glycated protein. The invention adds a whole blood mixing sequence before the sample is hemolyzed, and the whole blood mixing device includes, but is not limited to, a disk-shaped automatic mixing device, and the device is rotated to achieve the purpose of automatic mixing.
具体实施方式Detailed ways
下面结合具体实施例进一步阐述本发明，但并不仅限于下述实施例。The invention is further illustrated by the following specific examples, but is not limited to the following examples.
本发明所述的高活性果糖缬氨酸氧化酶为ZL200810166329.x中记载的核苷酸序列号为SEQ ID.No.1、氨基酸序列号为SEQ ID.No.2的高活性果糖缬氨酸氧化酶。The high-activity fructose proline oxidase of the present invention is a highly active fructose proline having the nucleotide sequence number of SEQ ID. No. 1, and the amino acid sequence number of SEQ ID. No. 2, as described in ZL200810166329.x. Oxidase.
实施例1：Example 1:
以蒸馏水为溶剂，按照常规配制试剂方法配制糖化血红蛋白在线溶血酶法检测试剂盒各配方，如表1所示。The formula of the glycated hemoglobin online hemolysis enzyme detection kit was prepared by using distilled water as a solvent according to the conventional preparation reagent method, as shown in Table 1.
表1 糖化血红蛋白在线溶血酶法检测试剂盒各配方Table 1 Glycosylated hemoglobin online hemolytic enzyme assay kit
Figure PCTCN2017108541-appb-000001
Figure PCTCN2017108541-appb-000001
Figure PCTCN2017108541-appb-000002
Figure PCTCN2017108541-appb-000002
实施例2：Example 2:
从患者采集EDTA抗凝新鲜全血，放置于全血混匀装置混匀5min内即可混匀，然后取2uL混匀的全血样本与120uL试剂R1和50uL试剂R2混合，于37℃温浴3分钟，读取吸光度A1。再向反应体系中加入70uL试剂R3，于37℃温浴2分钟，读取吸光度A2。ΔA＝A2-A1。测定时可选择751nm和571nm处的吸光值。其中，Hb浓度可由波长751nm处的吸光度求出，HbA1c浓度可由波长571nm处的吸光度求出。本发明上述的操作过程通过本发明三试剂的试剂盒配方调整后分析灵敏度提高，不需要红细胞沉降回收，省略这一耗时、繁琐的步骤，从而达到一个快速、简便的检测效果。Collect EDTA anticoagulated fresh whole blood from the patient, mix it in the whole blood mixing device for 5 minutes, then mix it, then take 2uL mixed whole blood sample and mix with 120uL reagent R1 and 50uL reagent R2, and warm at 37 °C. In minutes, read the absorbance A1. Further, 70 uL of the reagent R3 was added to the reaction system, and the mixture was incubated at 37 ° C for 2 minutes, and the absorbance A2 was read. ΔA = A2-A1. The absorbance at 751 nm and 571 nm can be selected for the measurement. Here, the Hb concentration can be obtained from the absorbance at a wavelength of 751 nm, and the HbA1c concentration can be obtained from the absorbance at a wavelength of 571 nm. The above operation process of the invention is improved by the formula of the kit of the three reagents of the invention, and the sensitivity of the analysis is improved, and the red blood cell sedimentation recovery is not required, and the time-consuming and cumbersome steps are omitted, thereby achieving a rapid and simple detection effect.
以上实施例是对专利的说明和进一步解释，而不是对本发明的限制，在本发明的精神和权利保护范围内所做的任何修改，都落入本发明的保护范围。 The above embodiments are illustrative of the invention and further explanations of the invention, and are not intended to limit the scope of the invention, and any modifications made within the spirit and scope of the invention are intended to fall within the scope of the invention.

Claims (3)

  1. 一种糖化血红蛋白在线溶血酶法检测试剂盒，其特征在于：该试剂盒由试剂R1、试剂R2、试剂R3组成，其中：The invention relates to a glycated hemoglobin online hemolysis enzyme detection kit, which is characterized in that the kit is composed of a reagent R1, a reagent R2 and a reagent R3, wherein:
    试剂R1为：Reagent R1 is:
    10-1000mmol/L吗啉代丙烷磺酸，10-1000 mmol/L morpholinopropanesulfonic acid,
    10-1000mmol/L N-环乙基-2氨基乙烷磺酸，10-1000 mmol/L N-cycloethyl-2-aminoethanesulfonic acid,
    1-200g/L聚氧乙烯十二烷基醚，1-200g/L polyoxyethylene lauryl ether,
    100-10000kU/L金属蛋白酶，100-10000kU/L metalloproteinase,
    0.1-10mmol/L 2-(碘苯基)-3-(2，4-二硝基苯)-5-(2，4-二磺苯基)-2H四唑钠盐；0.1-10 mmol/L 2-(iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium sodium salt;
    试剂R2为：0.1-10mmol/L吗啉代乙基磺酸，Reagent R2 is: 0.1-10 mmol / L morpholinoethyl sulfonic acid,
    0.5-50mmol/L CaCl2；0.5-50mmol/L CaCl2;
    试剂R3为：10-1000mmol/L Tris-HCl，Reagent R3 is: 10-1000 mmol/L Tris-HCl,
    1-150kU/L高活性果糖缬氨酸氧化酶，1-150kU/L high activity fructose proline oxidase,
    30-600kU/L过氧化物酶，30-600kU/L peroxidase,
    0.01-0.5mmol/L N-(羧甲基氨羰基)-4，4双偶(二甲胺)-二苯胺钠盐。0.01-0.5 mmol/L N-(carboxymethylaminocarbonyl)-4,4 bis(dimethylamine)-diphenylamine sodium salt.
  2. 根据权利要求1所述的糖化血红蛋白在线溶血酶法检测试剂盒，其特征在于：该试剂盒由试剂R1、试剂R2、试剂R3组成，其中：The glycated hemoglobin online hemolytic enzyme assay kit according to claim 1, wherein the kit comprises reagent R1, reagent R2, and reagent R3, wherein:
    试剂R1为：10-900mmol/L吗啉代丙烷磺酸，Reagent R1 is: 10-900 mmol/L morpholinopropanesulfonic acid,
    10-900mmol/L N-环乙基-2氨基乙烷磺酸，10-900 mmol/L N-cycloethyl-2-aminoethanesulfonic acid,
    1-150g/L聚氧乙烯十二烷基醚，1-150g/L polyoxyethylene lauryl ether,
    100-8000kU/L金属蛋白酶，100-8000kU/L metalloproteinase,
    0.5-10mmol/L 2-(碘苯基)-3-(2，4-二硝基苯)-5-(2，4-二磺苯基)-2H四唑钠盐；0.5-10 mmol/L 2-(iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium sodium salt;
    试剂R2为：0.5-10mmol/L吗啉代乙基磺酸，Reagent R2 is: 0.5-10 mmol / L morpholinoethyl sulfonic acid,
    0.5-40mmol/L CaCl2；0.5-40 mmol / L CaCl 2 ;
    试剂R3为：10-1000mmol/L Tris-HCl，Reagent R3 is: 10-1000 mmol/L Tris-HCl,
    1-100kU/L高活性果糖缬氨酸氧化酶，1-100kU/L high activity fructose proline oxidase,
    30-500kU/L过氧化物酶，30-500kU/L peroxidase,
    0.05-0.5mmol/L N-(羧甲基氨羰基)-4，4双偶(二甲胺)-二苯胺 钠盐。0.05-0.5 mmol/L N-(carboxymethylaminocarbonyl)-4,4 bis(dimethylamine)-diphenylamine Sodium salt.
  3. 根据权利要求2所述的糖化血红蛋白在线溶血酶法检测试剂盒，其特征在于：该试剂盒由试剂R1、试剂R2、试剂R3组成，其中：The glycated hemoglobin online hemolytic enzyme assay kit according to claim 2, wherein the kit comprises reagent R1, reagent R2, and reagent R3, wherein:
    试剂R1为：800mmol/L吗啉代丙烷磺酸，Reagent R1 is: 800 mmol / L morpholinopropane sulfonic acid,
    800mmol/L N-环乙基-2氨基乙烷磺酸，800 mmol/L N-cycloethyl-2-aminoethanesulfonic acid,
    100g/L聚氧乙烯十二烷基醚，100g/L polyoxyethylene lauryl ether,
    800kU/L金属蛋白酶，800kU/L metalloproteinase,
    5mmol/L 2-(碘苯基)-3-(2，4-二硝基苯)-5-(2，4-二磺苯基)-2H四唑钠盐；5 mmol/L 2-(iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium sodium salt;
    试剂R2为：5mmol/L吗啉代乙基磺酸，Reagent R2 is: 5mmol / L morpholinoethyl sulfonic acid,
    20mmol/L CaCl2；20mmol/L CaCl 2 ;
    试剂R3为：800mmol/L Tris-HCl，Reagent R3 is: 800mmol/L Tris-HCl,
    80kU/L高活性果糖缬氨酸氧化酶，80kU/L high activity fructose proline oxidase,
    250kU/L过氧化物酶，250kU/L peroxidase,
    0.5mmol/L N-(羧甲基氨羰基)-4，4双偶(二甲胺)-二苯胺钠盐。 0.5 mmol/L N-(carboxymethylaminocarbonyl)-4,4 bis (dimethylamine)-diphenylamine sodium salt.
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