CN101571489A - Method and kit for measuring glucose - Google Patents
Method and kit for measuring glucose Download PDFInfo
- Publication number
- CN101571489A CN101571489A CNA2008101053335A CN200810105333A CN101571489A CN 101571489 A CN101571489 A CN 101571489A CN A2008101053335 A CNA2008101053335 A CN A2008101053335A CN 200810105333 A CN200810105333 A CN 200810105333A CN 101571489 A CN101571489 A CN 101571489A
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- China
- Prior art keywords
- hole
- cholesterol
- add
- kit
- microplate reader
- Prior art date
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- 238000000034 method Methods 0.000 title claims abstract description 17
- 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 title abstract 3
- 239000008103 glucose Substances 0.000 title abstract 3
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 10
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 60
- 235000012000 cholesterol Nutrition 0.000 claims description 19
- 210000002966 serum Anatomy 0.000 claims description 13
- 210000002381 plasma Anatomy 0.000 claims description 9
- 238000002835 absorbance Methods 0.000 claims description 8
- 108010089254 Cholesterol oxidase Proteins 0.000 claims description 7
- 102000003992 Peroxidases Human genes 0.000 claims description 7
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000002965 ELISA Methods 0.000 claims description 6
- 238000005259 measurement Methods 0.000 claims description 6
- 108010055297 Sterol Esterase Proteins 0.000 claims description 5
- 102000000019 Sterol Esterase Human genes 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000012482 calibration solution Substances 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 3
- VADKRMSMGWJZCF-UHFFFAOYSA-N 2-bromophenol Chemical class OC1=CC=CC=C1Br VADKRMSMGWJZCF-UHFFFAOYSA-N 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract 1
- 238000000338 in vitro Methods 0.000 abstract 1
- 238000002372 labelling Methods 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 3
- -1 cholesteryl ester Chemical class 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 150000004060 quinone imines Chemical class 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102100024640 Low-density lipoprotein receptor Human genes 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- GZFGOTFRPZRKDS-UHFFFAOYSA-N 4-bromophenol Chemical compound OC1=CC=C(Br)C=C1 GZFGOTFRPZRKDS-UHFFFAOYSA-N 0.000 description 1
- 101150102415 Apob gene Proteins 0.000 description 1
- 208000000563 Hyperlipoproteinemia Type II Diseases 0.000 description 1
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- 201000004408 Hypobetalipoproteinemia Diseases 0.000 description 1
- 108010001831 LDL receptors Proteins 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- 206010029164 Nephrotic syndrome Diseases 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 206010045261 Type IIa hyperlipidaemia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 208000017580 chronic wasting disease Diseases 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 201000001386 familial hypercholesterolemia Diseases 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 208000003532 hypothyroidism Diseases 0.000 description 1
- 230000002989 hypothyroidism Effects 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 150000002561 ketenes Chemical class 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
- 235000000824 malnutrition Nutrition 0.000 description 1
- 230000006855 networking Effects 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- YLLIGHVCTUPGEH-UHFFFAOYSA-M potassium;ethanol;hydroxide Chemical compound [OH-].[K+].CCO YLLIGHVCTUPGEH-UHFFFAOYSA-M 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method and a kit for measuring glucose, belonging to the technical field of the in-vitro diagnostic reagent. The method and the kit for measuring glucose are used for detection with an enzyme-labeling instrument and are particularly applicable to urban hospitals, community hospitals and rural medical units.
Description
Technical field
The present invention relates to method for determination of cholesterol and measure kit, be used for measuring serum or blood plasma cholesterol level, belong to the external diagnosis reagent technical field.
Background technology
T-CHOL is measured the content that kit is used for quantitative measurement serum or blood plasma T-CHOL clinically.Under the pathological state, high T-CHOL has former and secondary two classes.Former as familial hypercholesterolemia (LDL receptor defective), familial apoB defective disease, the high T-CHOL of polyphyly (polygenic), Combination hyperlipoprotememia.Secondary see nephrotic syndrome, hypothyroidism, diabetes, gestation etc.Low T-CHOL mass formed by blood stasis also have former with secondary.The former is as the nothing or the hypobetalipoproteinemia of family; The latter such as hyperthyroidism, malnutrition, chronic wasting disease etc.
The main method of method of measuring T-CHOL in the market is broadly divided into 2 kinds.The 1st kind of normal hexane extracting L-B reaction solution method: make the haemocyanin sex change with potassium hydroxide ethanol liquid, and use cholesteryl ester to be hydrolyzed to cholesterol.Divide molten extracting with normal hexane after adding water, can from alkaline ethanol liquid, extract cholesterol (reaching 99.7%) quantitatively.Except that a small amount of other sterol, be substantially free of chaff interference in the cholesterol solution that extracts.So measurement result and isotopic dilution-gas chromatography-mass spectrography are approaching.The 2nd kind of T-CHOL enzyme colorimetric COD-POD enzyme process.The COD-POD enzyme process, the cholesterol in the serum about 1/3 is a free cholesterol, 2/3 is the cholesteryl ester that combines with fatty acid.Cholesteryl ester is hydrolyzed into free cholesterol by cholesterol ester hydrolase (CEH), the latter is oxidized to the black ketenes of courage and produces hydrogen peroxide by cholesterol oxidase (COD), and the hydrogen peroxide that is produced generates red quinone imines with developer under the effect of peroxidase (POD).According to quinone imines absorbance and institute test sample in this total cholesterol level be directly proportional.T-CHOL oxidation enzyme process is with low cost in these two kinds of methods, usable range more extensively, easier in clinical acceptance, therefore adopt T-CHOL oxidation enzyme process to carry out the development of T-CHOL kit.
The kit of measuring cholesterol level in the market all is applicable to biochemical instruments, the medical institutions of cities and towns, community do not possess biochemical instruments and possess microplate reader mostly, but cholesterol level is the physical signs that body weight for humans is wanted simultaneously, and the mensuration of cholesterol level has the important clinical meaning.
Summary of the invention
The present invention measures the method for cholesterol and measures kit, be applicable to microplate reader, because microplate reader is with low cost, equipment requirements is simple, need not to buy expensive full automatic biochemical apparatus, remedied the deficiency of semi-automatic biochemical analyzer simultaneously, significantly reduced workload and personal error than color comparison of naked eye hand-manipulated to the extensive detectability of same index; Experimental implementation is easy, once can detect nearly hundred increments originally, and dozens of minutes can obtain testing result.The measurement result convenience of calculation, data can keep or print, and are beneficial to laboratory standard management and networking construction.Therefore the objective of the invention is to develop the cholesterol level that is applicable to microplate reader and measure kit, to be applicable to rural area, cities and towns and community medicine.
Cholesterol level is measured the reaction principle that kit adopts the cholesterol oxidase method.
Cholesterol is after enzyme hydrolysis, and through the cholesterol oxidase oxidation, the hydrogen peroxide that is produced generates red quinone imines with developer under the effect of peroxidase (POD).Compare result of calculation simultaneously by standard items.
According to this reaction principle, cholesterol level is measured kit and is comprised ELISA Plate, R1 and titer, and R1 is made up of compositions such as cholesterol esterase, cholesterol oxidase, peroxidase, the amino antipyrine of 4-, p bromophenols.Titer is the cholesterol titer.
The assay method that adopts is, on ELISA Plate, establishes reagent blank hole, a hole, two hole gauge orifices, and all the other are for measuring the hole.Add R1:200 μ l, adding distil water 10 μ l in the blank well add calibration solution 10 μ l respectively in the gauge orifice, measure the hole and add test serum (blood plasma) 10 μ l respectively, and mixing was put 37 ℃ of incubations 10 minutes.In the microplate reader colorimetric, at predominant wavelength 492nm, commplementary wave length 620nm measures down, and the zeroing of reagent blank hole detects.Measurement result is calculated cholesterol level as follows: serum (blood plasma) cholesterol concentration=mensuration hole absorbance/gauge orifice absorbance * 100 (mg/dL).
Embodiment
Further specify the present invention with embodiment below.Embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.The experimental technique of unreceipted actual conditions in the following example carries out according to the normal condition of this area usually.
Embodiment one:
Consisting of of kit: R1 and titer.
Wherein R1's consists of the amino antipyrine 10mmol/L of cholesterol esterase 5KU/L, cholesterol oxidase 5KU/L, peroxidase 5KU/L, 4-, p bromophenol 5mmol/L.
Cholesterol titer cholesterol 100mg/dL
Embodiment two:
Assay method is as follows:
1. on 96 hole ELISA Plate, establish reagent blank hole, a hole, two hole gauge orifices, all the other are for measuring the hole.Add R1:200 μ l, adding distil water 10 μ l in the blank well add calibration solution 10 μ l in the gauge orifice, measure the hole and add test serum (blood plasma) 10 μ l respectively, and mixing was put 37 ℃ of incubations 10 minutes,
2. in the microplate reader colorimetric.Measure under predominant wavelength 492nm, commplementary wave length 620nm, the zeroing of reagent blank hole detects.
3. measurement result is calculated cholesterol level as follows:
Serum (blood plasma) cholesterol concentration=mensuration hole absorbance/gauge orifice absorbance * 100 (mg/dL).
Embodiment three:
The present invention measures kit according to embodiment one configuration, operates according to embodiment two.Accuracy: measured value is in quality controlled serum (quality controlled serum of Randox) normally reaches unusual quality controlled serum target value ± 20% scope.Reagent blank absorbance: A＜0.30 (predominant wavelength 492nm, commplementary wave length 620nm; 37 ℃).
Through clinical examination test, kit of the present invention compared with prior art correlativity can be applicable to clinically all greater than 0.9 there was no significant difference, and has the community of being applicable to, rural area, cities and towns medical system, has the wider scope of application.
Claims (3)
1, a kind of T-CHOL that is used for microplate reader is measured kit, and this kit comprises ELISA Plate, R1 and titer, and R1 is made up of compositions such as cholesterol esterase, cholesterol oxidase, peroxidase, the amino antipyrine of 4-, p bromophenols.Titer is the cholesterol titer.
2, a kind of method for determination of cholesterol that is used for microplate reader: in ELISA Plate, establish reagent blank hole, a hole, two hole gauge orifices, all the other are for measuring the hole.Add R1:200 μ l, adding distil water 10 μ l in the blank well add calibration solution 10 μ l in the gauge orifice, measure the hole and add test serum (blood plasma) 10 μ l respectively, and mixing was put 37 ℃ of incubations 10 minutes, in the microplate reader colorimetric.Measure under predominant wavelength 492nm, commplementary wave length 620nm, the zeroing of reagent blank hole detects.Measurement result is calculated cholesterol level as follows: serum (blood plasma) cholesterol concentration=mensuration hole absorbance/gauge orifice absorbance * 100 (mg/dL).
3, according to the claim 2 described method for determination of cholesterol that are used for microplate reader, it is characterized in that: on ELISA Plate, establish reagent blank hole, a hole, two hole gauge orifices, all the other are for measuring the hole.Add R1:200 μ l, adding distil water 10 μ l in the blank well add calibration solution 10 μ l respectively in the gauge orifice, measure the hole and add test serum (blood plasma) 10 μ l respectively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101053335A CN101571489A (en) | 2008-04-28 | 2008-04-28 | Method and kit for measuring glucose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101053335A CN101571489A (en) | 2008-04-28 | 2008-04-28 | Method and kit for measuring glucose |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101571489A true CN101571489A (en) | 2009-11-04 |
Family
ID=41230896
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008101053335A Pending CN101571489A (en) | 2008-04-28 | 2008-04-28 | Method and kit for measuring glucose |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101571489A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101975753A (en) * | 2010-09-17 | 2011-02-16 | 李立和 | Two-step enzymatic determination method for cholesterol ester in serum |
| CN105911047A (en) * | 2016-04-01 | 2016-08-31 | 河南工业大学 | Method for detecting cholesterol based on gold-silver core-shell nanoparticle colourimetry |
-
2008
- 2008-04-28 CN CNA2008101053335A patent/CN101571489A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101975753A (en) * | 2010-09-17 | 2011-02-16 | 李立和 | Two-step enzymatic determination method for cholesterol ester in serum |
| CN105911047A (en) * | 2016-04-01 | 2016-08-31 | 河南工业大学 | Method for detecting cholesterol based on gold-silver core-shell nanoparticle colourimetry |
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|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20091104 |