CN107153121A - Glycosylated hemoglobin, hemoglobin detection kit and its detection method - Google Patents
Glycosylated hemoglobin, hemoglobin detection kit and its detection method Download PDFInfo
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- CN107153121A CN107153121A CN201710237126.4A CN201710237126A CN107153121A CN 107153121 A CN107153121 A CN 107153121A CN 201710237126 A CN201710237126 A CN 201710237126A CN 107153121 A CN107153121 A CN 107153121A
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- 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
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
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Abstract
The invention discloses a kind of glycosylated hemoglobin, hemoglobin detection kit and its detection method of glycosylated hemoglobin detection technique field, particularly a kind of glycosylated hemoglobin based on boric acid affinity chromatography, hemoglobin detection kit and its detection method；The kit includes R1 reagents, R2 reagents, R3 reagents, R4 reagents, glycosylated hemoglobin-hemoglobin deposition film；The detection method is specifically a kind of while the method that can carry out glycosylated hemoglobin and hemoglobin detection.Compared with prior art, it is an advantage of the present invention that detection time, reduction blood sampling consumption, extension kit shelf life, increase detection accuracy, the mildness for improving testing conditions can be shortened using the detection kit and detection method of the present invention.
Description
Technical field
The invention belongs to glycosylated hemoglobin detection technique field, and in particular to a kind of glycosylated hemoglobin, hemoglobin
Detection kit and its detection method.
Background technology
Counted according to IDF, global diabetic there are about 4.15 hundred million people within 2015, and every 11 people just have 1 people to suffer from glycosuria
Disease.The year two thousand forty is predicted, the whole world will have 6.42 hundred million people with diabetes, and diabetes mellitus in China patient is also up to 1.51 hundred million, sugar
Sick early stage patient up to 2.24 hundred million people of urine.Planning commission and IDF statistics, China in 2015 18 years old and above maturity-onset diabetes are defended according to country
Illness rate is 10.7%, and pre-diabetic is up to 16%, and the diabetes of -80 years old 18 years old adults in the whole world in 2015 are ill
Rate is 9.1%.
Chinese diabetic is ranked the first in the world, and tends to rejuvenation, and tool DF is counted, and China has 1,300,000 people to die within 2015
Diabetes and its complication, wherein 40.8% people's age was less than 60 years old, and was presented young mouth radix greatly year by year, aging population
The factor such as aggravation and dietary structure, causes the people of China's diabetic's number about 1.5 hundred million, occupies the first in the world, China's glycosuria
Sick consumer group's scale of construction is big, and the trend that patient increases year by year is presented.
Glycosylated hemoglobin (glycated hemoglobin, or glycated hemoglobin, or glycosylated
Hemoglobin) (hereinafter sometimes called " HbA1c ") has been seen as screening diabetes, inspection and has been likely to become prediabetes
The glycemic control of the people of people or the glycemic control of monitoring patient and the useful tool for diagnosing diabetes.In erythrocyte
In normal 120 day life-span, glucose molecule reacts with hemoglobin, forms glycosylated hemoglobin.Once haemoglobin molecule
It is saccharified, it keeps the state being saccharified.Therefore, endoerythrocytic glycosylated hemoglobin increases the life reflected in cell
The average level for the glucose that cell is exposed in cycle.Adjusted by monitoring long serum glucose, measure HbAle
Albumen can assess the therapeutic effect of diabetes.HbA1c levels are directly proportional to preceding surrounding to trimestral mean blood glucose concentrations.According to
This, American-European countries regard this as diabetes curative effect and the goldstandard of adjustment therapeutic scheme.
At present, detect that the common methods of glycosylated hemoglobin mainly there are two major classes in preceding clinical labororatory：One class method base
, such as ion chromatography, electrophoresis method different from the electric charge of both the non-glycated hemoglobin institute band in glycosylated hemoglobin；Another kind of side
Design feature of the method based on the group that is saccharified on hemoglobin, such as affinity chromatography, Ion capture and immunization.Wherein, it is based on
The glycosylated hemoglobin of boric acid affinity chromatography, hemoglobin detection receive much concern.
Found through retrieval, Authorization Notice No. discloses a kind of hemoglobin detection examination for CN100593117C patent of invention
Agent box, including：The perforated membrane of the hemoglobin of precipitation can be retained；First reagent, including the zinc ion aqueous solution or can including water
Dissolubility zinc compound；Second reagent, including chromophore-boric acid conjugate；Include Aqueous wash reagent with optional；Although its skill
Art scheme solves the problem of being presented unstable with the extension testing result of kit preservation time, but Clinical Practice is demonstrate,proved
Bright its does not play a part of Shelf-life inherently.Retrieval also found that application publication number is CN102652264A hair
Bright patent discloses a kind of for measuring the centrifugal microfluidic structure of glycosylated hemoglobin, the centrifugation for measuring glycosylated hemoglobin
Immunosorbent assay and affinity chromatography are measured phase by microfluidic devices and the method for measuring glycosylated hemoglobin, the technical scheme
With reference to, glycosylated hemoglobin is not only detected, glycosylated hemoglobin variant or interfering material is also detected, it is final to eliminate and/or mend
The mistake in glycosylated hemoglobin measurement is repaid or calibrates, so that the detection of glycated hemoglobin levels is more accurate.Although should
Technical scheme can accurately more determine the content of glycosylated hemoglobin relative to prior art to a certain extent, but its skill
Art scheme is complicated, and detection time is longer, is unfavorable for the exploitation of convenient household detection means.Applicant also retrieves, Shen
Please publication No. for CN105301261A patent of invention disclose it is a kind of detect glycosylated hemoglobin kit and its preparation side
Method and application method, wherein, the kit includes reaction solution, cleaning solution and reaction plate, but reaction solution and cleaning solution preserve bar
Part is strictly defined as 2-8 DEG C, limits the progress detected under natural conditions, is unfavorable for the popularization of convenience-for-people kit.
The content of the invention
For the drawbacks described above of prior art, examined it is an object of the invention to provide a kind of glycosylated hemoglobin, hemoglobin
Test agent box and its detection method, particularly a kind of glycosylated hemoglobin based on boric acid affinity chromatography, hemoglobin detection
Kit and its detection method, detect accurate to shorten detection time, reduction blood sampling consumption, extend kit shelf life, increase
Property, improve testing conditions mildness.
The purpose of the present invention is achieved by the following technical solution：
In a first aspect, the present invention provides a kind of glycosylated hemoglobin, hemoglobin detection kit, including R1 reagents, R2
Reagent, R3 reagents, R4 reagents, glycosylated hemoglobin-hemoglobin deposition film；
The R1 reagents, its pH is alkalescent, and solvent is the sodium-chloride water solution that concentration is less than or equal to 0.35wt%, solute
And its content is in the solvent：Surfactant 0.12-0.18%w/v；
The R2 reagents, its solvent is Glycine-NaOH buffer solution, solute and its content in the solvent
For：Chromophore-boric acid conjugate 0.001-0.003wt%, surfactant 0.12-0.18%w/v；
The R3 reagents, its pH is alkalescent, specially Aqueous wash reagent；
The R4 reagent solutions, its solvent is Glycine-NaOH buffer solution, solute and its containing in the solvent
Measure and be：Inorganic zinc salt 10-15mM, surfactant 0.1-0.2%w/v.
Preferably, it is 2 that the surfactant, which refers specifically to Tween20 and Triton X-100 mass ratioes,：1.
Preferably, the alkalescent refers specifically to pH for 7.2-7.5.
Preferably, the alkalescent refers specifically to pH for 7.35-7.45.
Preferably, the R1 reagents, in addition to：0.002%w/v sodium azide, 0.05%w/v glycerine, 0.005%
W/v skimmed milk power, 0.001%w/v casein, 0.01%w/v BSA.
Preferably, the R2 reagents, in addition to：2-8mM magnesium chloride hexahydrate, 1-1.5%v/v formamide.
Preferably, the R4 reagents, in addition to：10-15%v/v ethanol, 0.002-0.004%w/v Azide
Sodium, 0.1-0.3%w/w sodium chloride.
Preferably, the glycosylated hemoglobin-hemoglobin deposition film is selected from glass fibre membrane, borate filter membrane or fibre
Tie up plasma membrane.
Second aspect, the present invention provides a kind of glycosylated hemoglobin based on the detection kit, hemoglobin detection
Method, comprises the following steps：
Blood sample to be detected and R1 reagents are taken, mixes, obtains the blood sample after haemolysis；
R2 reagents will be added in blood sample after haemolysis, mixing obtains glycosylated hemoglobin detection sample；
Glycosylated hemoglobin detection sample is placed in glycosylated hemoglobin-hemoglobin deposition film, one end time is placed
Afterwards, R4 reagent solutions are added, then place a period of time；
The glycosylated hemoglobin-hemoglobin deposition film is cleaned with R3 reagents；
The sample sites in the glycosylated hemoglobin-hemoglobin deposition film are detected based on reflectance photometry method,
Glycosylated hemoglobin, content of hemoglobin in detection blood sample can be obtained respectively.
The mechanism of kit of the present invention and detection method is：In R1 reagents, glycosylated hemoglobin and hemoglobin quilt
Release, then glycosylated hemoglobin is combined with the boric acid in R2 reagents, is added after R4 reagents, and target proteinses are deposited, R3 reagents
After flushing, in addition to the target proteinses being deposited in glycosylated hemoglobin-hemoglobin deposition film, other detection sample component quilts
Wash away, by aforesaid operations, glycosylated hemoglobin and hemoglobin are deposited, detected that accuracy is high.
Compared with prior art, the present invention possesses following beneficial effect：
Its constituent species and content are based on using the detection kit of the present invention, detection time, reduction blood sampling can be shortened
Consumption, extension kit shelf life, increase detection accuracy, the mildness for improving testing conditions.
In addition, the kit of the present invention is that a kind of base glycosylated hemoglobin and hemoglobin are detected on integral detection reagent
Box, is applicable not only to prevention, the detection of diabetes, prevention, the detection of anaemia is applied also for, relative to showing for single Protein Detection
Have for kit it is more convenience-for-people it is practical, advantageously in marketing.
Furthermore, reagent constituents and species based on the present invention are preferred, detection method optimization, in HbAle egg of the present invention
The glycosylated hemoglobin that is deposited in vain-hemoglobin deposition film, hemoglobin distribution of particles are very uniform, and granular size is fitted
In, it is 2-5 μm, such sedimentary characteristic is advantageously in the accuracy of testing result.
Brief description of the drawings
Fig. 1 is examined for the glycosylated hemoglobin produced respectively using kit described in embodiment and Bio-Rad companies of the U.S.
Test agent box detects the testing result comparison diagram of identical sample.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that to the ordinary skill of this area
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
Embodiment 1
Embodiment 1 provides a kind of glycosylated hemoglobin, hemoglobin detection kit, including R1 reagents, R2 reagents, R3
Reagent, R4 reagents, glycosylated hemoglobin-hemoglobin deposition film；Wherein,
R1 reagents：Its pH be 7.2-7.5, solvent be concentration be equal to 0.35wt% sodium-chloride water solution, solute and its
Content is in the solvent：Surfactant 0.12%w/v；The surfactant refers specifically to Tween20 and Triton X-
100 mass ratioes are 2：1；
R2 reagents：Its solvent is Glycine-NaOH buffer solution, and solute and its content in the solvent are：Two
Toluol-benzene amine-phenylboric acid conjugate 0.001wt%, surfactant 0.12%w/v；The surfactant is referred specifically to
Tween20 and Triton X-100 mass ratioes are 2：1；
R3 reagents：Specially physiological saline；
R4 reagents：Its solvent is Glycine-NaOH buffer solution, and solute and its content in the solvent are：Chlorine
Change zinc 10mM, surfactant 0.1%w/v；The surfactant refers specifically to Tween20 and Triton X-100 mass ratioes
For 2：1；
Glycosylated hemoglobin-hemoglobin deposition film：Fiber plasma membrane.
Embodiment 2
Embodiment 2 provides a kind of glycosylated hemoglobin, hemoglobin detection kit, including R1 reagents, R2 reagents, R3
Reagent, R4 reagents, glycosylated hemoglobin-hemoglobin deposition film；Wherein,
R1 reagents：Its pH is 7.2-7.5, and solvent is the sodium-chloride water solution that concentration is 0.20wt%, solute and its in institute
Stating content in solvent is：Surfactant 0.18%w/v；The surfactant refers specifically to Tween20 and Triton X-100
Mass ratio is 2：1；
R2 reagents：Its solvent is Glycine-NaOH buffer solution, and solute and its content in the solvent are：Two
Toluol-benzene amine-phenylboric acid conjugate 0.003wt%, surfactant 0.18%w/v；The surfactant is referred specifically to
Tween20 and Triton X-100 mass ratioes are 2：1；
R3 reagents：Specially Glycine-NaOH buffer solution；
R4 reagents：Its solvent is Glycine-NaOH buffer solution, and solute and its content in the solvent are：Sulphur
Sour zinc 15mM, surfactant 0.2%w/v；The surfactant refers specifically to Tween20 and Triton X-100 mass ratioes
For 2：1；
Glycosylated hemoglobin-hemoglobin deposition film：Glass fibre membrane.
Embodiment 3
Embodiment 3 provides a kind of glycosylated hemoglobin, hemoglobin detection method, specifically based on embodiment 1 and implementation
What the kit that example 2 is provided was carried out, specifically include following steps：
2.5 μ L blood samples to be detected and 100 μ LR1 reagents are taken, 3s is shaken, obtains the blood sample after haemolysis；
Blood sample after haemolysis is added to isometric R2 reagents, 3s is shaken, glycosylated hemoglobin detection sample is obtained；
Glycosylated hemoglobin detection sample is placed in glycosylated hemoglobin-hemoglobin deposition film, stood after 5s, then add
Enter R4 reagent solutions, then place 5s；
The glycosylated hemoglobin-hemoglobin deposition film is cleaned with R3 reagents；
The sample sites in the glycosylated hemoglobin-hemoglobin deposition film are detected based on reflectance photometry method,
It is respectively 616nm, 415nm using wavelength, you can obtain glycosylated hemoglobin, content of hemoglobin in detection blood sample respectively.
During the present embodiment is detected, in order to embody the advantage of kit of the present invention, the present embodiment is also based on
Same blood sample to be detected has carried out high performance liquid chromatography detection, and is detected using commercially available biochemical reagents are bought, every kind of
Method in triplicate, the results are shown in Table 1.
Table 1 repeats to detect the result of three times using different kits, method to same sample
| For the first time | Second | Second | |
| Embodiment 1 | 5.7 | 5.7 | 5.7 |
| Embodiment 2 | 5.7 | 5.7 | 5.7 |
| HPLC | 5.7 | 5.8 | 5.8 |
| Biochemical reagents | 5.4 | 5.3 | 5.4 |
Based on the result described in above-mentioned table 1 it is seen that, Example 1 and Example 2 of the present invention provide detection kit
Testing result is significantly better than commercially available biochemical reagents box extremely close to HPLC.Further, compared with HPLC, embodiment 1 and reality
There is following advantage in the kit for applying example 2：The kit user of the present invention need not possess the medical knowledge on testing of specialty, as long as
Indicated to complete according to the specification of kit, the whole detection method of skilled operation will not surpass 90s, and repeatability is good；
The kit of the embodiment of the present invention is i.e. operable at room temperature, and environment is disturbed it and its faint.
In above-described embodiment implementation process, inventor is found surprisingly that：
(1) restriction of the present invention is made to R1 reagents has unexpected effect, is specifically, the R1 reagents pair of the restriction
Glycosylated hemoglobin, the release of hemoglobin have acceleration in blood, can thoroughly be released in very short time (most 3s)
Put, can not only so increase the accuracy of testing result, and remarkable effect can also be played to shortening detection cycle indirectly；(2)
Although it is common technology means to increase surfactant in cell pyrolysis liquid, inventor has found the kind of Surfactant
In the case that class and its consumption are optimized, contractile effect of erythrocyte breakdown products state is uniform and stable, be affected by temperature less, without using
Person's examinations at 2-8 DEG C, in addition, in kit storage process, directly by its naturally occurring, without being store to it
Temperature is deposited to be defined；(3) during being optimized to R2 reagents, inventor has found, by being coupled to chromophore-boric acid
Thing and surface-active contents, the optimization of species so that color group-boric acid conjugate and saccharification after haemolysis blood sample is added into
Hemoglobin combination speed is accelerated, and is being mixed after thing moved in glycosylated hemoglobin-hemoglobin deposition film, sugar
Change hemoglobin to be evenly distributed, granular size is moderate (2-5 μm of diameter), is firmly combined with deposition film, is avoided that HbAle egg
It is white to be lost in, increase detection accuracy；(4) during being optimized to R4 reagents, inventor has found, although zinc ion is as heavy
Shallow lake agent is that this area is common, but its concentration is optimized and limits the deposition for being particularly conducive to accelerate hemoglobin, and is sunk
Product granular size is moderate (2-5 μm of diameter), even particle distribution, on the basis of kinds of surfactants and content optimization, R4
Reagent is placed directly within room temperature also without being stored in 2-8 DEG C.The basis optimized in mentioned reagent species and content
On, each detection is accelerated, and detection can not only be completed in the case of less than 90s, and storage condition is gentle, goods
Detected any time in 1 year frame phase and be able to ensure that result accuracy.
Embodiment 4
Embodiment 4 is the preference of embodiment 1, and its preferred part is solute and its content in R1 reagents, specifically such as
Under：
R1 reagents：Its pH be 7.35-7.45, solvent be concentration be equal to 0.35wt% sodium-chloride water solution, solute and its
Content is in the solvent：Surfactant 0.12%w/v；The surfactant refers specifically to Tween20 and Triton
X-100 mass ratioes are 2：1；In addition, R1 reagents also including 0.002%w/v sodium azide, 0.05%w/v glycerine,
0.005%w/v skimmed milk power, 0.001%w/v casein, 0.01%w/v BSA；It should be noted that R1 reagents are also
The degreasing of the glycerine, 0.003-0.006%w/v of sodium azide, 0.04-0.06%w/v including 0.001-0.003%w/v
It can still realize that technology is imitated in the case of milk powder, 0.001-0.003%w/v casein, 0.01-0.03%w/v BSA
Really；Simply in R1 the reagents also sodium azide including 0.002%w/v, 0.05%w/v glycerine, 0.005%w/v defatted milk
Powder, 0.001%w/v casein, 0.01%w/v BSA effect it is optimal；
R2 reagents：Its solvent is Glycine-NaOH buffer solution, and solute and its content in the solvent are：Two
Toluol-benzene amine-phenylboric acid conjugate 0.001wt%, surfactant 0.12%w/v；The surfactant is referred specifically to
Tween20 and Triton X-100 mass ratioes are 2：1；Also include 5mM magnesium chloride hexahydrate, 1.2%v/v formamide；Need
Illustrate, the content of magnesium chloride hexahydrate can be achieved in the content of 2-8mM, formamide under conditions of 1-1.5%v/v；
R3 reagents：Specially physiological saline；
R4 reagents：Its solvent is Glycine-NaOH buffer solution, and solute and its content in the solvent are：Chlorine
Change zinc 10-15mM, surfactant 0.1%w/v；The surfactant refers specifically to Tween20 and Triton X-100 matter
Amount is than being 2：1；Also include：10-15%v/v ethanol, 0.002-0.004%w/v sodium azide, 0.1-0.3%w/w
Sodium chloride；
Glycosylated hemoglobin-hemoglobin deposition film：Fiber plasma membrane.
Inventor is found surprisingly that, after being optimized to kit reagent, and its resistance to overturning is dramatically increased, specifically such as
Under：
Processing 1：The kit of the room temperature embodiment of 4 years 4；
Processing 2：The kit of the room temperature embodiment of 2 years 4；
Processing 3：The kit of embodiment 4 of current configuration；
Processing 4：The kit of embodiment 1 of current configuration；
It is as a result as shown in table 2 below with the same blood sample of aforementioned four processing detection：
Table 2
| Processing 1 | Processing 2 | Processing 3 | Processing 4 | |
| Saccharification hemoglobin content | 5.7 | 5.7 | 5.7 | 5.7 |
The kit of embodiment 4 can be seen that after storing 4 years by the result of above-mentioned table 4, to blood testing
Accuracy is not reduced, it is seen that the present embodiment is optimized by component, and finally shelf life is obviously improved.Although many existing
Technology is made that many effort to glycosylated hemoglobin detection kit Shelf-life, but the time limit of extension still extremely office
Limit, and the present invention finally significantly improves the shelf life of kit, this is people in the art by the optimization of reagent constituents
Member is unexpected.
In addition to it there is the effect of being obviously improved in above-mentioned improvement, inventor has also been found to exist following beneficial to kit shelf life
Effect：
(1) analyst coverage has been lifted
In order to determine the detection range of the kit of embodiment 4, inventor's saccharification blood according to technical configuration commonly used in the art
Lactoferrin sample gradient solution (being 0,0.5%, 3%, 5%, 10%, 12%, 15%, 20%, 25%), using above-mentioned detection side
Method is detected to glycosylated hemoglobin sample gradient solution, as a result as shown in table 3 below：
Table 3
By table 3, by the optimization to reagent component and content, kit of the invention is further in detection range
On lifted, the sample of 0.5% content rank is capable of detecting when, while can also detect the sample of 25% content rank.This
Also illustrate, the optimization of reagent component and content improves the sensitivity of kit.
(2) lot stability has been lifted
In order to determine the lot stability of the kit of embodiment 4, inventor's saccharification according to technical configuration commonly used in the art
Hemoglobin samples solution (5%), selective examination five batches kit sample is detected based on above-mentioned detection method, each
Batch spot-check 10 kits, finally counts the testing result average value of each batch, concrete outcome is shown in Table 4：
Table 4
| First batch | Second lot | 3rd batch | 4th batch | 5th batch | |
| Embodiment 1 | 5.1% | 5% | 5% | 4.9% | 5% |
| Embodiment 4 | 5% | 5% | 5% | 5% | 5% |
By table 4, by the optimization to reagent component and content, kit of the invention is further in batch quality
Lifted in stability, this explanation, the optimization of component and content contributes to stabilization of the reagent in physicochemical properties, so that
Ensure the stability of its testing result.
(3) equivalence experiment (carrying out methodology Determination with goldstandard high performance liquid chromatography)
To assess the glycosylated hemoglobin detection kit (particulate chromatography) and detection method described in above-described embodiment
Accuracy, the goldstandard high performance liquid chromatography used is diagnosed with current glycosylated hemoglobin and carries out methodology Comparability test.
Totally 100 human blood of clinical laboratory normal person and diabetes patient are randomly selected, everyone takes two parts of samples, immediately censorship.
Experimental procedure：
1) kit of embodiment 1：Tested according to the experimental implementation flow of the kit described in above-described embodiment；
2) high performance liquid chromatography：The glycosylated hemoglobin detection kit produced using Bio-Rad companies of the U.S. is (high
Effect liquid phase chromatogram method), tested according to its specification operating process.
Statistical method：Blood sample testing result to 100 people carries out correlation analysis, and seeks its coefficient correlation.
Final conclusion：Fig. 1 is referred to, data carry out correlation analysis, the two testing result height correlation, its coefficient correlation
It is 0.999, shows that the kit testing result of above-described embodiment 1 and goldstandard testing result are equivalent.But high performance liquid chromatography is examined
Survey cost high, professional too strong, big by environmental disturbances, stability is poor, and applies the kit described in above-described embodiment
HbA1c is detected, it is with low cost, it is simple to operate, it is quick, sensitive.
(4) performance test can be interrupted
In most cases, to the instantaneity that is detected as of glycosylated hemoglobin, i.e., sampling, which is finished, at once detects, inspection every time
Surveying the personnel operated, external condition may be different, therefore causes error unavoidably, ultimately results in the result comparison of METHOD FOR CONTINUOUS DETERMINATION not
By force；It is not to have adopted after blood sample to have the time or facilitate i.e. every time for family expenses detection kit user
Carve detection.In order to verify that can the kit of the present invention overcome above-mentioned technical problem, inventor has carried out following checking：
10 Healthy Volunteers of clinical laboratory are randomly selected, blood sample are extracted, and blood sample is handled as follows：
Processing 1：Blood sample is added to the R1 reagents of embodiment 4 immediately, detected immediately, gained glycosylated hemoglobin contains
Amount is respectively A1, A2, A3, A4, A5, A6, A7, A8, A9, A10；
Processing 2：Blood sample is added to the R1 reagents of embodiment 4 immediately, normal temperature is preserved 1 month, then carries out subsequent detection, gained
The content of glycosylated hemoglobin is respectively B1, B2, B3, B4, B5, B6, B7, B8, B9, B10；
Processing 3：Blood sample is added to the R1 reagents of embodiment 4 immediately, normal temperature is preserved 2 months, then carries out subsequent detection, gained
The content of glycosylated hemoglobin is respectively C1, C2, C3, C4, C5, C6, C7, C8, C9, C10；
Processing 1, processing 2,3 gained corresponding datas of processing are finally respectively compared, see whether data fluctuations are obvious.
As a result show, the data of A1, B1, C1 tri- are not fluctuated substantially, other nine groups of data are as the same.It can be seen that, R1 of the invention
Reagent has quality assurance effect to sample.
In order to illustrate the mechanism of above-mentioned beneficial effect, inventor sets following control, and the control is pair of above-mentioned processing 3
According to control part is：
Control 1：The kit of use is transformation, and transformation part is the surface-active in the R1 reagents by embodiment 4
Agent species is changed to Tween20 single kinds, while the content of surfactant is revised as 0.2%w/v；
Control 2：The kit of use is transformation, and transformation part is the 0.5%w/v in the R1 reagents by embodiment 4
Sodium azide, 0.05%w/v BSA；
Control 3：The kit of use is transformation, and transformation part is six water chlorinations in the R2 reagents by embodiment 4
Zinc chloride 20mM of the content of magnesium in 10mM, R4 reagents.
To control 1, control 2, compare 3 data count, presentation as a result, control 1 and compare 2 data it is notable
Be worse than processing 2, and compare 3 slightly poorer to processing 2.The result indicates why the kit provided in the present invention is handling blood
Detection can be postponed after sample, main reason is that reagent R1 constituent species and content, auxiliary reason are R2 reagents and R4
The synergy of reagent.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.
Claims (10)
1. a kind of glycosylated hemoglobin, hemoglobin detection kit, it is characterised in that including R1 reagents, R2 reagents, R3 examinations
Agent, R4 reagents, glycosylated hemoglobin-hemoglobin deposition film；
The R1 reagents, its pH be alkalescent, solvent be concentration be less than or equal to 0.35wt% sodium-chloride water solution, solute and its
Content is in the solvent：Surfactant 0.12-0.18%w/v；
The R2 reagents, its solvent is Glycine-NaOH buffer solution, and solute and its content in the solvent are：Hair
Color group-boric acid conjugate 0.001-0.003wt%, surfactant 0.12-0.18%w/v；
The R3 reagents, specially Aqueous wash reagent；
The R4 reagent solutions, its solvent is Glycine-NaOH buffer solution, and solute and its content in the solvent are：
Inorganic zinc salt 10-15mM, surfactant 0.1-0.2%w/v.
2. glycosylated hemoglobin according to claim 1, hemoglobin detection kit, it is characterised in that the surface
It is 2 that activating agent, which refers specifically to Tween20 and Triton X-100 mass ratioes,：1.
3. glycosylated hemoglobin according to claim 1, hemoglobin detection kit, it is characterised in that the R1 examinations
In agent, alkalescent refers specifically to pH for 7.2-7.5.
4. glycosylated hemoglobin according to claim 3, hemoglobin detection kit, it is characterised in that the weak base
Property refer specifically to pH for 7.35-7.45.
5. glycosylated hemoglobin according to claim 1, hemoglobin detection kit, it is characterised in that the R1 examinations
Agent, in addition to：0.001-0.003%w/v sodium azide, 0.04-0.06%w/v glycerine, 0.003-0.006%w/v
Skimmed milk power, 0.001-0.003%w/v casein, 0.01-0.03%w/v BSA.
6. glycosylated hemoglobin according to claim 5, hemoglobin detection kit, it is characterised in that the R1 examinations
Agent, in addition to：0.002%w/v sodium azide, 0.05%w/v glycerine, 0.005%w/v skimmed milk power, 0.001%w/
V casein, 0.01%w/v BSA.
7. glycosylated hemoglobin according to claim 1, hemoglobin detection kit, it is characterised in that the R2 examinations
Agent, in addition to：2-8mM magnesium chloride hexahydrate, 1-1.5%v/v formamide.
8. glycosylated hemoglobin according to claim 1, hemoglobin detection kit, it is characterised in that the R4 examinations
Agent, in addition to：10-15%v/v ethanol, 0.002-0.004%w/v sodium azide, 0.1-0.3%w/w chlorination
Sodium.
9. glycosylated hemoglobin according to claim 1, hemoglobin detection kit, it is characterised in that the saccharification
Hemoglobin-hemoglobin deposition film is selected from glass fibre membrane, borate filter membrane or fiber plasma membrane.
10. a kind of glycosylated hemoglobin based on any one of claim 1 to 9 detection kit, hemoglobin detection side
Method, it is characterised in that comprise the following steps：
Blood sample to be detected and R1 reagents are taken, mixes, obtains the blood sample after haemolysis；
R2 reagents will be added in blood sample after haemolysis, mixing obtains glycosylated hemoglobin detection sample；
Glycosylated hemoglobin detection sample is placed in glycosylated hemoglobin-hemoglobin deposition film, placed after a period of time, then
R4 reagent solutions are added, then place a period of time；
The glycosylated hemoglobin-hemoglobin deposition film is cleaned with R3 reagents；
The sample sites in the glycosylated hemoglobin-hemoglobin deposition film are detected based on reflectance photometry method, that is, obtained
Glycosylated hemoglobin in blood sample, content of hemoglobin must be detected.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110346580A (en) * | 2018-04-04 | 2019-10-18 | 南京东纳生物科技有限公司 | A kind of glycosylated hemoglobin fluorescence is affine immunochromatographytest test kit and detection method |
| CN110967488A (en) * | 2019-12-20 | 2020-04-07 | 深圳优迪生物技术有限公司 | Test strip, kit and method for measuring glycosylated hemoglobin |
| CN113219075A (en) * | 2018-12-29 | 2021-08-06 | 江山德瑞医疗科技有限公司 | Kit for determining anticoagulated venous whole blood glycosylated hemoglobin |
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| US5242842A (en) * | 1989-05-11 | 1993-09-07 | Axis Research As | Glycosylated haemoglobin assay |
| CN1531654A (en) * | 2001-04-24 | 2004-09-22 | ������˹-ϣ���¹�˾ | Haemoglobin assay |
| CN104897907A (en) * | 2015-05-21 | 2015-09-09 | 广东优尼德生物科技有限公司 | Kit for detecting glycosylated hemoglobin and detection method thereof |
| CN105301261A (en) * | 2015-10-12 | 2016-02-03 | 广州江元医疗科技有限公司 | Kit for detecting HbAlc (Glycosylated Hemoglobin), and preparation method and using method of kit |
| CN105606418A (en) * | 2015-12-30 | 2016-05-25 | 李新华 | Integral kit detecting glycosylated hemoglobin and detection method thereof |
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| US5242842A (en) * | 1989-05-11 | 1993-09-07 | Axis Research As | Glycosylated haemoglobin assay |
| CN1531654A (en) * | 2001-04-24 | 2004-09-22 | ������˹-ϣ���¹�˾ | Haemoglobin assay |
| CN104897907A (en) * | 2015-05-21 | 2015-09-09 | 广东优尼德生物科技有限公司 | Kit for detecting glycosylated hemoglobin and detection method thereof |
| CN105301261A (en) * | 2015-10-12 | 2016-02-03 | 广州江元医疗科技有限公司 | Kit for detecting HbAlc (Glycosylated Hemoglobin), and preparation method and using method of kit |
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| CN110346580A (en) * | 2018-04-04 | 2019-10-18 | 南京东纳生物科技有限公司 | A kind of glycosylated hemoglobin fluorescence is affine immunochromatographytest test kit and detection method |
| CN113219075A (en) * | 2018-12-29 | 2021-08-06 | 江山德瑞医疗科技有限公司 | Kit for determining anticoagulated venous whole blood glycosylated hemoglobin |
| CN113219075B (en) * | 2018-12-29 | 2023-03-21 | 江山德瑞医疗科技有限公司 | Kit for determining anticoagulated venous whole blood glycosylated hemoglobin |
| CN110967488A (en) * | 2019-12-20 | 2020-04-07 | 深圳优迪生物技术有限公司 | Test strip, kit and method for measuring glycosylated hemoglobin |
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| CN107153121B (en) | 2019-03-08 |
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