CN109991426A - A kind of glycated albumin detection kit - Google Patents
A kind of glycated albumin detection kit Download PDFInfo
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- CN109991426A CN109991426A CN201910263767.6A CN201910263767A CN109991426A CN 109991426 A CN109991426 A CN 109991426A CN 201910263767 A CN201910263767 A CN 201910263767A CN 109991426 A CN109991426 A CN 109991426A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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- G—PHYSICS
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
- G01N2333/765—Serum albumin, e.g. HSA
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Abstract
The present invention is for defect existing for the existing serum glycated albumin technology of detection, it is designed to provide a kind of serum glycated albumin detection reagent box, it is characterized in that directly acting on the glycosylated albumin in sample using fructosamine -6- kinases when glycosylated albumin content in detection serum.The kit is made of glycosylated albumin detection part, total albumin detection part, calibration object and quality-control product form, and mainly includes four detection reagents.Compared with existing commercial reagent box (Ketoamine oxidase method), detection reagent in kit provided by the present invention can be done directly on the glycosylated albumin in serum, it can avoid using a series of problems brought by protease, have the characteristics that at low cost, accuracy is high, property is stable, convenient for being widely applied.
Description
Technical field
The invention belongs to in-vitro diagnosis field, specifically a kind of glycated albumin detection agent box is used for human body blood
The quantitative detection of clear glycosylated albumin.
Background technique
Glycated albumin be in blood albumin and glucose occur non-enzymatic saccharification react caused by early stage it is sugared
Base product.It has been generally acknowledged that glycated albumin measurement can accurately reflect human body 2-3 weeks glycemic control situation, great Liang Yan
Study carefully and shows its testing result in terms of the confirmation for the treatment of diabetes effect and the adjustment of clinical dosage than blood sugar test " gold
Standard " glycosylated hemoglobin has advantage.In addition, as early stage glycation product in diabetic's body, glycosylated albumin energy
Inner skin cell function is destroyed, oxidative stress and inflammatory reaction, the stimulation smooth muscle cell proliferation and migration of vascular wall are caused, these
Pathologic process will lead to the formation and development of atherosclerosis and diabetic vascular complications.So glycated albumin
This index of content has great importance in terms of the glycemic control to diabetes patient.Therefore, glycated albumin has been
Through as clinically important one of blood glucose target.
Currently, clinically the conventional method of glycated albumin detection is Ketoamine oxidase method, detection process is depended on
Ketoamine oxidase is reacted with the fructosyl amino acid residue being saccharified.However, Ketoamine oxidase is difficult to directly act on the white egg of saccharification
It is white, so needing in advance to be decomposed into glycosylated albumin amino acid " fragment " using a large amount of protease in the detection process, recycle
Ketoamine oxidase height is selectively reacted with wherein fructosyl amino acid, quantitative hydrogen peroxide is generated by this process, in turn
Pass through Trinder ' s reaction assay and goes out glycosylated albumin content (CGA).At the same time, seralbumin is measured using bromocresol purple
Total amount (CALB), then by calculating the ratio measured twice, obtain the percentage composition (C of glycated albuminGA/CALB* 100%).
So glycated albumin assay kit is usually by glycosylated albumin reagent (call in the following text GA measurement reagent) and always
Albumin reagent (calling ALB measurement reagent in the following text) two parts composition (such as in patent document CN 102565420 A and US
The method that 8105800 B2 are announced).In GA measurement reagent, the main component of the first reagent (R1) be Ketoamine oxidase,
Trinder ' s reaction substrate, anti-interference substance；The main component of second reagent (R2) is that protease and Trinder ' s react bottom
Object.Ideally, sample is uniformly mixed with R1, after eliminating interfering substance, R2, which is added, keeps protease anti-with glycosylated albumin
It answers, causes above-mentioned Trinder ' s reaction chain, to measure the glycosylated albumin content in sample.However in fact, protease pair
All proteins all have identical reactivity, that is to say, that after R2 is added, protease can make all albumen in reaction system
Matter is degraded simultaneously, can thus inhibit the progress of subsequent reactions to a certain extent.To guarantee that measurement result is correct, it is necessary to
Other enzymes, such as Ketoamine oxidase, catalase are largely added in kit, make the increased costs of kit.In addition, egg
White enzyme itself is also protein, is saved it in liquid reagent, its own can also degrade, and causes active reduction, is shortened
The service life of kit.
During developing glycated albumin detection kit, it has been found that fructosamine -6- kinases can be direct
It is reacted with the epsilon-amino glycosyl on glycosylated albumin lysine residue, is translated into fructosamine -6- phosphoric acid, and the reaction meeting
Adenosine diphosphate (ADP) (ADP) is converted by atriphos (ATP) simultaneously.According to this discovery, it can develop and a kind of use fructose
Amine -6- kinases is the glycated albumin kit of core material.It is worth noting that, white with existing measurement saccharification serum
Protein process (Ketoamine oxidase method) is different, on the glycosyl for occur directly in glycosylated albumin, i.e., continuous mode of the invention is
In testing without using protease " cutting " glycosylated albumin, the addition in measurement process due to protease is avoided in this way
And bring increased costs and various problems.
Summary of the invention
The present invention provides a kind of detection of glycated albumin and determines agent box, it is characterised in that is using fructosamine -6- kinases
Core reaction substance is directly reacted with the glycosyl on the glycosylated albumin in sample, to reach the quantitative determination saccharification white egg of serum
White purpose.
So the contents of the present invention include:
[1] agent box is determined in a kind of glycated albumin detection, and kit measures part (C by glycosylated albuminGAPart is measured,
I.e. following middle reagent R1 and reagent R2), total albumin measuring part (CALBMeasure part, i.e., following middle reagent R3 and reagent R4),
Standard items and quality-control product composition, it is characterized in that in the C of kitGAIt measures and uses fructosamine -6- kinases in reagent；It needs especially to say
Bright, reagent R1 described in the content of present invention, reagent R2, reagent R3 and reagent R4 are only to state conveniently, and be not specific to reagent
A certain component part in box；
[2] C described in [1]GAMeasuring reagent includes two reagents, and wherein reagent R1 includes following component: 0.2 ~ 1U/ml
Bilirubin oxidase, 0.1 ~ 0.5U/ml ascorbic acid oxidase, 10 ~ 40U/ml Ketoamine oxidase, 2 ~ 10mM TOOS, 0.5 ~
10U/ml Catalase, 10 ~ 15mM phosphoenolpyruvate, 10 ~ 40U/ml pyruvate kinase, 10 ~ 15mM ATP, 10 ~ 40U/
Ml pyruvate oxidase, 0.01 ~ 0.02% gentamicin sulphate, 50 ~ 200mM HEPES buffer solution, pH value are 6.5 ~ 8.0；Reagent
R2 includes following component: 30 ~ 50U/ml fructosamine -6- kinases, 25 ~ 60U/ml horseradish peroxidase, 10 ~ 15mM NaN3、5~
20mM 4-AA, 50 ~ 200mM HEPES buffer solution, pH value are 6.5 ~ 8.0；
[3] C described in [1]ALBMeasuring reagent includes two reagents, and wherein R3 includes following component: 20 ~ 100mM acetic acid-vinegar
Sour sodium buffer, 0.1% ~ 2% Brij-35, pH value are 4.5 ~ 5.5；R4 includes following component: 20 ~ 100mM Acetic acid-sodium acetate is slow
Fliud flushing, 0.01% ~ 0.5% bromocresol purple, 10 ~ 15mM NaN3, pH value is 4.5 ~ 5.5；
[4] standard items and quality-control product of glycated albumin assay kit described in [1] be with human serum albumins with
A series of albumin/glycosylated albumin mixed solution prepared by glucose response certain time, wherein real content saccharification is white
Protein content and albumin content are determined by HPLC method.
Specifically, the use of kit of the invention includes that glycosylated albumin detection and albumin detect two steps,
Wherein, glycosylated albumin detects: sample being mixed with R1, it is dry that the fructosyl amino acid to dissociate in sample is eliminated using ketone ammonia oxidase
It disturbs, while eliminating the bilirubin in serum and vitamin C, and eliminate the hydrogen peroxide that above-mentioned reaction generates with Catalase；This
When R2 is added, wherein NaN3By the Catalase activity in inhibition system, fructosamine -6- kinases and the saccharification in reaction system are white
Albumen reaction, while ADP is converted by ATP, the ADP of generation will activate pyruvate kinase to react generation with phosphoenolpyruvate
Pyruvic acid reuses pyruvate oxidase and pyruvate oxidation is generated to quantitative hydrogen peroxide, reacts eventually by Trinder ' s
Measure the glycosylated albumin content in sample (standard items), i.e. CGAValue.
Albumin detection: bromocresol purple can be formed multiple under the action of Brij-35 in conjunction with the albumin in measurement sample
Object is closed, absorption is generated in 603 nm of wavelength, albumin content in sample, i.e. C can be measured using the reactionALBValue.
Finally, passing through CGAAnd CALBRatio determine the glycosylated albumin percentage composition of sample.
Detailed description of the invention
Fig. 1 relevance detection results figure
Specific embodiment
The following is specific embodiments of the present invention, and technical scheme of the present invention will be further described, but the present invention
It is not limited to these examples.
The as shown in table 1 of reagent used in case study on implementation
Table 1: reagent source in embodiment
Embodiment 1: the preparation of glycated albumin detection kit
Glycated albumin detection kit in the present invention is prepared as follows:
[1] C is prepared by table 2GAMeasure the first reagent of reagent, i.e. R1 in glycated albumin kit.
Table 2:CGAMeasure R1 formula in the first reagent of reagent, that is, glycated albumin kit
And its pH value is adjusted to 7.20.
[2] C is prepared by table 3GAMeasure the second reagent of reagent, i.e. R2 in glycated albumin kit.
Table 3:CGAMeasure R2 formula in the second reagent of reagent, that is, glycated albumin kit
And its pH value is adjusted to 7.20.
[3] in 50mM acetic acid-sodium acetate buffer solution addition 1% Brij-35 as CALBMeasure the pretreatment liquid of reagent
R3, and its pH value is adjusted to 5.20；
Use the bromocresol purple of 50mM acetic acid-sodium acetate buffer solution preparation 0.01% as CALBThe measurement reagent R4 of reagent is measured, and
Its pH value is adjusted to 5.20；
[4] standard items and the Quality Control for reacting certain time reagent preparation box is stirred at room temperature with glucose using human serum albumins
Product, and it is quantitative to wherein albumin and glycosylated albumin with HPLC method.
The R1 that will be prepared according to the above scheme, R2, R3, R4, calibration object and quality-control product combine as saccharification of the invention
Seralbumin detection kit.
Embodiment 2: glycated albumin detection kit correlation detection
According to the following steps, using kit measurement human serum glycated albumin array content prepared in embodiment 1.
One, using glycosylated albumin content (C in biochemical instruments measurement serumGA)
[1] the first reagent of glycosylated albumin reagent (R1) 270ul is added;
[2] sample (standard items) 5ul is added, in 37 DEG C of reaction 5min；
[3] it is master/slave that the second reagent of glycosylated albumin reagent (R2) 90ul, start recording 546/700(is added) wavelength absorbance,
Stop reading after reaction 5min, calculates absorbance change difference；
[4] sample is compareed with the absorbance difference of standard items, calculates glycosylated albumin concentration.
Two, Human Serum Albumin content (C is measured using full automatic biochemical apparatusALB)
[1] albumin reagent pretreatment liquid (R3) 240ul is added;
[2] sample (standard items) 3ul is added, in 37 DEG C of reaction 5min；
[3] it is master/slave that albumin measuring reagent (R4) 80ul, start recording 603/660(is added) wavelength absorbance, reacts 5min
Stop reading later, calculates absorbance change difference；
[4] sample is compareed with the absorbance difference of standard items, calculates albumin concentration.
Three, sample (standard items) glycosylated albumin content (%) is calculated as follows
Serum glycated albumin content (GA%)=CGA/CALB*100%
Use the sugar in the serum glycated albumin detection reagent box (Ketoamine oxidase method) and the present invention of commercially available Asahi KASEI
Change seralbumin kit, while measuring the glycosylated albumin content of 100 serum samples, it is provided by the present invention to detect
The reliability of kit.Experimental result as shown in Figure 1, kit provided by the invention detected to commercially available contrast agent box it is related
Coefficients R2It is 0.9859, has good correlation, testing result is reliable.
Embodiment 3: glycated albumin detection kit precision detection
3 batches of glycated albumin detection kits are prepared by embodiment 1, using this 3 batches of kits by the step in embodiment 2
Suddenly, low value Quality Control sample (HPLC definite value C is measured interior on the same dayGA/CALB4.45%) and high level Quality Control sample (HPLC definite value for
CGA/CALBIt is 16.28%) each 20 times, calculates batch interior CV value, then use same sample METHOD FOR CONTINUOUS DETERMINATION 20 days, CV in calculating batch
Value, as a result as shown in table 4 below:
Table 4: kit precision testing result
The results show that glycated albumin kit precision of the invention is preferable, to low value quality-control product and high-quality quality-control product
Detected value and HPLC detected value it is very close, testing result is reliable, also, kit batch in, interassay coefficient of variation it is equal
It is able to satisfy clinical detection demand.
Embodiment 4: glycated albumin detection kit interference free performance is investigated
In kit high level quality-control product, (HPLC measures CGA/CALBValue is 16.28%, wherein CGAValue is 0.62g/dL, wherein CALBValue
For 3.81g/dL), the bilirubin and vitamin C of different content is added, while isometric physiological saline is added with quality-control product and is made
On the basis of, it is detected using three batches of kits prepared in embodiment 3, as a result as shown in table 5 below:
Table 5: the anti-interference testing result of kit
The results show that kit of the invention has strong anti-interference ability, sample mesobilirubin content be less than 25mg/dL,
Measurement result deviation of the Vitamin C content less than 40mg/dL is in ± 5%, on testing result almost without influence.
It should be pointed out that above-described embodiment is only rather than the limitation of the present invention to a specific embodiment of the invention,
Protection scope of the present invention should be defined by the scope defined by the claims..It without departing from the scope of the present invention, can be with
Several improvement are made, these improvement also should be regarded as protection scope of the present invention.
Claims (3)
1. a kind of serum glycated albumin detection reagent box, it is characterized in that being swashed when measuring glycosylated albumin using fructosamine -6-
Enzyme.
2. according to fructosamine -6- kinases described in claim [1], it is characterized in that can on glycosylated albumin lysine residue
The reaction of epsilon-amino glycosyl, and convert adenosine diphosphate (ADP) (ADP) for atriphos (ATP) simultaneously.
3. serum glycated albumin detection reagent box described in claim [1], which is characterized in that each group of the kit
It is as follows at the ingredient of part:
Reagent R1:
0.2 ~ 1U/ml of bilirubin oxidase
0.1 ~ 0.5U/ml of ascorbic acid oxidase
10 ~ 40U/ml of Ketoamine oxidase
TOOS 2~10mM
Catalase 0.5~10U/ml
10 ~ 15mM of phosphoenolpyruvate
10 ~ 40U/ml of pyruvate kinase
ATP 10~15mM
10 ~ 40U/ml of pyruvate oxidase
Gentamicin sulphate 0.01 ~ 0.02%
50 ~ 200mM of HEPES buffer solution
Reagent R2:
30 ~ 50U/ml of fructosamine -6- kinases
25 ~ 60U/ml of horseradish peroxidase
NaN3 10~15mM
5 ~ 20mM of 4-AA
50 ~ 200mM of HEPES buffer solution
Reagent R3:
Brij-35 0.1%~2%
20 ~ 100mM of acetic acid-sodium acetate buffer solution
Reagent R4:
Bromocresol purple 0.01% ~ 0.5%
NaN3 10~15mM
20 ~ 100mM of acetic acid-sodium acetate buffer solution.
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| WO2007072941A1 (en) * | 2005-12-22 | 2007-06-28 | Kyowa Medex Co., Ltd. | Method for determination of glycosylated protein |
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