CN101135688A - Method for measuring fructosamine NBT in blood serum - Google Patents
Method for measuring fructosamine NBT in blood serum Download PDFInfo
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- CN101135688A CN101135688A CNA2007100715555A CN200710071555A CN101135688A CN 101135688 A CN101135688 A CN 101135688A CN A2007100715555 A CNA2007100715555 A CN A2007100715555A CN 200710071555 A CN200710071555 A CN 200710071555A CN 101135688 A CN101135688 A CN 101135688A
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- 210000002966 serum Anatomy 0.000 title claims abstract description 55
- IXZISFNWUWKBOM-ARQDHWQXSA-N fructosamine Chemical compound NC[C@@]1(O)OC[C@@H](O)[C@@H](O)[C@@H]1O IXZISFNWUWKBOM-ARQDHWQXSA-N 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 23
- 238000003556 assay Methods 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 16
- 238000002835 absorbance Methods 0.000 claims description 13
- 239000011159 matrix material Substances 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 8
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- 238000005352 clarification Methods 0.000 claims description 8
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 8
- 108010088751 Albumins Proteins 0.000 claims description 6
- 102000009027 Albumins Human genes 0.000 claims description 6
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- 230000002335 preservative effect Effects 0.000 claims description 5
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 4
- 108090000854 Oxidoreductases Proteins 0.000 claims description 4
- 102000004316 Oxidoreductases Human genes 0.000 claims description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000012856 packing Methods 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- JAJWGJBVLPIOOH-IZYKLYLVSA-M sodium taurocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 JAJWGJBVLPIOOH-IZYKLYLVSA-M 0.000 claims description 4
- YMTZCQOAGFRQHV-UHFFFAOYSA-N 3-methyl-4,5-dihydro-1,2-thiazole Chemical group CC1=NSCC1 YMTZCQOAGFRQHV-UHFFFAOYSA-N 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 102000004895 Lipoproteins Human genes 0.000 claims description 2
- 108090001030 Lipoproteins Proteins 0.000 claims description 2
- QNEFNFIKZWUAEQ-UHFFFAOYSA-N carbonic acid;potassium Chemical compound [K].OC(O)=O QNEFNFIKZWUAEQ-UHFFFAOYSA-N 0.000 claims description 2
- 239000000470 constituent Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 7
- 238000012360 testing method Methods 0.000 abstract description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 abstract 2
- 239000007853 buffer solution Substances 0.000 abstract 1
- 230000009977 dual effect Effects 0.000 abstract 1
- 229910000027 potassium carbonate Inorganic materials 0.000 abstract 1
- 239000012086 standard solution Substances 0.000 abstract 1
- JPXMTWWFLBLUCD-UHFFFAOYSA-N nitro blue tetrazolium(2+) Chemical compound COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 JPXMTWWFLBLUCD-UHFFFAOYSA-N 0.000 description 28
- 206010012601 diabetes mellitus Diseases 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 4
- 108010014663 Glycated Hemoglobin A Proteins 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
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- 238000002474 experimental method Methods 0.000 description 3
- 108091005996 glycated proteins Proteins 0.000 description 3
- ODUCDPQEXGNKDN-UHFFFAOYSA-N nitroxyl Chemical compound O=N ODUCDPQEXGNKDN-UHFFFAOYSA-N 0.000 description 3
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 2
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 2
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 2
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
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- 230000002596 correlated effect Effects 0.000 description 2
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- 229940116269 uric acid Drugs 0.000 description 2
- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
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- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
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- 150000008065 acid anhydrides Chemical class 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The method comprises: its standard solution uses the healthy human blood serum as base substance; the fructosamine measuring reagent box uses a combination of dual reagents R1 and R2; said R1 comprises NBT reaction component, pH is neutrality; said R2 is alkalinity potassium carbonate buffer solution; the ratio of R1,P2 to the sample under test is 40-50: 8-12: 3.
Description
Technical field
The present invention relates to fructosamine NBT assay method in a kind of serum.
Background technology
(Glucosylated Serum Protein GSP) is the product that non-slowly enzymatic saccharification react takes place for range protein and glucose in the human serum to the saccharification haemocyanin.Wherein main is albuminous saccharification, so GSP also can be described as glycosylated albumin.Because the half life period of albumin in human body be 17 days, thereby measure GSP and can reflect and measure preceding 2～3 all interior average blood sugar levels, is the good index of diabetic's control of blood sugar.Therefore, the mensuration of GSP is all significant to diagnosis of diabetes, prevention, treatment, especially to the prevention of the various complication of diabetes with treat even more important.
The method of measuring GSP is a lot, divides chemical method and chromatography two big classes; Chemical method is estimated the level of saccharification by the ketoamine (product) on the glycated protein in the mensuration serum.The chromatography rule is quantitative again behind elder generation's separation glycated protein, and method is loaded down with trivial details, is difficult to conventional the application.In recent years, occurring the enzymatic assays technology aspect the saccharification haemocyanin, effect is fine, but price is too high, also is difficult to widespread usage.Chemical method is commonly used thiobarbituricacid method and nitro blue tetrazolium (NBT) reducing process.The former poor performance is seldom used, and at present popular mainly is the NBT reducing process.
The principle that the NBT method is measured is: the protein glycation product is a ketoamine, has reductibility, and it can reduce NBT colour generation in alkaline solution, and the absorbance that records at wavelength 550nm place variation is directly proportional with amount, but colorimetric assay.A kind of material 1-deoxidation-1 morpholine fructose (DMF) of synthetic claims fructosamine again, and its structure proximate ketoamine is widely used as being color standards.The NBT method is fairly simple, easy operating, it is than functionally similar glycosylated hemoglobin (GHb) observation period short (about 100 days blood sugar level of GHb reflection) in addition, the speed of finding change of illness state is fast, also have advantages such as the expense of checking is low, it is fast that this method is accepted, popular wide, the existing nearly 20 years history of coming out.But find successively that in recent years the NBT method also has many problems and shortcoming, conclude bibliographical information and can be divided into three major types substantially:
1. the aqueous solution of fructosamine is stable inadequately in the titer, has matrix effect in the use, causes the muddiness of system.
2. the interference of other non-specific reducing substances in the serum, beta lipoprotein for example, the light key uric acid of immunoglobulin A, vitamin C and some can produce the medicine of reductivemetabolism thing etc.
3.NBT the problem etc. of separating out in the single reagent alkaline environment.
In recent years, many news appear in the diabetes control field, but the improvement aspect of NBT method, make progress unhappyly, people expect that the NBT method has significant innovation for this reason, further outstanding advantage, improve shortcoming, make it to benefit numerous diabetics, the present invention is born under this background.
Summary of the invention
The technical problem to be solved in the present invention is, overcome above-mentioned weak point, and provide the NBT assay method of fructosamine in a kind of serum, this assay method can effectively reduce the interference of various non-specific reducing substances, eliminate the matrix effect of titer and fat is turbid, turbid phenomenon, solve the separate out problem of NBT in storage, measured value is accurate.
Technical solution of the present invention is, fructosamine NBT assay method in the serum, and step is:
A. provide titer, fructosamine to measure kit R1, R2 reagent and sample to be measured;
B. get 15 μ l sample to be measured and R1 reagent 200～280 μ l mixings, put 30-45 ℃ and hatched 3～5 minutes;
C. get R2 reagent 40～70 μ l and above-mentioned mixed liquor mixing, hatch 180～240 seconds after, blank pipe zeroing is read absorbance A and is read absorbance A 2 after 1,90 second, wherein, the predominant wavelength that measures absorbance is 500～600nm, commplementary wave length 650～750nm;
D. according to formula: serum fructosamine concentration (mmol/L)=Δ A measures * normal concentration/Δ A standard and obtains serum fructosamine concentration;
It is matrix that described titer adopts the healthy human serum of removing lipoprotein; Described fructosamine is measured kit and is adopted double reagent R1, R2 combination, and described R1 comprises the NBT reacted constituent, and pH is neutral, and described R2 is an alkaline carbonic acid potassium damping fluid.
Described titer, its preparation method is: 1. use healthy people's pooled serum, multigelation is for several times to the upper strata clarification, and the upper strata liquid of getting clarification is used as the matrix of titer after the filtrator of 0.15～0.30um filter membrane filters; Survey its albumin content earlier, add bovine serum albumin(BSA) then, make the concentration that reaches about 4.0wt%, add 0.05～0.1wt% biological preservative at last, mixing, stand-by;
2. add disodium ethylene diamine tetraacetate (EDTA-Na2) 2g/L to above-mentioned refining serum, lactose 30g/L shakes up, and is stand-by;
3. survey the fructosamine concentration of above-mentioned serum, then add fructosamine dry powder, make its concentration reach 4mmol/L, fully 2～8 ℃ of refrigerator overnight of mixing postposition;
4. packing behind the 24h, after send freeze-drying;
5. redissolve, directly work as standard items and use, or its fructosamine value is transferred to through in the healthy people's pooled serum that filters repeatedly.
The described R1 component of fructosamine NBT assay method is in the serum of the present invention:
Kaliumphosphate buffer 50～the 100mmol/L of pH6～8
Potassium chloride 50～100mmol/L
NBT 0.5～1.5mmol/L
Polyvinyl alcohol (PVA) list alkane ether 50～100mmol/L
Sodium taurocholate 5～15mmol/L
Uricase 500～1000 μ mol/L
Vitc oxidase 200～500 μ mol/L;
The R2 component is
Sal tartari 0.1～1.0mmol/L
Saleratus 0.1～0.3mmol/L;
The ratio of described R1, R2 and sample to be measured is 40～56: 8～12: 3.
Measure kit and be designed to double reagent, R1 includes whole compositions that GSP reacts, and adopts kaliumphosphate buffer, and PH 6-8, neutral PH help the stable of NBT and other reactant and preserve.R2 is the stronger sal tartari damping fluid of alkalescence, PH 10.4 (25 ℃), the environmental baseline of reacting for fructosamine.
The NBT assay method of fructosamine in the serum of the present invention, this assay method can effectively reduce the interference of various non-specific reducing substances, eliminate the matrix effect of titer and fat is turbid, turbid phenomenon, solve the separate out problem of NBT in storage, have extensive market prospects.
Embodiment
Embodiment 1
Fructosamine NBT assay method in the serum, its concrete steps are:
1. titer, R1, R2 reagent and sample to be measured are provided;
The preparation method of described titer is as follows: 1. use healthy people's pooled serum, multigelation to the upper strata clarification, is got the upper strata liquid (lower floor discards) of clarification for several times.After the filtrator of 0.15～0.30 μ m filter membrane filters, be used as the matrix of titer.Survey earlier its albumin content, add bovine serum albumin(BSA) then, make to reach about 4.0% weight percent concentration, add 0.05% percentage by weight biological preservative (as methyl-isothiazoline PC-300 etc.) at last, mixing, stand-by;
2. add disodium ethylene diamine tetraacetate (EDTA-Na2) 2g/L to above-mentioned refining serum, lactose 30g/L shakes up, and is stand-by;
3. survey the fructosamine concentration of above-mentioned serum, then add the fructosamine dry powder that Sigma company buys, make its concentration reach 4mmol/L, fully 2～8 ℃ of refrigerator overnight of mixing postposition;
4. next day is by the packing of every bottle 0.5ml serum, after send freeze-drying;
5. every bottle of dried frozen aquatic products is redissolved with the 1.0ml deionized water, dried frozen aquatic products can be directly uses when standard items, more be that its fructosamine value is transferred to through in the healthy people's pooled serum that filters repeatedly.Transfer is that method is: with the standard items calibration that is matrix of above-mentioned refining serum, be that the standard items to be measured of matrix work as sample through the healthy people's pooled serum that filters repeatedly, treat the survey standard items by the definite value requirement and carry out definite value that this value is exactly a branch value.
Described R1 component is:
The kaliumphosphate buffer 100mmol/L of PH7.5
Potassium chloride 100mmol/L
NBT 1.5mmol/L
Polyvinyl alcohol (PVA) list alkane ether 100mmol/L
Sodium taurocholate 15mmol/L
Uricase 1000 μ mol/L
Vitc oxidase 500 μ mol/L;
Described R2 component is:
Sal tartari 1.0mmol/L
Saleratus 0.3mmol/L.
Described R1 and R2 all are solvent with water.
2. get 15 μ l sample to be measured and R1 reagent 240 μ l mixings, put 37 ℃ and hatched 3 minutes;
3. get R2 reagent 60 μ l and above-mentioned mixed liquor mixing, hatch 210 seconds after, blank pipe zeroing is read absorbance A and is read absorbance A 2 after 1,90 second.Wherein, the predominant wavelength that measures absorbance is 546nm, commplementary wave length 700nm, Δ A=A2-A1;
4. according to formula:
Obtain serum fructosamine concentration.
Embodiment 2
Fructosamine NBT assay method in the serum, its concrete steps are:
1. titer, R1, R2 reagent and sample to be measured are provided;
The preparation method of described titer is as follows: 1. use healthy people's pooled serum, multigelation to the upper strata clarification, is got the upper strata liquid (lower floor discards) of clarification for several times.After the filtrator of 0.15-0.30um filter membrane filters, be used as the matrix of titer.Survey earlier its albumin content, add bovine serum albumin(BSA) then, make to reach about 4.0% weight percent concentration, add the biological preservative (as methyl-isothiazoline PC-300 etc.) of 0.1% percentage by weight at last, mixing, stand-by;
2. add disodium ethylene diamine tetraacetate (EDTA-Na2) 2g/L to above-mentioned refining serum, lactose 30g/L shakes up, and is stand-by;
3. survey the fructosamine concentration of above-mentioned serum, then add the fructosamine dry powder that Sigma company buys, make its concentration reach 4mmol/L, fully 2～8 ℃ of refrigerator overnight of mixing postposition;
4. next day is by the packing of every bottle 0.5ml serum, after send freeze-drying;
5. every bottle of dried frozen aquatic products is redissolved with the 1.0ml deionized water, dried frozen aquatic products can be directly uses when standard items, more be that its fructosamine value is transferred to through in the healthy people's pooled serum that filters repeatedly.Transfer is that method is: with the standard items calibration that is matrix of above-mentioned refining serum, be that the standard items to be measured of matrix work as sample through the healthy people's pooled serum that filters repeatedly, treat the survey standard items by the definite value requirement and carry out definite value that this value is exactly a branch value.
Described R1 component is:
The kaliumphosphate buffer 62.5mmol/L of pH7.5
Potassium chloride 62.5mmol/L
NBT 0.75mmol/L
Polyvinyl alcohol (PVA) list alkane ether 70mmol/L
Sodium taurocholate 10mmol/L
Uricase 650 μ mol/L
Vitc oxidase 350 μ mol/L.
Described R2 component is:
Sal tartari 0.65mmol/L
Saleratus PH 10.4 (25 ℃) 0.22mmol/L.
Described R1 and R2 all are solvent with water.
2. get 15 μ l sample to be measured and R1 reagent 200 μ l mixings, put 37 ℃ and hatched 5 minutes;
3. get R2 reagent 70 μ l and above-mentioned mixed liquor mixing, hatch 210 seconds after, blank pipe zeroing is read absorbance A and is read absorbance A 2 after 1,90 second.Wherein, the predominant wavelength that measures absorbance is 546nm, commplementary wave length 700nm, Δ A=A2-A1;
4. according to formula:
, obtain serum fructosamine concentration.
Below announce the experimental result that obtains according to embodiment:
1. several the technical indicators that fructosamine NBT assay method reaches in the serum of the present invention:
A. test reference value scope: 1.58～2.14mmol/L (350 routine non-diabetics, healthy people).
B. the range of linearity: 0～6mmol/L, judgment basis r
2〉=0.995.
C. precision: in n=21 criticizes
CV=0.9%;
Between batch
CV=2.9%.
D. the recovery: sample concentration is 2.28 o'clock, the recovery is＜and ± 10%.
E. accuracy: relative deviation≤± 15%.
2.R1 the stability of reagent under storage requirement
R1 reagent is placed under 24～26 ℃, 2～8 ℃ environment the following index of repetition measurement after a year respectively:
The stability of table 1 R1 reagent under different storage requirements
| Fresh | 24～26 ℃ 1 year | 2～8 ℃ 1 year | |
| Blank absorbency | 0.0009 | 0.007 | 0.024 |
| Uricase activity among the R1 | 100％ | 77％ | 99％ |
Uricase is a material least stable in this kit, and we are benchmark with initial enzymatic activity 100%.Reach a conclusion from table 1: reagent R1 store 1 year relatively stable.
3. measure the stability (in the industry these several are indexs of kits for evaluation stability) of K factor and high, medium and low three horizontal quality controlled serums of kit maximum linearity range, calibration:
Table 2 kit stability test result
| Fresh | 24～26 ℃ 1 year | 2～8 ℃ 1 year | ||
| Maximum linearity range (mmol/L) | 6.00 | 6.11 | 6.13 | |
| The K factor of calibration | 0.0500 | 0.0518 | 0.0519 | |
| Quality controlled serum (mmol/L) | High | 4.25 | 4.15 | 4.33 |
| In | 3.82 | 3.87 | 3.94 | |
| Low | 2.13 | 2.12 | 2.24 | |
As can be seen from Table 2: it all is stable observing kit from above three aspects.
4. with the correlativity of the classical determination method of Luo Shi: this reagent and Luo Shi classical approach relatively, with two methods with 110 parts of test sample product, related coefficient γ=0.989, linear fit Y=1.03X-13.2, two methods are correlated with well.
5. about interference: as follows by this kit being carried out anti-interference experiment measured result:
Jaundice: bilirubin concentration＜85.5umol/L.
Piarhemia: be equivalent to triglyceride＜2000mg/dL.
Haemolysis: be equivalent to haemoglobin＜500mg/dL
Hyperglycemia: concentration of glucose＜900mg/dL
Vitc concentration:＜220umol/L
Uric acid concentration:＜40mg/DL
Experimental result shows: have only when the content among the patients serum surpasses above-mentioned these values and just can cause certain influence to test value, the patients serum of normal conventional is noiseless.Also once to the medicine commonly used of kind more than 20, as vitamin C, stone revolves sugared acid anhydride to the inventor, and left-handed dopa etc. carried out experiment external, had only left-handed dopa when therapeutic dose, can cause fructosamine and raise.
Do not have complete specialization owing to measure foreign peoples's group of glycated protein and the positive reaction of NBT, for caution's sake, the inventor adheres to evaluation result close combination with clinical and other relevant experiments (as HbAlc) etc.:
6.GSP correlativity with fasting blood-glucose
GSP and HbAlc are as follows in the related data of 10 first visit diabetes patients' (to clinical accidental sampling) measured value:
The correlativity of table 3 GSP and fasting blood-glucose
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| Sex | The man | The man | The man | The man | The woman | The man | The woman | The woman | The man | The woman |
| Age | 54 | 50 | 62 | 52 | 48 | 35 | 61 | 49 | 48 | 59 |
| Fasting blood-glucose (mmol/L) | 12.38 | 8.7 | 7.53 | 12.67 | 9.82 | 10.4 | 9.33 | 8.52 | 14.36 | 15.08 |
| Glycosylated hemoglobin (%) | 15.7 | 12.1 | 6.9 | 8.1 | 12.3 | 13.4 | 12.7 | 8.1 | 11.9 | 14.2 |
| Fructosamine (mmol/L) | 3.5 | 3.5 | 2.2 | 3.3 | 2.5 | 3.3 | 3.8 | 2.2 | 2.8 | 3.8 |
The standard that the reference value upper limit of these three projects is announced according to the clinical examination third edition: fasting blood-glucose: 6.1mmol/L, HbAlc:6.0%, GSP:2.15mmol/L, the above value of the control reference as a result upper limit, meet diagnosis of diabetes fully, being correlated with between them is very good.
Test findings to sum up illustrates that fructosamine NBT assay method accepted standard liquid and kit are stable in the serum of the present invention, and test accurately.
The foregoing description is used for the present invention that explains, rather than limits the invention, and in the protection domain of spirit of the present invention and claim, any modification and change to the present invention makes all fall into protection scope of the present invention.
Claims (6)
1. fructosamine NBT assay method in the serum, step is:
A. provide titer, fructosamine to measure kit R1, R2 reagent and sample to be measured;
B. get 15 μ l sample to be measured and R1 reagent 200～280 μ l mixings, put 30-45 ℃ and hatched 3～5 minutes;
C. get R2 reagent 40-70 μ l and above-mentioned mixed liquor mixing, hatch 180～240 seconds after, blank pipe zeroing is read absorbance A and is read absorbance A 2 after 1,90 second, wherein, the predominant wavelength that measures absorbance is 500～600nm, commplementary wave length 650～750nm;
D. according to formula: serum fructosamine concentration (mmol/L)=Δ A measures * normal concentration/Δ A standard and obtains serum fructosamine concentration;
It is characterized in that: it is matrix that described titer adopts the healthy human serum of removing lipoprotein; Described fructosamine is measured kit and is adopted double reagent R1, R2 combination, and described R1 comprises the NBT reacted constituent, and pH is neutral, and described R2 is an alkaline carbonic acid potassium damping fluid.
2. fructosamine NBT assay method in the serum according to claim 1 is characterized in that: described R1 component is
Kaliumphosphate buffer 50～the 100mmol/L of pH6～8
Potassium chloride 50～100mmol/L
NBT 0.5～1.5mmol/L
Polyvinyl alcohol (PVA) list alkane ether 50～100mmol/L
Sodium taurocholate 5～15mmol/L
Uricase 500～1000 μ mol/L
Vitc oxidase 200～500 μ mol/L.
3. fructosamine NBT assay method in the serum according to claim 1 and 2 is characterized in that: described R2 component is
Sal tartari 0.1～1.0mmol/L
Saleratus 0.1～0.3mmol/L.
4. fructosamine NBT assay method in the serum according to claim 1 is characterized in that: the ratio of described R1, R2 and sample to be measured is 40～56: 8～12: 3.
5. fructosamine NBT assay method in the serum according to claim 1, it is characterized in that: the preparation method of described titer is:
1. use healthy people's pooled serum, multigelation is for several times to the upper strata clarification, and the upper strata liquid of getting clarification is used as the matrix of titer after the filtrator of 0.15～0.30um filter membrane filters; Survey its albumin content earlier, add bovine serum albumin(BSA) then, make the concentration that reaches about 4.0wt%, add 0.05～0.1wt% biological preservative at last, mixing, stand-by;
2. add disodium ethylene diamine tetraacetate (EDTA-Na2) 2g/L to above-mentioned refining serum, lactose 30g/L shakes up, and is stand-by;
3. survey the fructosamine concentration of above-mentioned serum, then add fructosamine dry powder, make its concentration reach 4mmol/L, fully 2～8 ℃ of refrigerators of mixing postposition;
4. packing behind the 24h, after send freeze-drying;
5. redissolve, directly work as standard items and use, or its fructosamine value is transferred to through in the healthy people's pooled serum that filters repeatedly.
6. fructosamine NBT assay method in the serum according to claim 5 is characterized in that: described biological preservative is methyl-isothiazoline.
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