CN1896740B - Colloidal golden test-paper strip for fastly testing oxidative low-density fatty protein by semi-quantitative method and its production thereof - Google Patents
Colloidal golden test-paper strip for fastly testing oxidative low-density fatty protein by semi-quantitative method and its production thereof Download PDFInfo
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- CN1896740B CN1896740B CN2005102003946A CN200510200394A CN1896740B CN 1896740 B CN1896740 B CN 1896740B CN 2005102003946 A CN2005102003946 A CN 2005102003946A CN 200510200394 A CN200510200394 A CN 200510200394A CN 1896740 B CN1896740 B CN 1896740B
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- oxldl
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Abstract
This invention provides a rapid diagnoses test paper to detect oxide low-density lipoprotein rapidly, it is quick, simple ,convenient , sensitive , economical , distinctive , and not require specially appointed auxiliary instrument, it can build the relation of color and thickness according to the capture antigen amount of the antibody of oxide low-density lipoprotein building, determining the result of qualitative and semi-quantitative detect of OXLDL in human plasma (the thickness that higher or equal to or lower than the standard oxide low-density lipoprotein sample) ,the early phase auxiliary diagnose of atherosclerosis cardiovascular disease (ASCVD) comes true, it also provides the production and assemble method of test paper.
Description
Technical field: the immunity colloidal gold test paper strip that the present invention relates to OxLDL ELISA (OXLDL) content in a kind of sxemiquantitative fast detecting human plasma.
Background technology:
Cardiovascular and cerebrovascular disease is to cause human dead the first reason; And atherosclerotic (As) is the pathologic basis in early stage that most of cardiovascular and cerebrovascular diseases take place, so study arteriosclerotic pathogenesis, diagnose and prevent and treat the focus that method has become medical circle research.Epidemiology survey and medicine clinical research are all found and confirm to have confidential relation between hyperlipidemia and the atherosclerotic
[1]As everyone knows, the formation that the topmost pathology sign of atherosclerotic is a foam cells, and OXLDL is the key that foam cells forms.Recently research shows, in atherosclerotic focus, has found the deposition of OXLDL, and has not had in the normal arterial wall
[2,3], belonging to the endemic element in the AS focus, blood plasma OXLDL increases and the incidence and the mortality ratio of atherosclerotic cardiovascular disease (ASCVD) have substantial connection.Because OXLDL is a distinctive composition in the As focus; Content in patient's body is far above the normal person; Its concentration and extent of disease are proportional, the auxiliary diagnosis of ASCVD are worth be superior to TC (T-CHOL), TG (triglyceride), AP0-B (apolipoprotein B) and LP (a) (lipoprotein (a))
[4]Detect the level of OXLDL clinically, 1. can reflect patient's lesion degree indirectly. along with the development of As, SABC shows that OXLDL is deposited in the extracellular matrix more and more, especially fibrous cap district and lipid core, atheonecrosis district.Detection by quantitative result also shows: at As different times OXLDL content significant difference is arranged.Pathology is more to late period, and the content of OXLDL is high more.The auxiliary diagnosis that 2. will help arteriosclerotic disease.Raise and clinically do not occur cardiovascular artery sclerosis symptom person as yet for those OXLDL values. can play certain forewarning function
[5]3. measuring the level of oxidized low-density lipoprotein in the blood, is the important evidence of anti-oxidant and anti-low-density lipoprotein (LDL) modified therapeutic, also is the result of treatment monitoring index, for preventing and treating cardiovascular disease and found a new measure
[6]Clinically think that OXLDL content is thought unusual in the healthy human body between 200-500 μ g/L when detecting OXLDL content greater than 600ug/L clinically
[7,8]Because OXLDL is distinctive composition in the As focus, it is better than conventional blood fat project to the specificity of ASCVD auxiliary diagnosis, so the detection of OXLDL is extremely important to the early stage auxiliary diagnosis of ASCVD.
The method that detects OXLDL is a lot
[9,10], the most frequently used method is to adopt OXLDL monoclonal antibody or many anti-enzyme linked immunosorbent assays (ELISA) to detect the content of serum OXLDL
[10,11]The defective of this method is: need special instrument and equipment such as ELIASA to be used; The detecting operation personnel need pass through professional training; Operating process is relatively complicated, and it is higher to detect required expense, can not realize that single branch detects.Along with medical diagnosis on disease develops towards easy, quick, sensitive direction, a lot of diseases have realized detecting with gold-labelled rapid diagnosis reagent.But most colloidal gold strips all are qualitative detection, do not realize half-quantitative detection.The colloidal gold strip of fastly testing oxidative low-density fatty protein by semi-quantitative had both had the high sensitivity of ELISA enzyme process, had the sxemiquantitative function again, can replace conventional machines and detect quantitative status, significantly reduced and detected cost.Content for OxLDL ELISA in the external fast detecting blood plasma is highly significant.
List of references:
1, Guo Gang etc. the separation of low-density lipoprotein is purified and is identified. 1997 the 15th the 1st phases of volume of Tianjin Medical College's journal, 16-19.
2、Yla?Herttuala?s，Palinski?W，Rosenfeld?M?E，et?al.Lipoproteins?in?hormoland?atherosclerosic?aorra.Eur?Heart?J，1990，11[Suppl?E]：88-99.
3、Boyd?HC，Gown?AM，Wodlbaner，et?al.Direct?evidence?for?a?protein?recognizedby?a?monnoclonnal?antibody?against?oxidatively?modified?LDL?in?atherosclerosislesions?from?a?Watanabe?heritable?hyperlipidemic?rabbits.Am?J?Pathol，1989，136：815.
4、Yla?Herttuala?S，Palinski?W，Rosenfeld?M?E，et?al.Evidence?for?the?presenceof?oxidatively?modified?low?density?lipoprotein?in?atherosclerotic?lesions?ofrabbit?and?man.J?CIin?Invest，1993，84：1086.
5、Boyd?HC，Gown?AM，Wodfbaner，et?al.Direct?evidence?for?protein?recognizedby?a?monocolonal?antibody?against?oxidatively?modified?LDL?in?atheroselerosislesions?from?a?watanable?heritable?hyperlipidemic?rabbits.Am?J?Pathol，1992，36：815.
6, Ding Zhenhua. the change of the oxidative modification of low-density lipoprotein and physics and chemistry thereof, biological property. foreign medical science physiology, pathology and clinical fascicle, 1994,14:108.
7, Su Baoxin, Li Shucui etc. the quantitative examination of arteriosclerotic's oxidized low-density lipoprotein. Jiangxi medical test, 2000 the 18th the 1st phases of volume, 19-20.
8, Zhang Xiaogang, Chen Yunzhen etc. the clinical value that the blood plasma OxLDL ELISA is measured. clinical cardiovascular disease magazine, the 18th the 12nd phase of volume of Dec in 2002,613-616.
9, Yang Chengyu etc. the clinical progress of OxLDL ELISA. the foreign medical science clinical biochemistry with detect the credit volume, 2000 the 21st volume first phases, 18-19.
10、Rifai?N，Warnick?GR，Dorniniczax?MH.Handbook?of?lipproteintesting.Washington?DC：AACC，1997，357-372.
11, Wang Hualiang, Wang Xiaoli etc. the double antibodies sandwich method detects blood plasma oxidative modification type low-density lipoprotein. shanghai Medicine check magazine 1994 the 9th phases the 3rd volume, 135-136.
Summary of the invention: the object of the invention is exactly in order to remedy the deficiency of current detection technology; A kind of OxLDL ELISA gold mark fast diagnose test paper bar quick, easy, responsive, economic, special, that do not need particular instrument equipment to assist is provided; And can set up color to concentration relationship according to the amount of OxLDL ELISA antibody capture antigen; Judge the qualitative and half-quantitative detection result (being greater than or equal to or being lower than the concentration of standard contrast OxLDL ELISA sample) of OXLDL in the human plasma rapidly, realize early stage auxiliary diagnosis atherosclerotic cardiovascular disease (ASCVD).The ultimate principle of test paper and making and assembly method are with reference to Chinese invention patent application 2005102002271.The preparation assembly method of this test paper may further comprise the steps: and the righttest nanogold particle size of (1) selection institute mark monoclonal antibody (5nm～40nm).(2) required golden labelled protein is measured the righttest protein labeling amount.(3) prepare the nm of gold albumen colloidal sol of selected granularity in a large number.(4) cutting chromatographic film (the plain film of nitrocellulose filter or CAM or spun glass) becomes prescribed level, and middle intercropping 0.8～1mm narrow slit requires to be parallel to the film side, and seam is equidistance apart from both sides, the long 10～15mm of seam.(5) fixedly film is in the PVC plate, and fixing sponge sucks in water end is fixingly visited the appearance district sponge sucks in water pad.(6) point sample is at the detection zone and contrast Quality Control district of film.(7) will add the water-absorbing sponge fit on PVC plate that gold is marked albumen at last.Its detail and concrete making and assembly method are with reference to the embodiment of back, and its style is referring to Figure of description 1.
Compared with prior art, the present invention has outstanding advantage and practicality: 1. compatibility good (specificity is good, highly sensitive), and accuracy is high; 2. good stability, in batch/difference between batch is little, is easy to quality control; 3. be indicator with the collaurum, itself forms the color mark after the immunoassay process; 4. easy and simple to handle, need not instrument and equipment, realize half-quantitative detection; 5. the symbol display result carries the Quality Control contrast; 6. monomer operation, economical and practical; 7. break away from cold chain, be prone to transportation, storage.
Embodiment:
Embodiment one
The working method of the colloidal gold strip of fastly testing oxidative low-density fatty protein by semi-quantitative:
1, MONOCLONAL ANTIBODIES SPECIFIC FOR
With the immune small white mouse of OXLDL (available from Beijing three friendly scientific and technological development companies that coordinate); The splenocyte and the small white mouse myeloma cell that get immune mouse are hybridized fusion; The preparation hybridoma, the cell line that screening can stably excreting OXLDL monoclonal antibody prepares the monoclonal antibody the 1,2,1, the 2nd of anti-OXLDL, to different antigens determinant on the antigen; Therefore can not be bonded to each other, and measure tiring of monoclonal antibody.
2, the method for colloid gold label monoclonal antibody 1
Collaurum and OXLDL monoclonal antibody 1 be mixing by a certain percentage, makes collaurum and antibody form monoclonal antibody 1-colloid gold label thing.Select OXLDL monoclonal antibody 1 as golden labelled antibody, use the preparation of sodium citrate reducing process and measure its righttest marking nano gold grain size to be 10nm centrifugal 25 minutes of 7300rpm/min rotating speed, preparation liquid storage.10nm collaurum and mark monoclonal antibody are carried out albumen optimal dose mensuration; The pH9.0 borate buffer solution is made the mark monoclonal antibody gradient of 5～50ug/ml; Add the stable experiment of 10%NaCl do again after adding golden liquid storage; Centrifugal static survey OD580nm after 5 minutes, getting and getting protein concentration when optical density is stablized is 12ug/ml.After preparing, add PEG and make collaurum stabilizing solution.
3, gold being marked thing is sprayed on the pad
With polyglycol washing colloids Au composite, the centrifugal supernatant of removing gets the peony deposition.Deposition behind the purifying (being OXLDL monoclonal antibody 1-colloid gold label thing) is with the damping fluid dissolving, and the golden labeling antibody that mark is good is made colloid gold label thing pad with the aseptic water-absorbing sponge absorption of the long 0.08cm thickness of the wide 1.5cm of 1cm, and vacuum is drained.
4, nitrocellulose filter encapsulates
With the setting-out on nitrocellulose filter of special spraying equipment, the width of line is 1～1.5mm with OXLDL monoclonal antibody 2 solution that are prepared into accurate concentration behind the purifying, and the nitrocellulose filter that encapsulates seals with the 10%BSA solution spraying.Produce the aseptic cellulose nitrate film of choosing 0.22um, it is wide to be cut into 1cm, the rectangular strip that 5cm is long, and middle intercropping 0.8～1mm narrow slit requires to be parallel to the film side, and seam is equidistance apart from both sides, the long 10～15mm of seam.The detection zone band is visited appearance end 2.2cm apart from film, is in the narrow slit centre position.Detect the OXLDL monoclonal antibody 2 of band spraying 6ug/ml, the sxemiquantitative quality control band is coated with the OXLDL of 0.6ug/ml, two belt length 0.2cm, wide 0.05cm.4 ℃ of dark surrounds, CO
2Slowly dry up.
5, the assembling of test strips
Test strips is by sample pad, pad, and nitrocellulose filter, adsorptive pads and PVC backing are formed.Be divided into 4 parts: visit the appearance district, label land, comparison and detection district, suction zones.One slit is longitudinally arranged in the middle of the comparison and detection district; The test section and parallel reference region that are divided into the left side; With colloid gold label OXLDL monoclonal antibody 1, encapsulate in the label land, OXLDL monoclonal antibody 2 encapsulates in the detection of test section and is with; A certain amount of OxLDL ELISA encapsulates on the sxemiquantitative stripes of parallel reference region, and double as is accused band.This test strips is utilized immunochromatography technique and double antibodies sandwich method principle, judges the semi-quantitative results of detection through the test section and the power of parallel reference region color contrast, can judge having or not and the content height of determined antigen in the sample rapidly.Color contrast is strong more, and then OXLDL concentration is high more in the sample, and ill possibility is big more, and lesion degree is dark more.Thereby (ASCVD) carries out early stage auxiliary diagnosis to atherosclerotic cardiovascular disease.
Cutting is with the clean PVC plastic plate of nitrocellulose membrane width, and many 2cm use handle as detecting in the suction side in requirement, outside corresponding nitrocellulose membrane is visited the appearance district, extend 1.2cm more and with powerful double faced adhesive tape the nitrocellulose filter of handling well are fixed on the PVC plate.The nitrocellulose filter suction zones is with the aseptic water-absorbing sponge of the fixing long 0.25cm thickness of wide 1.5cm.Visit the fixedly long wide senior filter paper of 1cm of appearance district, distance is nitrocellulose membrane 0.2cm fixedly.The golden labeling pad that middle usefulness prepares is pressed in below filter paper and the nitrocellulose membrane by Z type mode; Ride over above the nitrocellulose membrane with sticker jail; The whole range request of crossing is avoided being infected with of other albumen; Promptly be prepared into the test strips of semi-quantitative determination oxidized low density lipoprotein, 4 ℃ of degree of aluminium foil bag sealing kept dry is subsequent use.
Embodiment two
The method of application of colloidal gold strip
1, sampling:
O'clock take out forearm blood on an empty stomach in morning 7～10, add anti-oxidant immediately,, get blood plasma after centrifugal and put-30 ℃ and examine OXLDL fully in case low-density lipoprotein is in external continuation oxidation.
2, detect
Before detection, test paper is connected packing and take out, room temperature was placed about 5 minutes.Take out test paper, will visit the appearance district and be dipped in the sample solution, sample is a blood plasma, and liquid level is not crossed and visited the appearance district.2-5 takes out after second, lies in a horizontal plane on the clean operator's console.Can see the result by naked eyes in 6～10 minutes.The sxemiquantitative band sees that red explanation testing result is negative if the detection band is not seen redness, and testing sample does not detect the OxLDL ELISA that can suspect sufferer, test strips device quality intact (shown in Figure 1 like signal in the accompanying drawing 2); If redness all appears in sample detection band and sxemiquantitative band, prove and detect the positive.Detection band color is higher than the OxLDL ELISA that exists in the sxemiquantitative carrying means sample and is higher than the 0.6ug/ml level, representes ill (shown in Figure 2 like signal in the accompanying drawing 2); The OxLDL ELISA that exists in the little then sample of color distortion approaches the critical level of 0.6ug/ml, maybe be ill; Be lower than the 0.6ug/ml level if detect the OxLDL ELISA of being with color to be lower than the sxemiquantitative band then existing in the surface sample, expression normal (shown in Figure 3) like signal in the accompanying drawing 2; The sxemiquantitative band is not seen red explanation test strips failure of apparatus yet if the detection band is seen redness, can not be used for detecting.
Claims (5)
1. an OxLDL ELISA (OXLDL) rapid semi-quantitative detects colloidal gold strip, and this test strips is divided into 4 parts, has comprised visiting the appearance district; The label land, comparison and detection district, suction zones; Adopt two monoclonal antibody sandwich mode to detect, the comparison and detection district comprises detects band and sxemiquantitative band, and colloid gold label OXLDL monoclonal antibody 1 is as detecting moving phase; Fixedly OXLDL monoclonal antibody 2 is as stationary phase, and the sxemiquantitative band is the OXLDL that can mark monoclonal antibody 1 specific bond with gold and encapsulate 0.6 μ g/ml, it is characterized in that; One vertical narrow slit is arranged in the middle of the comparison and detection district, detect band and sxemiquantitative band and be respectively in the narrow slit both sides side by side.
2. a kind of OxLDL ELISA according to claim 1 (OXLDL) rapid semi-quantitative detects colloidal gold strip, it is characterized in that, OXLDL monoclonal antibody l is the different antigenic determinants that are directed against OXLDL respectively with OXLDL monoclonal antibody 2.
3. detect colloidal gold strip according to the described a kind of OxLDL ELISA of claim 1 (OXLDL) rapid semi-quantitative, it is characterized in that, OXLDL monoclonal antibody 1 is with OXLDL monoclonal antibody 2 and adopts the preparation of mouse hybridoma cell method.
4. detect colloidal gold strip according to the described a kind of OxLDL ELISA of claim l (OXLDL) rapid semi-quantitative, it is characterized in that this test strips is used to detect plasma sample.
5. a kind of OxLDL ELISA according to claim 1 (OXLDL) rapid semi-quantitative detects colloidal gold strip, it is characterized in that, this test strips can be in the application in the early stage auxiliary diagnosis of atherosclerotic cardiovascular disease (ASCVD).
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101183108A (en) * | 2007-10-16 | 2008-05-21 | 李红玉 | Colloidal gold test paper for detecting trace quantity oxygenize low density lipoprotein by indirect competition method |
| CN102135535B (en) * | 2010-01-25 | 2015-06-24 | 常州博闻迪医药科技有限公司 | Immune colloidal metal detection technology capable of directly performing semi-quantitative analysis, preparation method and application |
| CN104597252A (en) * | 2015-01-23 | 2015-05-06 | 浙江卓运生物科技有限公司 | Kit for detecting oxidized low-density human serum lipoproteins by immunological turbidimetry |
| CN105158486B (en) * | 2015-08-21 | 2016-07-06 | 陈立国 | For detecting the enzyme linked immunological kit of people's OxLDL ELISA |
| CN110749731A (en) * | 2019-10-18 | 2020-02-04 | 北京协和洛克生物技术有限责任公司 | Time-resolved immunochromatography kit for quantitatively detecting oxidized low-density lipoprotein and application thereof |
| CN117192134A (en) * | 2023-09-14 | 2023-12-08 | 宁波美康盛德生物科技有限公司 | Detection kit and detection method for oxidized low-density lipoprotein |
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| DE10158147A1 (en) * | 2001-07-20 | 2003-01-30 | Imtec Immundiagnostika Gmbh | Determining autoantibodies against oxidized low-density lipoprotein, useful for diagnosis of e.g. arteriosclerosis, without interference from antibodies against native lipoprotein |
| CN1147729C (en) * | 1998-12-30 | 2004-04-28 | 卢氏实验公司 | Semi-quantitative one-step immunologic diagnosis method |
| CN1580776A (en) * | 2004-05-21 | 2005-02-16 | 王继华 | Test paper tape for detecting double-stranded DNA antibody by colloidal gold chromatographic analysis and its preparing method |
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2005
- 2005-07-15 CN CN2005102003946A patent/CN1896740B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1147729C (en) * | 1998-12-30 | 2004-04-28 | 卢氏实验公司 | Semi-quantitative one-step immunologic diagnosis method |
| DE10158147A1 (en) * | 2001-07-20 | 2003-01-30 | Imtec Immundiagnostika Gmbh | Determining autoantibodies against oxidized low-density lipoprotein, useful for diagnosis of e.g. arteriosclerosis, without interference from antibodies against native lipoprotein |
| CN1580776A (en) * | 2004-05-21 | 2005-02-16 | 王继华 | Test paper tape for detecting double-stranded DNA antibody by colloidal gold chromatographic analysis and its preparing method |
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