CN106706919A - Direct quantitative detection kit for alpha-ketoglutaric acid in human serum - Google Patents
Direct quantitative detection kit for alpha-ketoglutaric acid in human serum Download PDFInfo
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- CN106706919A CN106706919A CN201710019353.XA CN201710019353A CN106706919A CN 106706919 A CN106706919 A CN 106706919A CN 201710019353 A CN201710019353 A CN 201710019353A CN 106706919 A CN106706919 A CN 106706919A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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Abstract
The invention relates to the field of detection technology and particularly discloses a direct quantitative detection kit for alpha-ketoglutaric acid in human serum. The direct quantitative detection kit comprises a serum sub-packaging tube, a hepes buffer solution, a reducing coenzyme, an enzyme stabilizer, a fluorescent dye and an alpha-KG standard. The kit disclosed by the invention can quickly and efficiently detect alpha-ketoglutaric acid in the serum of patients suffering from liver cirrhosis and liver cancer, thereby providing a technical guarantee for detecting and screening patients suffering from early liver cirrhosis and liver cancer. Compared with the traditional chromatography coupled detection method, nuclear magnetic resonance method and the like, the kit disclosed by the invention is low in cost and simple to operate and has the characteristics of high speed and high throughput; the kit for detecting alpha-ketoglutaric acid in serum has specific selectivity and relatively high stability; and compared with a biological detection method, the kit is obviously improved in operation repeatability, can be used as an effective tool for diagnosing liver cirrhosis and liver cancer, is of great significance to early detection of liver diseases and evaluation of treatment effects, and has broad application prospects.
Description
Technical field
It is important small molecule biological marker in a kind of human serum specifically the present invention relates to detection technique field
The direct quantitative detection kit of thing KG.
Background technology
KG (α-Ketoglutaric acid, abbreviation α-KA, CAS:328-50-7) it is a kind of important biology
Compound, its form for generally existing is its anion, i.e. alpha-ketoglutarate (α-ketoglutarate, abbreviation α-KG).
KG is a key substance in tricarboxylic acid cycle, and position in the circulating cycle is located at after isocitric acid and amber
Before acyl coenzyme A.The amino of amino acid is incorporated on KG by transamination, and as blood is downloaded to liver
Dirty, ammonia enters urea cycle in liver, so KG is widely present in human serum.Existing result of study table
Bright, the KG change in concentration in blood is closely related with numerous diseases, and such as NASH and acute marrow are thin
The change in concentration of KG has important clinical diagnosis meaning (Weihai in born of the same parents' leukaemia etc., therefore detection serum
He,et al.,Nature,2004,429,188-193;E Rodriguez-Gallego et al.,Int.J.Obesity.,
2015,39,279-287;Perl,A.E.et al.,Blood,2012,121,4917-4924).
It is well known that cirrhosis is clinical common chronic progressive hepatopathy, by one or more cause of disease for a long time or repeatedly
Act on the diffusivity hepatic lesion for being formed.Statistics shows, every year about 300,000 people because cirrhosis it is dead, its incidence of disease is in the 4th
Position, is only second to malignant tumour, cardiovascular disease, cerebrovascular disease.Liver cancer is even more a kind of disease very big to harm, is also all
One of most fast malignant tumour of the death rate, death rate in cancer.China is always the district occurred frequently of liver cancer, due to initial symptoms not
Substantially, disease progression is fast, and malignancy is high, easily recurs and causes liver cancer patient poor prognosis, and its five year survival rate is very low.It is early
It was found that, early diagnosis, early treatment, be still the best approach for improving tumor prognosis.
The method of KG concentration is chromatograph joint used detection method and nuclear magnetic resonance method in traditional detection serum.But due to
The molecular weight of KG is smaller, it is difficult to directly carry out quantitative determination, it is necessary to serum to it using chromatograph joint used detection method
The pretreatment of complexity is carried out, such as the pretreatment of aromatic ring addition derivatization reaction is carried out to KG, its operating process is not only numerous
It is trivial, time-consuming, and being also easy to produce interference accessory substance causes test error big, poor sensitivity.Although magnetic nuclear resonance method is suitable for clinic
Non-invasive detection, but its expensive equipment, testing cost are higher, expend the time more long, it is difficult to realize quantitative determination.At present, favorably
With the report of KG change in concentration in the Fluorometric assay aqueous solution, but due to its cannot directly apply to serum in detect
KG, and there is the defects such as poor specificity, response speed be slow, significantly limit its in vitro in in-situ diagnostics should
With (Pengwei Jin, et al., Chem.Sci., 2014,5,4012-4016).Simultaneously as KG is in human body
Be widely present, had on the market dedicated for quantitatively or semi-quantitatively detecting the reagent of the KG of KG
Box, such as effect are preferably respectively the KG kits of Biovision and Sigma-Aldrich companies.Wherein
The detection range of the KG kit of Biovision companies is 0.01-10nmol (http://www.amyjet.com/
products/K677-100.shtml).The detection sensitivity of the KG kit of Sigma-Aldrich companies is
0.01-10nmol(http://www.sigmaaldrich.com/catalog/searchTerm=MAK054&interface
=All_ZH&N=0&mode=match%20partialmax&lang=zh&region=CN&foc us=product).
But these kits are expensive, often measuring 1 time is needed to spend 40 to 60 yuan, and complex pre- place is needed before test
Reason, and can there is a problem of much testing inaccurate.As shown by data kit at present on the market cannot be directly used to detection
KG in serum, and price is high, cost is too high, and test is inaccurate.
Therefore, development is needed badly a kind of with KG concentration in simple and quick efficient direct detection human serum in situ
Kit, be that early stage, quick diagnosis are carried in expecting its clinical diagnosis that can apply to relevant disease (such as cirrhosis, liver cancer)
For reliable important evidence.
The content of the invention
It is an object of the invention to provide a kind of direct quantitative detection kit of KG in human serum, it is intended to
Overcome in the prior art such as cannot direct measurement, detection method be cumbersome, deficiency with high costs, so as to greatly expand it face
Application in bed diagnosis.Human serum sample (73) is detected using the kit in the present invention, as a result shows to compare
In normal person, the concentration values of KG are significantly higher in cirrhosis and liver cancer sufferer blood, therefore in cirrhosis and liver
In the clinical diagnosis of cancer, the KG change in concentration in accurate measurement blood of human body can be as the weight of early clinical diagnosis
Will foundation.
A kind of the first aspect of the present invention, there is provided the direct quantitative detection kit of KG in human serum, bag
Include:Serum packing pipe, hepes buffer solutions, reducibility coenzyme, enzyme stabilizers, fluorescent dye and α-KG reference materials.
Wherein, described hepes pH of cushioning fluid is 7.0, and content is 50 μm of ol/L.
Described reducibility coenzyme includes reduced coenzyme Q 10, NADH, nicotinamide adenine two
One or several in nucleotide phosphodiesterase, content is 5 μm of ol/L.
Described enzyme stabilizers are comprising a kind of or several in trehalose, mannitol, glycerine, vitamin C, bovine serum albumin
Kind, content is 0.5g/L.
Described fluorescent dye is used to carry out KG in serum fluoroscopic examination, and content is 5 μm of ol/L, purchased from Thailand
Smooth Reagent Company.
Described α-KG reference materials content is 50 μm of ol/L, purchased from Tai Tan Reagent Companies.
Described serum packing pipe, its volume size is 1.5mL, purchased from Axygen companies of the U.S..
A kind of the second aspect of the present invention, there is provided detection side of the above-mentioned kit of application to KG in serum
Method, comprises the following steps:
A detection kit) is taken out from subzero 20 DEG C of refrigerators, while preparing human serum to be measured;
B above-mentioned serum) is dispensed into pipe, hepes buffer solutions, reducibility coenzyme, enzyme stabilizers, fluorescent dye, α -one penta 2
Sour reference material and human serum are added sequentially in the orifice plate of black sxemiquantitative 96, are placed in fluorescence intensity change in ELIASA;
C) fluorescence change read according to ELIASA, using the standard detecting method of KG, calibrates serum
The concentration of middle α-KG.
By KG kit successfully detect normal person, cirrhosis and in In Sera of Patients With Hepatocarcinoma α-KG it is dense
Degree change, shows that the kit has important clinical value in the clinical diagnosis of cirrhosis and liver cancer, for hepatopathy
Early detection and therapeutic effect evaluation it is significant.
The third aspect of the present invention, there is provided the direct quantitative of above-mentioned kit KG in human serum is prepared
Application in detection means.
The invention has the advantages that:
1st, the detection kit used in the inventive method can be other with KG in direct quantitative detection serum
Detection method does not possess on the market or in laboratory.
2nd, the fluorometric investigation instrument that the method for the present invention is used is ELIASA, with sensitivity and detection very high
Limit, belongs to simple to operate, quick, high-throughout detection method.
3rd, the invention belongs to external in situ detection, and with the feature such as quick, efficient, easy, its operability and safety
Property is secure.
4th, the testing result of human serum sample (73) shows, detects that normal person, liver are hard using kit of the present invention
Change and the KG concentration between hepatocarcinoma patient three has significant difference, wherein in cirrhosis and liver cancer patient's serum
KG concentration is apparently higher than normal population.
The α -one penta 2 in cirrhosis and In Sera of Patients With Hepatocarcinoma can be quickly and efficiently detected using kit of the invention
Acid concentration, ensures for detection screening early-phase hepatocirrhosis and liver cancer patient provide technology.It is simultaneously chromatograph joint used compared to traditional
Detection method, nuclear magnetic resonance method etc., the kit used in the present invention are with low cost, simple to operate, with fast high-flux feature;
And for detecting that the kit of KG in serum has specific selectivity and stability higher, and examined relative to biology
Survey method, it is significantly improved in operation repeatability.
Using detection kit of the invention without carrying out any pretreatment to serum sample, can directly, quickly, efficiently
The concentration of KG in ground detection serum, detection is limited to 0.1 μM.α in the direct detection serum that the inventive method is provided-
Ketoglutaric acid kit, with multiple advantages such as easy to operate, with low cost, specific good, direct detection, favorable repeatabilities.
Particularly 73 human serum pattern detections altogether are shown using detection kit of the invention, normal person and cirrhosis, liver
The concentration of KG has significant difference in cancer patients serum, shows that the kit can be as diagnosis cirrhosis, liver
The effective tool of cancer, the significant and wide application prospect of the evaluation of early detection and therapeutic effect for hepatopathy.
Brief description of the drawings
Fig. 1 be normal person, cirrhosis and in In Sera of Patients With Hepatocarcinoma α-KA concentration distribution (n representative samples number).
Specific embodiment
The specific embodiment that the present invention is provided is elaborated with reference to embodiment.
Embodiment 1
1st, the collection of blood serum sample and treatment
Cirrhosis and hepatocarcinoma patient early morning venous blood samples 5mL, not anti-freezing, are put into 0 DEG C of refrigerator and preserve 60min, take out
Be centrifuged 15 minutes in 5000rpm afterwards, take that supernatant is sub-packed in 1.5mL with serum packing pipe in.
2nd, detection of the KG kit to KG in human serum
A) parameter setting.The orifice plate of black 96 is put into support plate platform, Read → test mode is directly set
Fluorescence intensity → detection method terminal/kinetic measurement → optics type Filters → Ok → go out
Now new parameter meeting, i.e., property selective exitation wavelength 530nm, launch wavelength 590nm and increasing according to KG probe
Benefit value is 50.Start detection after setting completed.
B) KG concentration in detection serum.Blood serum sample, kit solution are thawed successively after etc. in hand-hole,
It is placed in the detection of support plate platform.By blood serum sample inject 50 microlitres in each hole, hepes buffer solutions, reducibility coenzyme, enzyme stabilizers
5 microlitres are injected in each Kong Zhongjun, fluorescent dye injects 10 microlitres in each hole, then is implanted sequentially KG reference material
0th, 5,10,15,20,25 microlitres.Three groups of data of parallel testing, preserve data and drawing curve, calculate α in serum-
The concentration of ketoglutaric acid, experimental result is as shown in Figure 1.
The concentration of KG is in 2.5-5 × 10 in Fig. 1 display normal human serums-6In the range of mol/L, cirrhosis is suffered from
The concentration of KG is in 8-32 × 10 in person's serum-6In the range of mol/L, KG is dense in liver cancer patient blood serum
Degree is in 12-58 × 10-6In the range of mol/L, the significant difference of KG concentration indicates KG between three
Kit can detect the concentration of KG in serum situ, have in the clinical diagnosis of cirrhosis and liver cancer important
Clinical value, indicating kit provided by the present invention has that detection speed is quick, simple to operate, the cost time
Less, do not need that large-scale instrument, serum requirement are few, have the advantages that clinical value and can the direct quantitative determination in serum,
This is the advantage that other detected not available for KG means or only possessed one.
Below the preferred embodiment to the invention is illustrated, but the invention be not limited to it is described
Embodiment, those of ordinary skill in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (9)
1. in a kind of human serum KG direct quantitative detection kit, it is characterised in that including:Serum is dispensed
Pipe, hepes buffer solutions, reducibility coenzyme, enzyme stabilizers, fluorescent dye and α-KG reference materials;Wherein, described reducibility coenzyme
It is a kind of or several in comprising reduced coenzyme Q 10, NADH, nicotinamide-adenine dinucleotide phosphate
Kind;Described enzyme stabilizers include one or several in trehalose, mannitol, glycerine, vitamin C, bovine serum albumin.
2. in human serum according to claim 1 KG direct quantitative detection kit, it is characterised in that
Described hepes pH of cushioning fluid is 7.0, and content is 50 μm of ol/L.
3. in human serum according to claim 1 KG direct quantitative detection kit, it is characterised in that
Described reducibility coenzyme content is 5 μm of ol/L.
4. in human serum according to claim 1 KG direct quantitative detection kit, it is characterised in that
Described enzyme stabilizers content is 0.5g/L.
5. in human serum according to claim 1 KG direct quantitative detection kit, it is characterised in that
Described fluorescent dye is used to carry out KG in serum fluoroscopic examination, and content is 5 μm of ol/L.
6. in human serum according to claim 1 KG direct quantitative detection kit, it is characterised in that
Described α-KG reference materials content is 50 μm of ol/L.
7. in human serum according to claim 1 KG direct quantitative detection kit, it is characterised in that
Described serum packing pipe, its volume size is 1.5mL.
8. a kind of kit applied as described in claim 1-7 is any is to the detection method of KG in serum, and it is special
Levy and be, comprise the following steps:
A described kit) is taken out from subzero 20 DEG C of refrigerators, while preparing human serum to be measured;
B) by described serum packing pipe, hepes buffer solutions, reducibility coenzyme, enzyme stabilizers, fluorescent dye, KG
Reference material and human serum are added sequentially in the orifice plate of black sxemiquantitative 96, are placed in fluorescence intensity change in ELIASA;
C) the fluorescence change read according to ELIASA, using the standard detecting method of KG, calibrate α in serum-
The concentration of KG.
9. the direct quantitative inspection of a kind of kit KG in human serum is prepared as described in claim 1-7 is any
The application surveyed in device.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN109748970A (en) * | 2019-01-24 | 2019-05-14 | 华东理工大学 | α-ketoglutaric acid optical probe and its preparation method and application |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN109748970A (en) * | 2019-01-24 | 2019-05-14 | 华东理工大学 | α-ketoglutaric acid optical probe and its preparation method and application |
CN109748970B (en) * | 2019-01-24 | 2022-07-05 | 华东理工大学 | Alpha-ketoglutaric acid optical probe and preparation method and application thereof |
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