CN107385012A - A kind of H2O2The method of the instruction system detectio sialic acid of coupling - Google Patents
A kind of H2O2The method of the instruction system detectio sialic acid of coupling Download PDFInfo
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- CN107385012A CN107385012A CN201710602044.5A CN201710602044A CN107385012A CN 107385012 A CN107385012 A CN 107385012A CN 201710602044 A CN201710602044 A CN 201710602044A CN 107385012 A CN107385012 A CN 107385012A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of H2O2The method of the instruction system detectio sialic acid of coupling, its principle of described method are based on utilizing H2O2The instruction system of coupling, sialic acid generation N acetylmannosamines are decomposed using neuraminidase, neuraminic acid aldolase, N acetylmannosamines are decomposed generation H by Ketoamine oxidase2O2, last H2O2Quinone imines color group is generated in the presence of peroxidase, 4 amino-antipyrines and phenol.Compared with conventional method, this method high sensitivity, stability is good, is worth further genralrlization to use.
Description
Technical field
The present invention relates to the detection technique field of sialic acid, more particularly, to a kind of H2O2The instruction system detectio saliva of coupling
The method of liquid acid.
Background technology
Sialic acid (Sialic acid, SA)：It is the derivative of 9- carbon monose.Generally with oligosaccharide, glycolipid or glycoprotein
Form exist.It is the important component of glycoprotein, it is relevant with many biological functions of organism, and and cell
Canceration, metastasis of cancer, infiltrate, lose inhibition contact, cell adhesion reduce and tumor antigenicity it is closely related.Determine serum
Sialic acid concentration, cancerous swelling diagnostic assistance index and parameters for observation on effect can be used as.
The significantly raised disease of sialic acid has：Acute leukemia, cancer of the esophagus, cardia cancer, stomach cancer, intestinal cancer, liver cancer, lung cancer with
And oophoroma etc., wherein using acute leukemic patient as highest.It is probably tumour cell saliva that Serum of Cancer Patients sialic acid, which increases,
Reflection of the change of glycoprotein synthesis increase and metabolism in blood, can be used for the transfer and recurrence of early detection tumour.
The conventional method of sialic acid detection technique has following several at present：Thiobarbituricacidα- method, R- reagents method, isophthalic two
Phenol-periodic acid/hydrochloric acid method, HPLC methods, TLC, enzyme process etc..Thiobarbituricacidα- method, R- reagents method, resorcinol-mistake
Acid iodide/hydrochloric acid method operation is complicated, is not suitable for automated analysis, HPLC methods, TLC instrument cost are higher, and enzyme process is universal
With NAD+- NADH indicate system measurement material concentration or activity, but many of NADH less stables, blood plasma using NADH as
The dehydrogenase of coenzyme, negative reaction is also easy to produce with NADH, the interference of endogenous pyruvic acid be present, therefore, a kind of degree of accuracy is high, precision
Good sialic acid detection method, it is the technical problem of this area urgent need to resolve.
The content of the invention
For overcome the deficiencies in the prior art, the invention provides a kind of H2O2The instruction system detectio sialic acid of coupling
Method, this method can exclude the interference of the material such as vitamin C, bilirubin in sialic acid sample to be tested, and the detection method has
The advantages of degree of accuracy is high, precision is good.
To achieve these goals, the technical solution adopted by the present invention is：A kind of H2O2The instruction system detectio saliva of coupling
The method of acid, it is characterised in that comprise the following steps：1. in testing sample add PH=6.5-7.5, concentration be 20~
100mmol/L Tris-HCL buffer systems, it is anti-interference agent to add 0.3~0.8g/L potassium ferrocyanides, and adds enzyme amount as 1
Mating type sialic acid in sample to be tested is resolved into N- acetyl nerve ammonia by~5KU/L neuraminidases, neuraminidase
Acid, its reaction equation are：
2. using N-acetyl-neuraminate as substrate, addition enzyme amount is that 1~5KU/L neuraminic acid aldolases make it that N- acetyl is refreshing
It is decomposed through propylhomoserin and generates ManNAc, its reaction equation is：
3. using ManNAc as substrate, addition enzyme amount is that 5~10KU/L Ketoamine oxidases are sugared by N- acetylated mannans
Amine decomposes generation H2O2, its reaction equation is：
4. in H2O20.8~1.2g/L tetrahydroxy benzenes sodium formate and 2~5g/L EDETATE SODIUMs are added in solution as stable
Agent, and it is 2~5KU/L peroxidase, 1-10g/L 4-AAs and 1g/L phenol to add enzyme amount so that H2O2
Quinone imines color group is generated in the presence of peroxidase, 4-AA and phenol, its reaction equation is：
5. the liquid and calibration object rolled into a ball containing quinone imines color are respectively placed in into dominant wavelength 510nm/ commplementary wave length 660nm sections to read
Take the value added Δ A of absorbanceTWith Δ AS, and according to formula：Sialic acid content (mg/dl)=Δ A in sampleT/ΔAS× calibration
Liquid concentration, calculate the sialic acid content in sample to be tested.
Using such scheme, the present invention decomposes the saliva in testing sample using neuraminidase, neuraminic acid aldolase
Liquid acid makes it generate ManNAc, and ManNAc is decomposed generation H by Ketoamine oxidase2O2, last H2O2In mistake
Quinone imines color group is generated in the presence of oxide enzyme, 4-AA and phenol.Quinone imines color group and calibration object are put respectively
The absorbance increase of each pipe is read under dominant wavelength 510nm/ commplementary wave lengths 660nm, and the increased degree of absorbance is with treating test sample
Sialic acid content in this is directly proportional；The present invention utilizes H2O2The instruction system detectio sialic acid of coupling, the detection method are being lifted
Simultaneously, it reacts obtained chromophore has preferable colour developing degree and solubility to precision, evaded NADH less stables,
Produce negative reaction, the problems such as endogenous pyruvic acid interference be present, by adding EDETATE SODIUM during it, improve H2O2Stabilization
Property, addition potassium ferrocyanide excludes the interference of the material such as vitamin C, bilirubin, further increased whole as anti-interference agent
The degree of accuracy of detection process.
The invention will be further described below in conjunction with the accompanying drawings.
Brief description of the drawings
Accompanying drawing 1 is schemed for the linearly related of low value of the present invention；
Accompanying drawing 2 is schemed for the linearly related of intermediate value of the present invention；
Accompanying drawing 3 is schemed for the linearly related of high level of the present invention；
Accompanying drawing 4 is the line of commercial reagent of the intermediate value of the present invention with indicating system detectio sialic acid method using NAD+-NADH
Property related figure.
Embodiment
The present invention illustrates technical scheme in conjunction with specific embodiments, and protection scope of the present invention is not limited to above-mentioned specific implementation
Mode, persons skilled in the art can use other a variety of embodiments to implement according to present disclosure
The present invention's, or every design structure and thinking using the present invention, simple change or change are done, both falls within the present invention's
Protection domain.
Embodiment 1 takes reagent component concentration low value, intermediate value respectively, and high level is added, and is used according to the invention described above
Technical scheme is operated, and the specific concentration that it is added is as follows：
Low value：Tris-HCL buffer solutions (PH6.5-7.5) 20mmol/L, neuraminidase 1KU/L, Ketoamine oxidase
5KU/L, potassium ferrocyanide 0.3g/L, tetrahydroxy benzene sodium formate 0.8g/L, neuraminic acid aldolase 1KU/L, peroxidase
2KU/L, EDETATE SODIUM 2g/L, 4-AA 1g/L, phenol 1g/L, solvent for use are purified water.It is (hereinafter referred to as low
Value).
Intermediate value：Tris-HCL buffer solutions (PH6.5-7.5) 60mmol/L, neuraminidase 3KU/L, Ketoamine oxidase
8KU/L, potassium ferrocyanide 0.5g/L, tetrahydroxy benzene sodium formate 1.0g/L, neuraminic acid aldolase 3KU/L, peroxidase
3KU/L, EDETATE SODIUM 3g/L, 4-AA 5g/L, phenol 1g/L, solvent for use are purified water.(in hereinafter referred to as
Value).
High level：Tris-HCL buffer solutions (PH6.5-7.5) 100mmol/L, neuraminidase 5KU/L, Ketoamine oxidase
10KU/L, potassium ferrocyanide 0.8g/L, tetrahydroxy benzene sodium formate 1.2g/L, neuraminic acid aldolase 5KU/L, peroxidase
5KU/L, EDETATE SODIUM 5g/L, 4-AA 10g/L, phenol 1g/L, solvent for use are purified water.It is (hereinafter referred to as high
Value).
Precision determines：Entered with reagent component concentration low value, intermediate value, the technical scheme that high level uses according to the invention described above
Row operation, is measured for 20 times to same sample continuous drawing, calculates mean, standard deviation and the coefficient of variation of measured value respectively,
The precision testing result of table 1
Coefficient of variation CV is generally used for weighing the precision of an assay method, and CV values are smaller, represent the assay method
As a result precision is better.For clinical chemistry test project, method precisions of the CV less than 5% is generally recognised as receiving
's.CV values are respectively less than 3% in table 1, show that the present invention has excellent precision.
Linear determination：With reagent component concentration low value, intermediate value, the technical scheme that high level uses according to the invention described above is carried out
Operation, does linear determination respectively.Reagent dilutions are detected 3 times into 5 gradient concentrations, each gradient concentration using deionized water,
Average, make regression analysis to measured value and desired value, (result is shown in Fig. 1, Fig. 2, Fig. 3, unit for calculating r values and relative deviation
mg/dl)。
The low value linear correlation detection result of table 2
| Diluted concentration | 30.00 | 50.00 | 100.00 | 150.00 | 200.00 |
| Measured value | 29.89 | 49.72 | 103.40 | 141.38 | 201.93 |
| 27.81 | 50.45 | 101.71 | 140.50 | 205.91 | |
| 25.93 | 52.31 | 98.06 | 142.61 | 209.18 | |
| Average | 28 | 51 | 101 | 141 | 206 |
| Absolute deviation | 0.48 | 2.20 | 1.75 | 8.49 | 5.01 |
| Relative deviation | 1.68% | - 4.53% | - 1.77% | 5.66% | - 2.50% |
Coefficient correlation is obtained by table 2：r2=0.9948, linear equation is：Y=1.0136x-2.0530, the results showed that
Low value correlation is good.
The intermediate value linear correlation detection result of table 3
Coefficient correlation is obtained by table 3：r2=0.9968, linear equation is：Y=0.9634x+3.0406, the results showed that
Intermediate value correlation is good.
The high level linear correlation detection result of table 4
| Diluted concentration | 30.00 | 50.00 | 100.00 | 150.00 | 200.00 |
| Measured value | 29.37 | 47.14 | 106.70 | 150.02 | 199.81 |
| 29.72 | 47.40 | 109.15 | 151.81 | 192.16 | |
| 28.38 | 48.27 | 111.24 | 158.62 | 195.38 | |
| Average | 29 | 48 | 109 | 153 | 196 |
| Absolute deviation | 2.24 | 3.69 | 7.99 | 2.69 | 4.76 |
| Relative deviation | 7.12% | 7.19% | - 7.91% | - 1.79% | 2.37% |
Coefficient correlation is obtained by table 4：r2=0.9942, linear equation is：Y=0.9950x+1.5424, the results showed that
High level correlation is good.
What the present invention of embodiment 2 was compareed with using the correlation research of NAD+-NADH instruction system detectio sialic acid methods
It is as follows using the commercial reagent of NAD+-NADH instruction system detectio sialic acid methods, its information：
Name of product：Application of Sialic Acid Measurement kit【Yonghe County's sunlight (Hunan) bio tech ltd】
Method：Enzyme process
Formulation：Liquid double reagent (3：1)
Reagent components：
Reagent 1：
Tris-HCl 100mmol/L pH 7.0
Neuraminidase 0.2KU/L
Lactic dehydrogenase (LDH) 2KU/L
Reagent 2：
Nicotinoyl adenine-dinucleotide (NADH) 0.13mmol/L
Neu 5 Ac aldolase (NANA- aldolases) 2KU/L
Analysis method：
Calculate：
Detecting instrument：AU480
Linear correlation measure：Concentration using reagent component is intermediate value, and the technical scheme used according to the invention described above is entered
Row operation, commercial reagent are operated by its specification, using AU480 automatic clinical chemistry analyzers, respectively to 50 parts of samples (bag
Containing normal and exceptional sample), it is measured by each autoregressive parameter, and (result is shown in Fig. 4, and X-axis represents to measured value progress correlation analysis
Be intermediate value of the present invention measured value, Y-axis represent is commercial reagent measured value).Coefficient correlation：r2=0.9930, linearly
Equation is：Y=1.005x+1.060, the results showed that intermediate value of the present invention is with using NAD+-NADH instruction system detectios sialic acid side
The commercial reagent correlation of method is good.
The linear correlation detection result of table 5
Precision determines：Concentration using reagent component is intermediate value, and the technical scheme used according to the invention described above is carried out
Operation, commercial reagent operated by its specification, and continuous drawing in same sample is measured for 20 times respectively, calculates measure
Mean, standard deviation and the coefficient of variation of value,
The precision testing result of table 6
Coefficient of variation CV is generally used for weighing the precision of an assay method, and CV values are smaller, represent the assay method
As a result precision is better.For clinical chemistry test project, method precisions of the CV less than 5% is generally recognised as receiving
's.The CV of intermediate value of the present invention is 2.90% in table 6, is compared using the commercially available of NAD+-NADH instruction system detectio sialic acid methods
Reagent has more preferable precision.
Accuracy determination：Concentration using reagent component is intermediate value, and the technical scheme used according to the invention described above is carried out
Operation, commercial reagent is operated by its specification, respectively with same quality-control product, replication 3 times, averaged, with Quality Control
Product target value relative deviation should be in the range of ± 15%.
The degree of accuracy testing result of table 7
Relative deviation in table 7 measured by intermediate value of the present invention is 4.49%, compares and indicates system detectio using NAD+-NADH
The commercial reagent of sialic acid method has the more preferable degree of accuracy.
Specific assay：Interference experiment measure is carried out, takes 5 parts of Quality Controls respectively, a copy of it is as control, other four parts points
Jia Ru not 4 kinds of potential interference things：Bilirubin, vitamin C, hemoglobin and lactic acid, respectively with intermediate value of the present invention and commercial reagent
Time-and-motion study is carried out, as a result such as table 8, shown in table 9：
The intermediate value specific detection result of the present invention of table 8
| Simulate chaff interference | Add concentration | Control group measured value | Measured value of the present invention | Accurately (CB%) |
| Bilirubin | 50mg/dl | 50mg/dl | 51.56mg/dl | 3.12% |
| Vitamin C | 4g/L | 50mg/dl | 51.01mg/dl | 2.02% |
| Hemoglobin | 500mg/dl | 50mg/dl | 52.41mg/dl | 4.82% |
| Lactic acid | 450mg/dl | 50mg/dl | 48.77mg/dl | - 2.46% |
The commercial reagent specific detection result of table 9
| Simulate chaff interference | Add concentration | Control group measured value | Commercial reagent measured value | Accurately (CB%) |
| Bilirubin | 50mg/dl | 50mg/dl | 53.21mg/dl | 6.42% |
| Vitamin C | 4g/L | 50mg/dl | 48.55mg/dl | - 2.90% |
| Hemoglobin | 500mg/dl | 50mg/dl | 54.23mg/dl | 8.46% |
| Lactic acid | 450mg/dl | 50mg/dl | 48.12mg/dl | - 3.76% |
For clinical chemistry test project, CB% is generally recognised as acceptable between ± 5%.Table 8, this in table 9
CB% measured by invention intermediate value is generally recognised as acceptable, relative usage NAD+-NADH instruction system inspections between being in ± 5%
Surveying the commercial reagent of sialic acid method has preferably specificity.
Claims (1)
- A kind of 1. H2O2The method of the instruction system detectio sialic acid of coupling, it is characterised in that comprise the following steps：1. to be measured PH=6.5-7.5 is added in sample, the Tris-HCL buffer systems that concentration is 20~100mmol/L, it is sub- to add 0.3~0.8g/L The potassium ferricyanide is anti-interference agent, and it is 1~5KU/L neuraminidases to add enzyme amount, and neuraminidase is by sample to be tested Mating type sialic acid resolve into N-acetyl-neuraminate, its reaction equation is：2. using N-acetyl-neuraminate as substrate, addition enzyme amount is that 1~5KU/L neuraminic acid aldolases cause N- acetyl nerve ammonia Acid, which is decomposed, generates ManNAc, and its reaction equation is：3. using ManNAc as substrate, addition enzyme amount is that 5~10KU/L Ketoamine oxidases divide ManNAc Solution generation H2O2, its reaction equation is：4. in H2O20.8~1.2g/L tetrahydroxy benzenes sodium formate and 2~5g/L EDETATE SODIUMs are added in solution as stabilizer, and Addition enzyme amount is 2~5KU/L peroxidase, 1-10g/L 4-AAs and 1g/L phenol so that H2O2In peroxidating Quinone imines color group is generated in the presence of thing enzyme, 4-AA and phenol, its reaction equation is：Inhaled 5. the liquid and calibration object rolled into a ball containing quinone imines color to be respectively placed in dominant wavelength 510nm/ commplementary wave length 660nm sections and read The value added Δ A of luminosityTWith Δ AS, and according to formula：Sialic acid content (mg/dl)=Δ A in sampleT/ΔAS× calibration solution is dense Degree, calculates the sialic acid content in sample to be tested.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110346316A (en) * | 2018-04-04 | 2019-10-18 | 浙江力耐得传动科技有限公司 | It is coupled the method and assay kit of pyruvate oxidation enzymatic determination sialic acid (SIA) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6112299A (en) * | 1984-06-28 | 1986-01-20 | Fujirebio Inc | Determination of sialic acid |
| EP0206316A2 (en) * | 1985-06-26 | 1986-12-30 | Kyowa Medex Co. Ltd. | Method and test composition for determination of hydrogen peroxide |
| CN102072953A (en) * | 2010-12-30 | 2011-05-25 | 北京九强生物技术有限公司 | Method and kit for stably detecting sialic acid by enzyme method |
| CN106092942A (en) * | 2016-05-26 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | Sialic test kit of a kind of mensuration and preparation method thereof |
-
2017
- 2017-07-21 CN CN201710602044.5A patent/CN107385012A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6112299A (en) * | 1984-06-28 | 1986-01-20 | Fujirebio Inc | Determination of sialic acid |
| EP0206316A2 (en) * | 1985-06-26 | 1986-12-30 | Kyowa Medex Co. Ltd. | Method and test composition for determination of hydrogen peroxide |
| CN102072953A (en) * | 2010-12-30 | 2011-05-25 | 北京九强生物技术有限公司 | Method and kit for stably detecting sialic acid by enzyme method |
| CN106092942A (en) * | 2016-05-26 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | Sialic test kit of a kind of mensuration and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 陈泰权和程腊梅 主编: "《医学检验专业试用教材 临床化学》", 30 April 1995, 湖南医学高等专科学校生物化学教研室 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110346316A (en) * | 2018-04-04 | 2019-10-18 | 浙江力耐得传动科技有限公司 | It is coupled the method and assay kit of pyruvate oxidation enzymatic determination sialic acid (SIA) |
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