CN101183108A - Colloidal gold test paper for detecting trace quantity oxygenize low density lipoprotein by indirect competition method - Google Patents
Colloidal gold test paper for detecting trace quantity oxygenize low density lipoprotein by indirect competition method Download PDFInfo
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- CN101183108A CN101183108A CNA2007102020847A CN200710202084A CN101183108A CN 101183108 A CN101183108 A CN 101183108A CN A2007102020847 A CNA2007102020847 A CN A2007102020847A CN 200710202084 A CN200710202084 A CN 200710202084A CN 101183108 A CN101183108 A CN 101183108A
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- oxldl elisa
- oxldl
- density lipoprotein
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Abstract
The present invention provides an indirect competition method for golden test paper for detecting micro-oxidation low density lipoprotein colloid. The competition method is applied to the test paper. A certain amount of golden dot antibody is covered on the golden dot antibody pad. The oxidation low density lipoprotein is covered on a detecting region. Through the observation of the color of the detecting region, whether the oxidation low density lipoprotein in the serum or the plasma of a human body being higher than a normal level can be detected. The present invention is convenient and quick; the detection of the oxidation low density lipoprotein can be directly carried out; and the present invention provides reliable evidence to the early diagnose of atherosclerosis.
Description
One. technical field:
The present invention relates to the detection of OxLDL ELISA, be specifically related to a kind of colloid gold test paper that detects OxLDL ELISA.
Two. background technology:
According to the american heart association recent statistics data of calendar year 2001, angiocardiopathy has become human No.1 killer.And atherosclerotic 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.
Lipoprotein is the main carrier of plasma cholesterol and triglyceride.Low-density lipoprotein (LDL) is by phosphatide, apolipoprotein (Apo) B polypeptied chain, cholesterol, triglyceride, compositions such as carbohydrates.OxLDL ELISA (oxLDL) forms through the LDL oxidative modification.It is found that in recent years oxLDL and atherosclerotic have confidential relation.OxLDL impels the formation of foam cells, and the death of foam cells will cause the deposition of lipid at arterial wall, and this is to cause atherosclerotic main diseases therefore.Studies show that recently, in atherosclerotic lesion, found the oxLDL deposition, and do not had in the normal arterial wall that this belongs to the endemic element of atherosclerotic lesion.Therefore, the oxLDL amount in patient's body is far above the normal person, and its concentration and extent of disease are ratio.Clinically think that the content of oxLDL is 200-500 μ g/L in the healthy human body, and when clinical detection oxLDL content be unusual during greater than 600 μ g/L.Therefore the detection of oxLDL is significant to the early warning and the early stage auxiliary diagnosis of atherosclerotic cardiovascular disease.
The method that detects oxLDL at present is a lot, and 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.The defective of this method is: the operating process relative complex, and need special instrument such as microplate reader to operate, need the reviewer of specialty to operate; Running time is longer, and whole process need was above one hour; Testing cost is higher, and can not realize single branch detection etc.
The colloidal gold immunochromatographimethod technology is characterized in detecting single the branch as a kind of quick clinical diagnosis technology, and is convenient and swift, need not any instrument and equipment except that commercially available reagent, and a few minutes get final product the visual inspection result, and long shelf-life.
Chinese patent 200510200394.6 discloses a kind of OxLDL ELISA colloidal gold strip, and its amount according to OxLDL ELISA antibody capture antigen is set up the relation of color to concentration.Yet there is certain limitation in naked eyes to distinguishing of shade, and therefore the sensitivity to this kind detection method sxemiquantitative function forms a kind of restriction, and may be different to distinguishing of shade according to the individual, produce individual difference.
The object of the present invention is to provide a kind of special instruments and equipment that do not need, measure the method for low-density oxidation lipoprotein effectively, fast and accurately, can judge simply directly by the method whether OxLDL ELISA is higher than normal level in the human body.Effectively reduce the detection cost simultaneously, simplified operating process, shortened detection time.
Three. summary of the invention:
The present invention sets up a kind of method of measuring OxLDL ELISA content in the human body by competition law.By to the having, do not have of detection zone color, judge whether OxLDL ELISA is higher than normal level in the human body.Thereby the early warning and the early stage auxiliary diagnosis of atherosclerotic angiocardiopathy have been realized.
The present invention includes basic supporting pieces, and the absorption of sample pad that on described basic supporting pieces, is arranged in order, golden labeling antibody pad, chromatography developing film and adsorptive pads.
Basic supporting pieces as the PVC plate, plays fixing other ingredients of test paper of supporting for simultaneously scribbling the toughness material that do not absorb water of adhesive sticker.
The absorption of sample pad is the plain film of nonwoven fabrics or glass fibre.
Gold labeling antibody pad is provided with the plain film of glass fibre, and the plain film of this glass fibre is coated with golden labeling antibody.Above-mentioned golden labeling antibody is the monoclonal antibody or the polyclonal antibody of a certain amount of mouse source anti-oxidation low-density lipoprotein.
The chromatography developing film is nitrocellulose filter or cellulose acetate membrane.Set up successively in the colour developing district and detect band and quality control band.Detecting band is coated with as the antigen OxLDL ELISA.Quality control band is coated with the IgG of second kind of anti-mouse of animal.
Adsorptive pads is the plain film of filter paper or glass fibre.
Principle of the present invention is that bag is detected with the OxLDL ELISA of going up the bag quilt and becomes competitive relation with OxLDL ELISA in the sample by a certain amount of golden labeling antibody on the plain film of glass fibre.OxLDL ELISA combines with golden labeling antibody in the testing sample, forms gold mark antigen-antibody bond.The golden labeling antibody of gold mark antigen-antibody and conjugated antigen not since capillary action along the chromatographic film chromatography to detecting band.Do not develop the color if detect band, prove that then the OxLDL ELISA in the sample has surpassed limit value, promptly golden labeling antibody all with the combination of the OxLDL ELISA in the sample and can't with detection with on OxLDL ELISA combine.If detect the band colour developing, prove that then the OxLDL ELISA concentration in the sample does not reach limit value, not in conjunction with the golden labeling antibody of OxLDL ELISA in the sample and detection with on OxLDL ELISA combine colour developing.
The present invention has the following advantages:
1. detect accuracy rate height, high specificity.
2. detect fast: only need to get final product in 5 minutes observations.
3. easy to carry, simple to operate: detect without any need for special instrument or equipment, operation steps is simple, is convenient to medical personnel and carries, and also can detect and monitoring for long-term patient oneself, thereby reach forewarning function.
4. OxLDL ELISA detection test paper manufacture craft is simple, and cost is low.
5. but colloid gold test paper normal temperature is preserved down, need not specific apparatus and equipment.Storage life reaches 1 year, good reproducibility.
Four. description of drawings:
Figure one is the front schematic view of structure of the present invention and side schematic view.1 is adsorptive pads among the figure one, and 2 is quality control band, and 3 for detecting band, and 4 is the chromatography developing film, and 5 is golden labeling antibody pad, and 6 is the absorption of sample pad, and 7 is basic supporting pieces, as the PVC plate etc.
Figure two is use presentation of results figure of the present invention.A quality control band colour developing and detect band and do not develop the color among the figure two, positive result shows that OxLDL ELISA content is greater than or equal to 600 μ g/L in the sample, and is ill; B quality control band and all colour developings of detection band, negative result shows that OxLDL ELISA content is lower than normal value μ g/L in the sample, and is normal; C quality control band and detection band all do not develop the color, and show that test strips lost efficacy.
Five. specific embodiments:
Embodiment one
The OxLDL ELISA MONOCLONAL ANTIBODIES SPECIFIC FOR
With oxLDL (available from Beijing three friendly scientific and technological development companies that coordinate) immune mouse.Get the splenocyte and the murine myeloma cell hybridization of immune mouse and merge the preparation hybridoma.Select positive high hybridoma and clone, the preparation monoclonal hybridoma.Continue to cultivate for 20 generations, but screening and the final hybridoma cell strain of setting up the stably excreting monoclonal antibody.
Adopt inoculation hybridoma to the method for mouse peritoneal to prepare ascites, the method for extracting antibody again from ascites prepares monoclonal antibody.
Embodiment two
The method of Preparation of Colloidal Gold and labeled monoclonal antibody
Adopt trisodium citrate reduction method to produce colloid gold particle.Get 0.01%HAuCl
4Aqueous solution 100ml, heated and boiled.Add the water-soluble 2ml of 1% citric acid trisodium as required rapidly, continue to boil about 5min, claret occurs.Leave standstill cooling, make the colloid gold particle of the about 20nm of diameter like this.
Adopt cohesion colorimetric method for determining collaurum the suitableeest in conjunction with protein content.With 0.1Mol/L K
2CO
3Regulating collaurum pH is 9.0, is sub-packed in ten pipes, every pipe 1ml.Behind the gradient dilution protein concentration, add respectively in above-mentioned ten pipes, every pipe adds 1ml.Behind the 5min, in above-mentioned each pipe, add 1ml 10%NaCl solution, leave standstill 2h behind the mixing, survey its OD value.Can observe to draw according to the OD value and stablize the needed antibody amount of 1ml collaurum.
Use the colloid gold particle labelled antibody.Collaurum and certain density antibody-solutions are respectively with 0.1Mol/L K
2CO
3Transfer pH to 9.0.The electromagnetic agitation antibody-solutions dropwise adds colloidal gold solution.Continue to stir 10min, the stabilizing agent bovine serum albumin(BSA) (BSA) of adding 5% precipitates to prevent antibody protein and collaurum polymerization, stirs 10min again.
Antibody with supercentrifugation purifying colloid gold label.With the centrifugal 20min of 1200r/min, remove big polymkeric substance (can according to circumstances omit this step).Use 13000g, 4 ℃ of centrifugal 40min, careful sucking-off supernatant, sediment (contains 0.02%NaN with the PBS liquid that contains 1%BSA
3) resuspended, centrifugal repeatedly 3 times.With precipitating resuspended is 1/10,4 ℃ of preservation of original volume.Can be stored in 20 ℃ of preservations more than 1 year as in bond, adding 50% glycerine.
Embodiment three
Detect the preparation and the assembling of OxLDL ELISA colloidal gold strip
The absorption of sample pad: it is standby that glass fibre is cut into 1.9cm * 30cm size.
Gold pad: the glass fibre that will be cut into 0.5mm * 30cm soaked in certain density golden labeling antibody solution 2 hours, 37 ℃ of dryings 1 hour, and sealing, 4 ℃ of preservations are standby.
Chromatographic film: adopt nitrocellulose filter or cellulose acetate membrane, be cut into 2.5cm * 30cm size.From 1cm place, film lower end bag by certain density OxLDL ELISA as detecting band, bandwidth is 2mm, upwards wrap by the IgG of the anti-mouse of 1mg/ml animal (mostly being sheep anti mouse) as quality control band at 0.5cm place at interval.Naturally after drying in the shade, with containing 1%BSA, 0.01mol/L, the PBS sealing 2h of pH7.4, room temperature is dried in the shade, and seals standby.
Adsorptive pads: glass fibre torn open to be cut into 4cm * 30cm size standby.
With adsorptive pads, chromatographic film, gold fills up and the absorption of sample pad is overlapping successively from top to bottom is affixed on the PVC plate, and about 2mm is wide for overlapping.And from chromatographic film lower end 2mm beginning, comprise the gold pad and partially absorb pad, with the fixing connection each other of adhesive tape, and stick MAX line labeling.Sticking colour code for then the adsorptive pads surface pastes.At last, with being cut into the test strips sealing of 4mm, put into drying agent and preserve.
Embodiment four
The service instruction of colloidal gold strip
Sampling: took out forearm blood on an empty stomach 7 to 10 o'clock mornings, add antioxidant immediately, in case low-density lipoprotein is oxidized in external continuation.Get blood plasma after centrifugal and be equipped with inspection.
Detect: before detecting test paper is connected packing and take out, room temperature is placed about 5min, takes out test paper.The absorption of sample pad is immersed in the sample solution, and liquid level does not surpass MAX line labeling, perhaps keeps flat test strips on the worktable of cleaning, drips sample solution to the absorption of sample pad.Visual inspection result after 10 minutes.
Observe: do not develop the color if detect band, the quality control band colour developing proves that then the OxLDL ELISA in the sample has surpassed limit value, and is positive.If detect the band colour developing, the quality control band colour developing proves that then the OxLDL ELISA concentration in the sample does not reach limit value, and is negative.All do not develop the color if detect band and quality control band, then prove the test strips actual effect.
Claims (4)
1. half-quantitative detection trace OxLDL ELISA colloidal gold strip, this test strips comprises basic supporting pieces, and the adsorptive pads that is arranged in order at basic supporting pieces, chromatographic film, gold pad and absorption of sample pad.It is characterized in that fixing can only in conjunction with the golden labeling antibody of about 600 μ g/L concentration on the gold pad, adopting the indirect competition method, the OxLDL ELISA that is fixed on the chromatographic film is developed the color with the competition of the OxLDL ELISA in the testing sample.
2. a kind of half-quantitative detection trace OxLDL ELISA colloidal gold strip according to claim 1 is characterized in that setting up successively in the colour developing district and detects band and quality control band.Detecting band is coated with as the antigen OxLDL ELISA.Quality control band is coated with the IgG of second kind of anti-mouse of animal.The gold labeling antibody is the monoclonal antibody or the polyclonal antibody of a certain amount of mouse source anti-oxidation low-density lipoprotein.
3. a kind of half-quantitative detection trace OxLDL ELISA colloidal gold strip according to claim 1 is characterized in that basic supporting pieces is for simultaneously scribbling the toughness material that do not absorb water of adhesive sticker, as the PVC plate.Adsorptive pads is the plain film of filter paper or glass fibre.Chromatographic film is nitrocellulose filter or cellulose acetate membrane.The gold pad is coated with golden labeling antibody for the plain film of glass fibre, the plain film of this glass fibre.The absorption of sample pad is the plain film of nonwoven fabrics or glass fibre.
4. a kind of half-quantitative detection trace OxLDL ELISA colloidal gold strip according to claim 1, but it is characterized in that this test strips half-quantitative detection human serum or blood plasma OxLDL ELISA content, thereby be applied to the early stage auxiliary clinical diagnosis of atherosclerotic cardiovascular disease.
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| CNA2007102020847A CN101183108A (en) | 2007-10-16 | 2007-10-16 | Colloidal gold test paper for detecting trace quantity oxygenize low density lipoprotein by indirect competition method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104101581A (en) * | 2013-04-11 | 2014-10-15 | 南昌大学 | Apparatus for rapidly detecting allergic peanut proteins in food, and making method thereof |
| CN104459152A (en) * | 2014-12-03 | 2015-03-25 | 青岛拓新生物科技有限公司 | Rapid sperm concentration detection method based on colloidal gold immunochromatograohic assay technology |
| 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 |
| CN106170699A (en) * | 2014-04-04 | 2016-11-30 | 田中贵金属工业株式会社 | Immunochromatographiassays assays method |
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| CN1523355A (en) * | 2003-02-20 | 2004-08-25 | 刘凤鸣 | Process for detecting grease peroxide |
| WO2004097419A1 (en) * | 2003-04-25 | 2004-11-11 | Biodigit Laboratories Corp. | Membrane strip biosensor system for point-of-care testing |
| CN1896740A (en) * | 2005-07-15 | 2007-01-17 | 兰州大学 | Colloidal gold test strip for semi-quantitatively and rapidly detecting oxidized low-density lipoprotein and preparation method thereof |
| CN101004421A (en) * | 2007-01-25 | 2007-07-25 | 兰州大学 | Test paper for semi quantitative detecting oxLDL |
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- 2007-10-16 CN CNA2007102020847A patent/CN101183108A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1523355A (en) * | 2003-02-20 | 2004-08-25 | 刘凤鸣 | Process for detecting grease peroxide |
| WO2004097419A1 (en) * | 2003-04-25 | 2004-11-11 | Biodigit Laboratories Corp. | Membrane strip biosensor system for point-of-care testing |
| CN1896740A (en) * | 2005-07-15 | 2007-01-17 | 兰州大学 | Colloidal gold test strip for semi-quantitatively and rapidly detecting oxidized low-density lipoprotein and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104101581A (en) * | 2013-04-11 | 2014-10-15 | 南昌大学 | Apparatus for rapidly detecting allergic peanut proteins in food, and making method thereof |
| CN106170699A (en) * | 2014-04-04 | 2016-11-30 | 田中贵金属工业株式会社 | Immunochromatographiassays assays method |
| CN104459152A (en) * | 2014-12-03 | 2015-03-25 | 青岛拓新生物科技有限公司 | Rapid sperm concentration detection method based on colloidal gold immunochromatograohic assay technology |
| 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 |
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Application publication date: 20080521 |