CN108287245A - Measure the kit and preparation method thereof of glycosylated hemoglobin - Google Patents
Measure the kit and preparation method thereof of glycosylated hemoglobin Download PDFInfo
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- CN108287245A CN108287245A CN201810095525.6A CN201810095525A CN108287245A CN 108287245 A CN108287245 A CN 108287245A CN 201810095525 A CN201810095525 A CN 201810095525A CN 108287245 A CN108287245 A CN 108287245A
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- reagent
- kit
- glycosylated hemoglobin
- surfactant
- tween
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—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
- G01N33/723—Glycosylated haemoglobin
-
- 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
Abstract
The present invention relates to biotechnologies, and in particular to a kind of glycosylated hemoglobin assay kit, the kit include reagent R1 and reagent R2, and reagent R1 includes latex, double buffering liquid, surfactant；Reagent R2 includes glycosylated hemoglobin monoclonal antibody, Dual Surfactants, buffer solution, and the kit measurement accuracy is high, at low cost, stability is good.
Description
Technical field
The invention belongs to glycosylated hemoglobin detection technique fields, and in particular to a kind of glycosylated hemoglobin detection reagent
Box.
Background technology
Glycosylated hemoglobin (HbAlc) is the product that hemoglobin is combined into glucose in blood, numerical value and blood
Sugared concentration is directly proportional, and is irreversible combination, withers away with red blood cell and disappears (120 days or so the lifetime of red blood cell).By
It is related to red blood cell life span and average blood glucose levels in glycosylated hemoglobin, be evaluation diabetic's long-term blood glucose control compared with
Ideal index can reflect 2~3 months in the past average blood glucose levels, not influenced by daily blood glucose fluctuation.With capilary and
Macrovascular complications are related closely.Glycated hemoglobin levels increase, diabetic retinopathy, renal lesions, god
Accordingly increase through lesion, cardiovascular event occurrence risk.Glycosylated hemoglobin has preferable predictive ability to diabetes.
Currently, the method for clinically measuring HbA1c has very much, more common is HPLC, latex immunoturbidimetry, enzyme process
Deng.HPLC methods are to measure the goldstandard of HbA1c, the method quickly, it is accurate, easy, but need special instrument and instrument, reagent are high
Expensive, testing cost is higher, it is difficult in basic hospital labs.Affinity chromatography take pre-treatment be more troublesome, take compared with
It is long；It is poor that enzyme process detects precision；Latex immunoturbidimetry does not need specific apparatus, can in biochemical instruments with other biochemical projects
It detects together, flux is big in the unit interval, and testing cost ratio HPLC methods are low, is suitable for batch detection.
Emulsion reagent is traditional three reagents on the market at present, and reagent 1 is control latex particle, and reagent 2 is HbAle
The monoclonal antibody of albumen (HbA1C), reagent 3 are corresponding secondary antibody sheep anti-mouse igg antibody.Because monoclonal antibody and secondary antibody are formed immune
Compound long-term storing stability is poor, and gradually inactivation causes to measure signal decline, so need to will be tried using preceding operating personnel
Agent 2 and reagent 3 mix, cumbersome, and the mixed stable reagent effect phase is shorter.
Simultaneously as latex immunoturbidimetry measurement result is to measure HbA1c to account for the content of total Hb albumen, therefore detecting
Susceptible when middle anemia sample keeps testing result relatively low.
Invention content
In view of the above-mentioned problems, the present invention provides a kind of double reagent glycosylated hemoglobin detection kit and its preparation sides
Method, to replace traditional detection kit for including three kinds of reagents.It is cumbersome unstable with reagent that the present invention solves aforementioned operation
The problem of and the relatively low problem of middle anemia sample measured value.
Specifically, the present invention provides a kind of for measuring the kit of glycosylated hemoglobin, it includes reagent R1 and
Reagent R2, wherein the reagent R1 includes latex, buffer solution A and surfactant A；And the wherein described reagent R2 includes
Glycosylated hemoglobin antibody, surfactant B and buffer B.
In some embodiments, buffer solution A includes any two component selected from the group below：Glycine, borate,
Tris, phosphate.
Preferably, component contained by buffer solution A is selected from the group：Glycine+borate, glycine+Tris or glycine+phosphorus
Hydrochlorate；Most preferably, group contained by buffer solution A is divided into glycine+borate.In some embodiments, the sweet ammonia in reagent R1
Acid concentration is 2mM-200mM, preferably 50-150mM, most preferably 100mM.In some embodiments, the boron in reagent R1
Hydrochlorate a concentration of 1mM-50mM, preferably 10-30mM, most preferably 20mM.
In some embodiments, the latex particle size in reagent R1 is 50nm-200nm.In a preferred embodiment,
The grain size of the latex is 80nm-150nm, most preferably 106nm.In some embodiments, the concentration range of the latex
For 0.2g/L-10g/L, it is preferable that the concentration range of the latex is 0.5g/L-2g/L, most preferably 1g/L.
In some embodiments, the surfactant A in reagent R1 is TWEEN Series surfactant, preferably tween
20(Tween-20).Preferably, a concentration of 0.01ml/L-1ml/L of surfactant A, preferably 0.01ml/L-0.1ml/L,
Most preferably 0.05ml/L.
In some embodiments, reagent R1 further includes preservative.Preferably, the preservative in reagent R1 can be
Proclin series preservative (such as PC150, PC200, PC300, PC950), CAA (2- chloroacetamides), NaN3, phenol, IZU
(imidazolidinyl urea), preferably Proclin series preservative or NaN3, most preferably PC950.In some embodiments, it tries
Concentration of preservatives in agent R1 is 0.01ml/L-5ml/L, preferably 0.2ml/L-2ml/L, most preferably 1ml/L.
In some embodiments, the pH value of reagent R1 is 5-9, such as 5.0,5.5,6.0,6.5,7.0,7.5,8.0,
8.5 or 9.0.Preferably, the pH value of reagent R1 be 8-8.5, most preferably 8.1.
In some embodiments, surfactant B described in reagent R2 is TWEEN Series surfactant+polyoxyethylene
Alkyl ether type nonionic surfactant.In some embodiments, the TWEEN Series surfactant is selected from the group：Tween
20 (Tween-20), tween 21 (Tween-21), polysorbate40 (Tween-40), polysorbate60 (Tween-60), Tween61 (Tween-
61), Tween 80 (Tween-80), sorbimacrogol oleate100 (Tween-81), polysorbate85 (Tween-85).In some preferred embodiments,
The TWEEN Series surfactant is Tween-20 or Tween-80, preferably Tween-20.
In some embodiments, the polyoxyethylene alkyl ether-type nonionic surfactant can be polyoxyethylene alkane
Base ether, polyoxyethylene lauryl ether, polyoxyethylene cetyl ether or diphenylethyllene phenol polyoxyethylene ether；Preferably
Diphenylethyllene phenol polyoxyethylene ether.
In some embodiments, a concentration of 0.05ml/L-2ml/ of TWEEN Series surfactant described in reagent R2
L, preferably 0.2ml/L-0.8ml/L, most preferably 0.5ml/L.In some embodiments, polyoxyethylene described in reagent R2
A concentration of 0.05ml/L-2ml/L of alkyl ether type nonionic surfactant, preferably 0.2ml/L-0.8ml/L；Most preferably
0.5ml/L。
In some embodiments, glycosylated hemoglobin antibody described in reagent R2 is glycosylated hemoglobin monoclonal antibody
(such as poly monoclonal antibody).In some embodiments, the glycosylated hemoglobin monoclonal antibody can be that mouse is anti-human
Or rabbit-anti human monoclonal antibodies.In some embodiments, a concentration of 0.01- of glycosylated hemoglobin antibody described in reagent R2
1mg/ml, preferably 0.03-0.2mg/ml, most preferably 0.1mg/ml.
In some embodiments, the buffer solution B in reagent R2 can be 2-morpholine ethane sulfonic acid (MES), 4- ethoxy piperazines
Piperazine ethanesulfonic acid (HEPES), glycine, borate or phosphate etc..In a preferred embodiment, buffer solution B is phosphate
Buffer solution.It is highly preferred that the phosphatic a concentration of 10-200mM, preferably 20-80mM, most preferably 50mM.
In some embodiments, reagent R2 also includes protective agent.Preferably, the protective agent is selected from the group：Mannitol,
Sucrose, sorbierite and trehalose.It is highly preferred that the protective agent is mannitol.In some embodiments, in reagent R2
Protective agent a concentration of 2ml/L-50ml/L, more preferably 5ml/L-30ml/L, most preferably 20ml/L.
In some embodiments, reagent R2 also includes protein stabiliser.Preferably, the protein stabiliser is BSA.
In some embodiments, protein stabiliser a concentration of 1g/L-50g/L, preferably 1g/L-10g/L in reagent R2, most preferably
5g/L。
In some embodiments, reagent R2 also includes inorganic salts.Preferably, the inorganic salts be sodium salt or sylvite, most
Preferably NaCl or KCl.In some embodiments, the inorganic salt concentration in reagent R2 is 1g/L-30g/L, preferably 5g/L-
15g/L, most preferably 9g/L.
In some embodiments, reagent R2 also includes preservative.Preferably, the preservative in reagent R2 can be
Proclin series preservative (such as PC150, PC200, PC300, PC950), CAA, NaN3, phenol, IZU, preferably IZU.
In some embodiments, the concentration of preservatives in reagent R2 is 0.5-5g/L, preferably 2-4g/L, most preferably 3g/L.
In some embodiments, reagent R2 also includes chelating agent.In some embodiments, the chelating agent is selected from down
Group：EDTA.2Na, EDTA.2K, EDTA.4Na, preferably EDTA.2Na.In some embodiments, the chelating agent in reagent R2
A concentration of 0.01g/L-4g/L, preferably 0.1g/L-1g/L, most preferably 0.5g/L.
In some embodiments, the pH value of reagent R2 is 4-9, such as 4.0,4.5,5.0,5.5,6.0,6.5,7.0,
7.5,8.0,8.5 or 9.0.Preferably, the pH value of reagent R2 be 5.5-6.5, most preferably 6.
In some preferred embodiments, the present invention provides a kind of kit for measuring glycosylated hemoglobin, institutes
It includes R1 and reagent R2 to state kit, and it includes following components：
Reagent R1：
Reagent R2：
In some preferred embodiments, the present invention provides a kind of kit for measuring glycosylated hemoglobin, institutes
It includes R1 and reagent R2 to state kit, and it includes following components：
Reagent R1：
Reagent R2：
Advantageous effect
Compared with prior art, the present invention has following beneficial advantage：Double buffering of the present invention employed in reagent R1
Liquid energy enough keeps pH value to stablize, and can eliminate the variation of latex charge during latex preserves and lead to latex agglutination and physical absorption
Ability changes, and keeps reagent stability more preferable.And the Dual Surfactants in reagent 2 keep the knot of antibody as suspending agent
Structure is stablized, while can further eliminate influence of the variation of latex charge during latex preserves to physical absorption, makes simultaneously
Antibody is not attracted on latex, promotes the specific reaction of antigen-antibody.Therefore, reagent of the invention can make latex physics
The ability of absorption non-specific adsorption hemoglobin and glycosylated hemoglobin maintains a suitable level, makes immobilised egg
White amount arrival is constant, because hemoglobin and saccharification hemoglobin content are much excessive in sample, is influenced by total hemoglobin
It is small, the influence of individual difference can be eliminated, anemia sample has more advantage in the assay.
Specific implementation mode
In order to make art technology field personnel more fully understand the technical solution in the application, below in conjunction with following knot
Closing embodiment, the invention will be further described, it is clear that and described embodiments are only a part of embodiments of the present application, without
It is whole embodiment.Based on the embodiment in the application, those of ordinary skill in the art are not before making creative work
The all other embodiment obtained is put, shall fall within the protection scope of the present application.
The testing principle of 1. glycosylated hemoglobin detection kit of the present invention of embodiment and use
Testing principle：Hemoglobin and glycosylated hemoglobin have identical non-specific suction with latex in reagent 1 in sample
Attached and immobilization forms glycosylated hemoglobin antigenantibody complex after mouse anti-human glycosylated hemoglobin antibody is added,
Agglutination amount is different due to the difference of the immobilised glycosylated hemoglobin amount of latex surface.By measure its absorbance and with saccharification blood
The standard curve of Lactoferrin percentage concentration compares, and can find out the glycosylated hemoglobin in sample and accounts for the percentage of total hemoglobin and contains
Amount.When the amount of total hemoglobin disclosure satisfy that latex adsorbance, testing result is unaffected, but when total hemoglobin reduces
When deficiency meets latex adsorbance, is reduced with the immune complex that the anti-glycosylated hemoglobin antibody in reagent 2 is formed, cause to examine
Surveying result reduces, while antibody can be attracted on latex, be unfavorable for the specific reaction of antigen-antibody.In clinical application,
Due to individual difference, some patients' sample total hemoglobin content is different, when hemoglobin concentration is reduced, then can influence to try
Agent box detects, and such as anaemia patient's sample, is clinically often replaced with hemoglobin (Hb) concentration.Standard according to China：Normally
People Hb contents are in 110-160g/L, anemia (90g/L-109g/L), anemia (60-89g/L), anemia (30-
59g/L)。
Here, giving the application method of an illustrative kit of the present invention：
1. embodiment any one of them component and content prepare the kit of the present invention as described above；
2. taking the whole blood sample of acquisition, 50 times of dilutions are carried out using purified water, are combined it with reagent 1 after haemolysis, sample
Middle hemoglobin and glycosylated hemoglobin and latex have an identical non-specific adsorption and immobilization；
3. being mixed with reagent R2, glycosylated hemoglobin monoclonal antibody is fully reacted with it in reagent 2, is saccharified in conjunction with being formed
Hemoglobin antigenantibody complex, agglutination amount because the immobilised glycosylated hemoglobin amount of latex surface it is different without
Together；
4. measuring the absorbance difference after reaction with automatic clinical chemistry analyzer；
5. calculating the percentage composition of HbA1c in sample according to absorbance change value.
It should be appreciated that the occupation mode of the kit of the present invention is not limited to example provided herein, step, dosage and
Other parameters can modify on the basis of without prejudice to inventive concept, as long as can achieve the effect that the present invention.
2. glycosylated hemoglobin detection kit of the present invention of embodiment influences and improves stability for reducing individual difference
Effect
(1)The preparation of kit of the present invention：According to component described in the following table 1 and content difference preparation experiment group 1 and experimental group 2
Kit.
The preparation of table 1. experimental group 1 and 2 kits
Note：aEmulgen A90, that is, surfactant diphenylethyllene phenol polyoxyethylene ether are Kao Corp
Emulgen series of products.
(2)Compliance test result method：
A. individual difference is verified：The same sample of 10 HbA1c concentration is measured, the difference is that hemoglobin (Hb) concentration is not
Together.10 sample HbA1c values are 5.2%, are measured using internationally recognized IFCC mass spectrometry methods, and this method has the spy of height
The opposite sex, numerical value can reflect its real content.10 sample Hb concentration be respectively 32g/L, 45g/L, 55g/L, 65g/L, 98g/L,
110g/L, 122g/L, 125g/L, 130g/L, 155g/L are Sysmex Hematometer measured value, and the distribution of Hb concentration in gradient can
To reflect Different Individual difference.Wherein：1-5# samples belong to anaemia sample, and Hb values are lower to illustrate that anaemia is more apparent, 6-10# samples
For conventional sample.This 10 samples are tested using the commercially available producer's reagent of test kit respectively, it can according to commercial reagent measured value
It is influenced by individual difference with carrying out judgement HbA1c detections, test reagent testing result is better than commercial reagent measured value, then illustrates to try
The effect for eliminating individual difference influence can be had by testing reagent.
B. stability is verified：Under 2~8 DEG C of storage requirement, the Quality Control of the project into measurement, is seen 0 month, March respectively
Examine the stability of reagent.
(3)Experimental result：
The kit of experimental group 1 and 2 and conventional reagents box (i.e. commercial reagent 1 and commercial reagent 2) have been carried out to having a competition
It tests, individual difference verification result and verification result more steady in a long-term are as shown in the following table 2 and 3：
(#1-#10 respectively represents pathology sample 1-10, HbA1c to the individual difference sample experimental result of 2. experimental group 1 and 2 of table
Value unit is %)
| Whole blood sample | 1# | 2# | 3# | 4# | 5# | 6# | 7# | 8# | 9# | 10# |
| Hb(g/L) | 32 | 45 | 55 | 65 | 98 | 110 | 125 | 122 | 130 | 155 |
| Mass spectroscopy (control) | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 |
| Commercial reagent 1 | 1.02 | 2.36 | 3.69 | 3.96 | 5.02 | 5.12 | 5.36 | 5.25 | 5.36 | 5.36 |
| Commercial reagent 2 | 1.53 | 2.93 | 3.54 | 3.87 | 4.89 | 5.13 | 5.42 | 5.27 | 5.29 | 5.26 |
| Experimental group 1 | 4.11 | 4.48 | 4.62 | 4.88 | 5.10 | 5.21 | 5.22 | 5.25 | 5.24 | 5.30 |
| Experimental group 2 | 4.12 | 4.43 | 4.63 | 4.96 | 5.15 | 5.15 | 5.23 | 5.23 | 5.26 | 5.29 |
The above result shows that：Conventional reagents box measures HbA1c in Hb ＞ 110g/L, and measurement result is more accurate；Work as Hb
When ＜ 110g/L, with the reduction of Hb values, measurement result and actual value difference are increasing, illustrate that the concentration of Hb is less than 110g/
L influences the detection of HbA1c, and Hb is lower, and HbA1c measured values are lower；Illustrate that individual difference sample influences detection HbA1c results.
Compared with the measurement result of commercial reagent, the measured value of kit of the present invention illustrates that it can disappear closer to actual value
Except individual difference is in glycosylated hemoglobin；On the basis of the measurement result of commercial reagent, measurement result closer to actual value,
Illustrate that effect is better.
And the HbA1c testing results of experimental group 1 and 2 compare conventional reagents box closer to actual value, then explanation is in elimination
Body difference sample influences HbA1c measured values upper better.On the basis of the measurement result of commercial reagent, measurement result more connects
It is bordering on actual value, illustrates that effect is better.
The long-time stability experimental result of 3. experimental group 1 and 2 of table
It can be seen that by the stability data of table 3：3 months Quality Control determination datas are compared with 0 month, measured value difference
Very little, deviation illustrate that the detection kit of the present invention is steady under 2~8 DEG C of storage requirement in Quality Control claimed range (± 7%)
Qualitative height.
The measure of merit of the glycosylated hemoglobin detection kit of the present invention of 3. various concentration value of embodiment
(1)The preparation of kit：According to component described in the following table 4 and the kit of content difference preparation experiment group 3-7.
The component table of 4. experimental group 3-7 of table
(2)Compliance test result method：
Individual difference is verified and stabilizing effect verification method is same as Example 2.
(3)Experimental result：
The kit of experimental group 3-7 and conventional reagents box (i.e. commercial reagent 1 and commercial reagent 2) have been carried out to having a competition
It tests, individual difference verification result and verification result more steady in a long-term are as shown in the following table 5 and 6：
(#1-#10 respectively represents pathology sample 1-10, HbA1c to the individual difference sample experimental result of 5. experimental group 3-7 of table
Value unit is %)
The above result shows that：Conventional reagents box is measuring HbA1c in Hb>When 110g/L, measurement result is more accurate；Work as Hb
When ＜ 110g/L, with the reduction of Hb values, measurement result and actual value difference are increasing, illustrate that the concentration of Hb is less than 110g/
L influences the detection of HbA1c, and Hb is lower, and HbA1c measured values are lower；Illustrate that individual difference sample influences detection HbA1c results.
Compared with the measurement result of commercial reagent, the measured value of kit of the present invention illustrates that it can disappear closer to actual value
Except individual difference is in glycosylated hemoglobin；On the basis of the measurement result of commercial reagent, measurement result closer to actual value,
Illustrate that effect is better.
And the kit of experimental group 3-7 compares commercial reagent closer to actual value to the testing result of HbA1c, especially when
Its measured value is substantially better than conventional reagents box when Hb ＜ 110g/L, illustrates that it is eliminating individual difference sample to the influence of HbA1c measured values
The effect of aspect, which has, to be obviously improved.By result as it can be seen that experimental group 3-6 measurement results have been considerably better than conventional reagents box, connect
It is bordering on actual value.And the testing result of experimental group 7 and the basic indifference of actual value, illustrate that it can completely eliminate the shadow of individual difference
It rings.
The long-time stability experimental result of 6. experimental group 3-7 of table
It can be seen that by the stability data of table 6：3 months Quality Control determination datas are compared with 0 month, measured value difference
Very little, deviation illustrate that the detection kit of the present invention can under 2~8 DEG C of storage requirement in Quality Control claimed range (± 7%)
Stablize.And experimental group 5-6 results are better than experimental group 3-4, most preferred formula is experimental group 7.
The concentration boundary of 4. glycosylated hemoglobin detection kit of the present invention of embodiment is tested
Embodiment 3 shows that the applicable concentration range of kit each component of the present invention, the present embodiment then test applicable model
Enclose influence of other the outer concentration for individual difference and stabilizing effect.
(1)The preparation of kit：According to component described in the following table 7 and the kit of content difference preparation experiment group 8-9.
The component table of 7. experimental group 8-9 of table
(2)Experimental result：
8 experimental group calibration curve of experimental group is poor, and low side sensitivity is poor, high-end no gradient, and calibration does not pass through.Experimental group
9 experimental group sensitivity are preferable, but high-side signal value transfinites, and calibration is also caused to fail, and effect is poor.
The measure of merit of Dual Surfactants component in double buffering liquid component, reagent R2 in the replacement reagent of embodiment 5. R1
(1)The preparation of kit：According to component described in the following table 8 and the kit of content difference preparation experiment group 3-7.
The component table of 8. experimental group 10-12 of table
(2)Compliance test result method：
Individual difference is verified and stabilizing effect verification method is same as Example 2.
(3)Experimental result：
The kit of experimental group 10-12 and conventional reagents box (i.e. commercial reagent 1 and commercial reagent 2) have been carried out to having a competition
It tests, individual difference verification result and verification result more steady in a long-term are as shown in the following table 9 and 10：
9. experimental group 10-12 of table individual difference sample experimental result (#1-#10 respectively represents pathology sample 1-10,
HbA1c value units are %)
| Whole blood sample | 1# | 2# | 3# | 4# | 5# | 6# | 7# | 8# | 9# | 10# |
| Hb(g/L) | 32 | 45 | 55 | 65 | 98 | 110 | 125 | 122 | 130 | 155 |
| Mass spectroscopy (control) | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 |
| Experimental group 10 | 2.52 | 2.92 | 4.12 | 4.55 | 5.12 | 4.89 | 5.11 | 4.98 | 4.78 | 4.96 |
| Experimental group 11 | 2.53 | 2.92 | 4.13 | 4.50 | 5.25 | 4.78 | 5.33 | 4.85 | 5.06 | 5.06 |
| Experimental group 12 | 2.56 | 2.63 | 4.26 | 4.53 | 5.36 | 4.93 | 5.36 | 4.96 | 5.13 | 4.76 |
| Commercial reagent 1 | 1.02 | 2.36 | 3.69 | 3.96 | 5.02 | 5.12 | 5.36 | 5.25 | 5.36 | 5.36 |
| Commercial reagent 2 | 1.53 | 2.93 | 3.54 | 3.87 | 4.89 | 5.13 | 5.42 | 5.27 | 5.29 | 5.26 |
Test result shows：Commercial reagent 1 and commercial reagent 2 measure HbA1c in Hb ＞ 110g/L, and measurement result is more
Accurately；As Hb ＜ 110g/L, with the reduction of Hb values, measurement result and actual value difference are increasing, illustrate the concentration of Hb
Less than 110g/L, the detection of HbA1c is influenced, and Hb is lower, HbA1c measured values are lower；Illustrate that individual difference sample influences detection
HbA1c results.
After having carried out reagent as described above and having replaced, the HbA1c of three groups of test kits is for sample 1#-5# (Hb ＜
When 110g/L) testing result and actual value have larger difference, illustrate that measured value is not accurate enough.
The long-time stability experimental result of 10. experimental group 10-12 of table
Stability data shows：0 month Quality Control determination data, deviation is larger, and the measurement of Quality Control in 3 months is not requiring
In range (± 7%), illustrate that 2-8 DEG C of stability of reagent is bad.
Embodiment 6. alternatively tries the measure of merit of component
The present embodiment then tests some influences of replaceable component to effect in reagent R1 and R2.
(1)The preparation of kit：According to component described in the following table 11 and the kit of content difference preparation experiment group 13-14.
11. experimental group 13-14 of table individual difference sample experimental result (#1-#10 respectively represents pathology sample 1-10,
HbA1c value units are %)
(2)Compliance test result method：
Individual difference is verified and stabilizing effect verification method is same as Example 2.
(3)Experimental result：
The kit of experimental group 13-14 and conventional reagents box (i.e. commercial reagent 1 and commercial reagent 2) have been carried out to having a competition
It tests, individual difference verification result and verification result more steady in a long-term are as shown in the following table 12 and 13：
12. experimental group 13-14 of table individual difference sample experimental result (#1-#10 respectively represents pathology sample 1-10,
HbA1c value units are %)
| Whole blood sample | 1# | 2# | 3# | 4# | 5# | 6# | 7# | 8# | 9# | 10# |
| Hb(g/L) | 32 | 45 | 55 | 65 | 98 | 110 | 125 | 122 | 130 | 155 |
| Mass spectroscopy (control) | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 | 5.2 |
| Experimental group 13 | 4.02 | 4.45 | 4.72 | 4.99 | 5.11 | 5.21 | 5.22 | 5.23 | 5.27 | 5.25 |
| Experimental group 14 | 4.10 | 4.36 | 4.69 | 4.98 | 5.14 | 5.23 | 5.25 | 5.25 | 5.31 | 5.26 |
| Commercial reagent 1 | 1.02 | 2.36 | 3.69 | 3.96 | 5.02 | 5.12 | 5.36 | 5.25 | 5.36 | 5.36 |
| Commercial reagent 2 | 1.53 | 2.93 | 3.54 | 3.87 | 4.89 | 5.13 | 5.42 | 5.27 | 5.29 | 5.26 |
Test result shows：Conventional reagents box measures HbA1c in Hb ＞ 110g/L, and measurement result is more accurate；Work as Hb
When ＜ 110g/L, with the reduction of Hb values, measurement result and actual value difference are increasing, illustrate that the concentration of Hb is less than 110g/
L influences the detection of HbA1c, and Hb is lower, and HbA1c measured values are lower；Illustrate that individual difference sample influences detection HbA1c results.
For kit measurement value of the present invention than commercial reagent measurement result closer to actual value, explanation can eliminate individual difference
In glycosylated hemoglobin；On the basis of the measurement result of commercial reagent, measurement result illustrates that effect is got over closer to actual value
It is good.
Upper table result can be seen that using above-mentioned replacement (such as with Tween-80 replace Tween-20, with A-60 replace A-
90) it, still can achieve the effect that the present invention reduces individual difference and influences.
The long-time stability experimental result of 13. experimental group 13-14 of table
Stability data shows：3 months Quality Control determination datas are compared with 0 month, measured value difference very little, and deviation is equal
In Quality Control claimed range (± 7%), illustrate that the detection kit of the present invention can be stablized under 2~8 DEG C of storage requirement.
Upper table result can be seen that using above-mentioned replacement (such as with Tween-80 replace Tween-20, with A-60 replace A-
90) it, still can reach the stabilizing effect of the present invention.
It should be appreciated that the present invention is not limited only to specific method, scheme and the substance of description, because these can be
Change in the case of not departing from present subject matter.It will also be understood that embodiment as described herein is merely for the sake of description specific embodiment party
The purpose of case, and be not intended to limit the scope of the invention, the scope of the present invention is defined only by the following claims.
Those skilled in the art, which will also be appreciated that or be able to confirm that, uses no more than routine experiment, institute herein
Many equivalents of the specific embodiment of the present invention stated.These equivalents are also contained in the attached claims.
Claims (10)
1. a kind of kit for measuring glycosylated hemoglobin, it includes reagent R1 and reagent R2, wherein the reagent R1 packets
Containing latex, buffer solution A and surfactant A；And the wherein described reagent R2 includes glycosylated hemoglobin antibody, surfactant
B and buffer B；The wherein described buffer solution A includes any two component selected from the group below：Glycine, borate, Tris, phosphoric acid
Salt, the surfactant B are TWEEN Series surfactant+polyoxyethylene alkyl ether-type nonionic surfactant.
2. the kit of claim 1, wherein the pH value of the reagent R1 is 5-9.
3. the kit of claims 1 or 2, wherein the TWEEN Series surfactant is selected from the group：Polysorbas20, tween 21,
Polysorbate40, polysorbate60, Tween61, Tween 80, sorbimacrogol oleate100 and polysorbate85.
4. the kit of claims 1 or 2, wherein the polyoxyethylene alkyl ether-type nonionic surfactant is selected from the group：
Polyoxyethylene alkyl ether, polyoxyethylene lauryl ether, polyoxyethylene cetyl ether and diphenylethyllene phenol polyoxyethylene
Ether.
5. the kit of any one of claim 1-4, wherein the latex particle size in the reagent R1 is 50nm-200nm.
6. the kit of any one of claim 1-4, wherein the R1 also includes preservative.
7. the kit of any one of claim 1-6, wherein the pH value of the reagent R2 is 4-9.
8. the kit of any one of claim 1-7, wherein the reagent R2 also includes as follows one or more：Protective agent,
Protein stabiliser, inorganic salts, preservative, chelating agent.
9. a kind of for measuring the kit of glycosylated hemoglobin, the kit includes R1 and reagent R2, respectively contain as
Lower component：
Reagent R1：
Reagent R2：
10. a kind of for measuring the kit of glycosylated hemoglobin, the kit includes R1 and reagent R2, respectively contain as
Lower component：
Reagent R1：
Reagent R2：
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110514848A (en) * | 2019-08-21 | 2019-11-29 | 深圳上泰生物工程有限公司 | A kind of glycosylated hemoglobin antibody complex and glycosylated hemoglobin detection kit |
| CN111057150A (en) * | 2019-12-30 | 2020-04-24 | 深圳开立生物医疗科技股份有限公司 | Latex microsphere, application thereof and glycosylated hemoglobin detection kit |
| CN112034186A (en) * | 2020-09-07 | 2020-12-04 | 南京立顶医疗科技有限公司 | Glycosylated hemoglobin kit based on biotin-streptavidin amplification and preparation method thereof |
| CN112198123A (en) * | 2020-10-12 | 2021-01-08 | 青岛汉唐生物科技有限公司 | Detection system and detection method for correcting influence of anemia on glycated hemoglobin measurement value |
| 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110514848A (en) * | 2019-08-21 | 2019-11-29 | 深圳上泰生物工程有限公司 | A kind of glycosylated hemoglobin antibody complex and glycosylated hemoglobin detection kit |
| CN111057150A (en) * | 2019-12-30 | 2020-04-24 | 深圳开立生物医疗科技股份有限公司 | Latex microsphere, application thereof and glycosylated hemoglobin detection kit |
| CN112034186A (en) * | 2020-09-07 | 2020-12-04 | 南京立顶医疗科技有限公司 | Glycosylated hemoglobin kit based on biotin-streptavidin amplification and preparation method thereof |
| CN112198123A (en) * | 2020-10-12 | 2021-01-08 | 青岛汉唐生物科技有限公司 | Detection system and detection method for correcting influence of anemia on glycated hemoglobin measurement value |
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