CN108359710B - The enzyme colour developing quantitative determination reagent kit and measuring method of glucosephosphate isomerase - Google Patents
The enzyme colour developing quantitative determination reagent kit and measuring method of glucosephosphate isomerase Download PDFInfo
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Abstract
The present invention relates to the enzyme of glucosephosphate isomerase colour developing quantitative determination reagent kit and measuring methods, D-Fructose -6- disodium hydrogen phosphate generates G-6-P under glucosephosphate isomerase effect, G-6-P and nicotinamide adenine dinucleotide generate reduced nicotinamide adenine dinucleotide (NADH) under glucose-6-phosphate dehydrogenase (G6PD) effect, NADH is reacted with water-soluble tetrazolium, reaction product has maximum absorbance at 550nm, and absorbance value is directly proportional to GPI concentration.The indices of the enzyme colour developing method for quantitatively determining of glucosephosphate isomerase of the invention meet clinical requirement, can be used for the detection of clinical glucose phosphoric acid isomerase content.
Description
Technical field
The present invention relates to the enzyme of glucosephosphate isomerase colour developing quantitative determination reagent kit and methods, belong to clinical detection skill
Art field.
Background technique
Rheumatoid arthritis (rheumatoid arthritis, RA) is that a kind of cause of disease not yet illustrates, completely to invade four
Autoimmune disease based on primary minimum joint, RA early stage can produce irreversible bone erosion and destruction of joint, if failed
It gets timely medical treatment, RA patient is about 60%-70% in the disability rate of 2-3, therefore early diagnosis and the Normalized Treatment of RA
It is particularly important.Its pathogenesis is inquired into for the diagnosis and treatment of RA and is prevented most important.
Glucosephosphate isomerase (GPI) is a kind of wide expression and the protein with multiple functions, it is energy
Essential cytoplasm enzyme during cyclic process and sugar decomposition, while it is used as extracellular signal transduction molecule again.GPI is certainly
Molility factor and the nerve cell factor are secreted, may be played a role in terms of tumour and autoimmunity.Research in recent years hair
Existing, glucosephosphate isomerase (GPI) can be used as autoantigen and autoimmunity disease especially rheumatoid arthritis is closely related,
And it is positively correlated with the pain of patients with active and swollen joint number.Research shows that GPI may be by activating with antibody complex
Complement alternative route induces arthritis.GPI can induce C57BL/10 mouse and generate arthritis, and the generation of disease and II type are main
Histocompatibility complex is related.About the diagnostic value of GPI and its antibody in RA, most research results show that GPI is anti-
The sensing G T.GT.GT 65% of original diagnosis RA, specificity >=90% have higher diagnostic value.As GPI detection project is answered in clinic
With quick development, for detection technique quick and precisely and efficiently put forward new requirements.It can be fast so urgent need is a kind of
The method of fast accurate and sensitivity and the high detection glucosephosphate isomerase of precision meets the clinical diagnosis of RA.
Chinese patent document CN102323401A discloses a kind of glucose-6-phosphate isomerase (GPI) antigen in vitro inspection
Test agent box, including detection plate, goat-anti GPI antibody, colloidal gold conjugate, confining liquid, cleaning solution, positive reference product and negative ginseng
Examine product.Using the GPI antigen in double-antibody sandwich colloid gold immune absorption method principle detection human serum, by goat-anti GPI antibody packet
By in nitrocellulose filter, solid phase antibody is made, to capture human peripheral it is clear in GPI antigen that may be present.Again by colloid
Gold is marked on goat-anti GPI antibody, forms colloidal gold conjugate as tracer, contains GPI antigen as being tested in human serum, then shape
At solid phase goat-anti GPI antibody-GPI antigen-colloidal gold conjugate.However, this method can only detect the protein content of GPI, without
Its enzymatic activity height can be detected.Current clinical research confirmation GPI activity is more significant in Diagnosis of Rheumatoid Arthritis.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of enzyme colour developing quantitative determination reagent of glucosephosphate isomerase
Box and method.
Technical scheme is as follows:
A kind of enzyme colour developing quantitative determination reagent kit of glucosephosphate isomerase, including reagent R1, reagent R2And diluent A；
The reagent R1It is formed including following concentration of component: D-Fructose -6- disodium hydrogen phosphate (F6P) 1250-1300mg/L,
Nicotinamide adenine dinucleotide (NAD) 450-500mg/L, glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) 4-6mg/L；
The reagent R2It is formed including following concentration of component: water-soluble tetrazolium (WST-3) 18-22mg/L, azophenlyene
Methylsulfate (PMS) 1-3mg/L, nonylphenol polyoxyethylene ether (NP-40) 990-1010mg/L；
The diluent A includes following group and is grouped as: Tris 1200-1210mg/L, EDTA2Na 370-375mg/
L, KCl 3720-3730mg/L, MgCl21015-1020mg/L；
The reagent R1It is dissolved with diluent A, the reagent R2It is dissolved with water.
, according to the invention it is preferred to, the reagent R1It is formed including following concentration of component: F6P 1200mg/L,
NAD400mg/L, G6PDH 5mg/L；
The reagent R2It is formed including following concentration of component: WST-3 20mg/L, PMS 2mg/L, NP-401000mg/
L；
The diluent A includes following group and is grouped as: Tris 1210mg/L, EDTA2Na 372.24mg/L,
KCl3725mg/L, MgCl2 1016.5mg/L。
, according to the invention it is preferred to, the diluent A lemon acid for adjusting pH of 1mol/L to 8.05.
According to the present invention, the kit is placed on 2~8 DEG C of preservations.
According to the present invention, using mentioned reagent box to the enzyme colour developing method for quantitatively determining of glucosephosphate isomerase, including
Steps are as follows:
(1) by reagent R1With reagent R2It is uniformly mixed, obtains mixed liquor；
(2) sample to be tested is added into mixed liquor, 37 DEG C of reaction 10min obtain reaction solution；
(3) absorbance of the measurement reaction solution at 550nm, according between the concentration and absorbance of the formulation of GPI standard items
Standard curve y=0.0429x+0.1083, y indicate the concentration of GPI in sample, and x indicates absorbance, obtains GPI in sample to be tested
Concentration.
, according to the invention it is preferred to, reagent R in step (1)1With reagent R2It is mixed according to 1:1 volume ratio.
, according to the invention it is preferred to, the volume ratio of mixed liquor and sample to be tested is 10:1 in step (2).
The principle of the present invention:
The present invention generates G6P under glucosephosphate isomerase effect according to F6P, G6P again with NAD G6PDH effect
Color reaction occurs for lower generation NADH, NADH and WST-3, have at 550nm maximum absorbance, absorbance value and GPI concentration at
Direct ratio.The concentration of substrate that the present invention is generated by measurement enzymic catalytic reaction, and then indirectly embody glucosephosphate isomerase
Active height.Display substrate concentration is higher, and the activity for illustrating glucosephosphate isomerase is higher.
Beneficial effects of the present invention are as follows:
1, the present invention using enzyme development process to GPI quantitative detection, it is easy to operate quickly, examined compared to existing quantitative detection product
The survey time is short, and the detection of single sample can be completed in 10 minutes, can sensitive, accurately and rapidly reflect glucose phosphorus
The activity of acid isomer enzyme.
2, the linear model of detection of the invention be 0.00082-2.5mg/L, in the linear range have preferable precision and
Accuracy.Stability test shows that detectable concentration is the GPI quality-control product of 0.05mg/L, 0.3mg/L, 0.6mg/L in 12 months,
Each concentration relative deviation can control within allowed band, it is believed that reagent validity period > 12 month.Chaff interferent bilirubin
(200mg/L), hemoglobin (5g/L), triglycerides (15g/L) do not influence reagent detection.Testing result is different with glucose phosphate
The measurement result of structure enzyme (GPI) ELISA detection kit has good correlation, and two methods measurement result deviation is without system
Meter learns meaning (P > 0.05).
3, the method for the present invention is suitable for local basic hospital without being equipped with large-scale instrument and equipment, use cost relative moderate
Using.
Detailed description of the invention
Fig. 1 is the standard curve of GPI enzyme determination of color in embodiment.
Specific embodiment
Below by specific embodiment and in conjunction with attached drawing, the invention will be further described, but not limited to this.
Raw materials used and equipment is regular market purchase product in embodiment.
1. Specimen origin: RA group chooses in December, 2015 to 2016 Nian9Yue Shandong Qilu Hospital rheumatism and section's door is immunized
Examine with inpatient 39, wherein male 13, women 26, the age 20-83 years old, average age (60 ± 15) year, all trouble
Person meets the RA diagnostic criteria that American Rheumatism Association (ACR) combines European wind resistance diseases caused by dampness alliance (EULAR) revision in 2009.
Non- RA group chooses the same period in our hospital's outpatient clinic and be hospitalized patient 52, wherein male 18, and women 34, age 19-83
Year, average age (54 ± 17) year, meet domestic or international corresponding diagnostic criteria.Wherein Patients with SLE 23
Example is mixed connective tissue disease 2, ankylosing spondylitis 6, Sjogren syndrome 10, undifferentiated connective tissue disease 3, more
Hair property myositis/dermatomyositis 5, SpA 2, enteropathic arthritis 1.Normal group is chosen at our hospital's physical examination health
Person 32, wherein male 10, women 22, the age 26-81 years old, average age (53 ± 16) year.All cases are adopted on an empty stomach
Collect venous blood 3ml, separate serum, -80 DEG C of refrigerators are spare.It is measured after the freeze thawing of all samples.
2. instrument and reagent: Infinite M200 Pro multi-function microplate reader is bought from Tecan company, Switzerland, PHS-3E
The purchase of type pH meter is bought from Lei Ci company, Tris from sigma company, the U.S., EDTA.2Na, KCl, MgCl2Buy Gansu Province chemical industry westerly
Limited liability company, D-Fructose -6- disodium hydrogen phosphate (F6P), nicotinamide adenine dinucleotide (NAD), G-6-P are de-
From Roche company, Switzerland, water-soluble tetrazolium (WST-3) is purchased for hydrogen enzyme (G6PDH), the purchase of glucosephosphate isomerase standard items
It buys from Nanjing optically-active Science and Technology Ltd., citric acid, phenazine methosulfate (PMS) are bought from Aladdin company, polyoxyethylene nonyl phenyl
Vinethene (NP-40) is bought from Ann Kyrgyzstan company.Glucosephosphate isomerase (GPI) ELISA detection kit is bought from Shanghai
North plus biochemical reagents Co., Ltd.
Standard curve in embodiment between GPI concentration and absorbance is y=0.0429x+0.1083, R2=0.9923,
As described in Figure 1.The preparation process of standard curve is as follows:
By the GPI standard items of 0.75mg/L, it is diluted to 0.6mg/ respectively with 0.05mol/L Tris buffer (pH=7.6)
L, 0.45mg/L, 0.3mg/L, 0.15mg/L, 0.05mg/L, Tris buffer itself are used as blank calibration object, measure GPI standard
Relation curve between product concentration and absorbance.
Embodiment 1
A kind of enzyme colour developing quantitative determination reagent kit of glucosephosphate isomerase, comprising:
Prepared and diluted liquid A: wherein Tris 1210mg/L, EDTA.2Na 372.24mg/L, KCl 3725mg/L, MgCl2
1016.5mg/L, with the lemon acid for adjusting pH of 1mol/L to 8.05.
Reagent R1: F6P 1200mg/L, NAD 400mg/L, G6PDH 5mg/L；It is dissolved with diluent A.
Reagent R2: WST-3 20mg/L, PMS 2mg/L, NP-40 1000mg/L is dissolved with water.
Mentioned reagent, which prepares, is placed on 2~8 DEG C of preservations.
Embodiment 2
Using kit described in embodiment 1 to the enzyme colour developing method for quantitatively determining of glucosephosphate isomerase, including step
It is rapid as follows:
Experiment first 10 minutes first by reagent R1With reagent R2It is uniformly mixed by 1:1 volume ratio, obtains mixed liquor, every hole is drawn
100uL mixed liquor；
10ul sample to be tested is added into mixed liquor, 37 DEG C of reaction 10min obtain reaction solution；
With absorbance of the Infinite M200 Pro measurement reaction solution at 550nm, formulated according to GPI standard items dense
The standard curve of degree and absorbance, obtains the concentration of GPI in sample to be tested.
The precision and accuracy of test example 1, GPI enzyme colour developing method for quantitatively determining
The detection reagent of two batches is extracted, respectively to the GPI Quality Control of concentration 0.05mg/L, 0.30mg/L, 0.60mg/L
Product are measured, and each concentration is repeated 20 times, calculate batch in and batch between each concentration test result CV, and count each concentration determination
As a result bias situation.Using twice match liquid measure simultaneously, each Quality Control replication 20 times, batch in and criticize between each detectable concentration CV
<10%, and t is examined and is shown that each concentration test result bias is subjected to (P>0.05).It is shown in Table 1.
1 two batches reagent accurate degree of table and accuracy analysis result
As shown in Table 1, the preci-sion and accuracy CV value of reagent of the present invention is both less than 10%, is fully able to meet clinical inspection
The requirement of survey.
Test example 2, interference experiment
Clinically mainly there are turbid rouge, jaundice and haemolysis for the interference of serum measurement.Experiment is using the side for adding chaff interferent
Formula carries out, and adds the interfering substance of various concentration in high, medium and low value serum respectively, and the difference of front and back result is added in measurement.
Respectively detection sample addition the bilirubin of 200mg/L, the hemoglobin of 5g/L, 15g/L triglycerides front and back
Result difference, as a result relative deviation thinks that chaff interferent does not influence sample test within ± 10%.It is shown in Table 2.
The measurement result of front and back is added in each chaff interferent of table 2
Above-mentioned 2 result of table as it can be seen that the reagent in bilirubin concentration≤200mg/L, hemoglobin concentration≤5g/L, glycerol three
In the case where ester concentration≤15g/L, the average deviation of each concentration range is in 5%, according to clinical criteria requirement of experiment deviation
It should be within ± 10%, in the tolerance interval that clinically uses completely.
Test example 3, stability experiment result
Reagent is placed in 2~8 DEG C of preservations, tests respectively 14 months (12 months reagent validity periods+extension 2 months) interior every month
Concentration is the GPI quality-control product of 0.05mg/L, 0.30mg/L, 0.60mg/L, and each concentration is repeated 10 times, testing result x ± s table
Show, test result relative deviation thinks that reagent stability is good within ± 20%.
The results are shown in Table 3 for stability test, and detectable concentration is 0.05mg/L, 0.3mg/L, 0.6mg/L in 14 months
GPI quality-control product, each concentration relative deviation is within ± 20%, reagent validity period > 12 month.It is shown in Table 3.
The stability test result of the enzyme development process quantitative determination of 3 glucosephosphate isomerase of table
Note: ± the 20% of 0.05mg/L, 0.3mg/L, 0.6mg/L concentration be respectively 0.04~0.06mg/L, 0.24~
0.36mg/L, 0.48~0.72mg/L.
As shown in Table 3, kit of the present invention is with good stability.
Test example 4, determination of recovery rates
Analyze 1 concentration 0.3mg/L of sample and analysis 2 concentration 0.6mg/L replication of sample 5 times, averagely recycling concentration point
Not Wei 0.31mg/L and 0.59mg/L, the rate of recovery is respectively 103.3% and 98.3%, average recovery rate 100.8%.Such as table 4.
4 determination of recovery rates of table
Test example 5, the comparison of clinical samples testing result
123 parts of samples are divided into 3 groups according to RA group (n=39), non-RA group (n=52) and control group (n=32), detection knot
Fruit is shown in Table 5.
This 123 parts of serum specimens are detected with enzyme development process and ELISA kit respectively, obtain 123 parts of valid data.It will
This two groups of data carry out correlation analysis, and two groups of data are in RA group (n=39), non-RA group (n=52) and control group (n as the result is shown
=32) each group is without notable difference, the comparison about two methods to clinical samples detection data, and rank sum test result is aobvious
Show: Z=﹣ 1.38, P=0.122 > 0.05, between the two no difference of science of statistics.
5 GPI enzyme development process testing result of table and ELISA detection kit testing result compare
As shown in Table 5,123 parts of clinical serum samples are detected with the method for the present invention, with GPI ELISA kit testing result
Compare, there is preferable consistency.
Using 19.0 fit standard curvilinear equation of SPSS, r >=0.99 indicates linear good.Each group of data is indicated with x ± s.
Reagent accuracy Bias analysis uses the t method of inspection.The correlation of two methods clinic quantitative detection result uses SPSS unitary
Linear regression analysis, related coefficient (r) >=0.98 indicate that correlation is good, and variance analysis is using pairing rank sum test.
Claims (2)
- The quantitative determination reagent kit 1. a kind of enzyme of glucosephosphate isomerase develops the color, which is characterized in that the kit includes reagent R1, reagent R2And diluent A；The reagent R1It is formed including following concentration of component: D-Fructose -6- disodium hydrogen phosphate (F6P) 1200mg/L, nicotinoyl amine gland Purine dinucleotides (NAD) 400mg/L, glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) 5mg/L；The reagent R2It is formed including following concentration of component: water-soluble tetrazolium (WST-3) 20mg/L, phenazine methosulfate (PMS) 2mg/L, nonylphenol polyoxyethylene ether (NP-40) 1000mg/L；The diluent A includes following group and is grouped as: Tris 1210mg/L, EDTA2Na 372.24mg/L, KCl 3725mg/L, MgCl21016.5mg/L；The reagent R1It is dissolved with diluent A, the reagent R2It is dissolved with water；The diluent A is with the lemon of 1mol/L Acid for adjusting pH is to 8.05.
- The quantitative determination reagent kit 2. enzyme of glucosephosphate isomerase according to claim 1 develops the color, which is characterized in that The kit is placed on 2 ~ 8 DEG C of preservations.
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109593825A (en) * | 2018-12-17 | 2019-04-09 | 浙江达美生物技术有限公司 | It is a kind of detect glucose-6-phosphate isomerase kit and kit application method |
| CN109900686A (en) * | 2019-02-27 | 2019-06-18 | 山东大学齐鲁医院 | A kind of method, system and terminal improving sxemiquantitative poisonous substance detection accuracy |
| CN111562374A (en) * | 2020-06-12 | 2020-08-21 | 上海怡珏生物科技有限公司 | New application of glucose 6 phosphate isomerase kit |
Citations (3)
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|---|---|---|---|---|
| CN1257128A (en) * | 1998-12-11 | 2000-06-21 | 协和梅迪克斯株式会社 | Method for quantifying 1,5-anhydrosorbierite and quantitative reagent |
| CN1419604A (en) * | 2000-03-28 | 2003-05-21 | 生命扫描有限公司 | Reagents and methods for detecting a reduced cofactor |
| CN1214117C (en) * | 2000-02-25 | 2005-08-10 | 生命扫描有限公司 | Diagnosis based on tetrazotized zole compound |
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2018
- 2018-02-11 CN CN201810142833.XA patent/CN108359710B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1257128A (en) * | 1998-12-11 | 2000-06-21 | 协和梅迪克斯株式会社 | Method for quantifying 1,5-anhydrosorbierite and quantitative reagent |
| CN1214117C (en) * | 2000-02-25 | 2005-08-10 | 生命扫描有限公司 | Diagnosis based on tetrazotized zole compound |
| CN1419604A (en) * | 2000-03-28 | 2003-05-21 | 生命扫描有限公司 | Reagents and methods for detecting a reduced cofactor |
Non-Patent Citations (2)
| Title |
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| 类风湿性关节炎中葡萄糖-6-磷酸异构酶的检测及其临床意义;张明梅等;《同济大学学报》;20071231;第28卷(第6期);105-108 * |
| 葡萄糖-6-磷酸异构酶研究进展;韩龙等;《药物生物技术》;20121231;第19卷(第6期);561-564 * |
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