CN101140286B - Colloid selenium test paper of semi-quantitative determination oxidized low density lipoprotein - Google Patents
Colloid selenium test paper of semi-quantitative determination oxidized low density lipoprotein Download PDFInfo
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- CN101140286B CN101140286B CN200710202086.6A CN200710202086A CN101140286B CN 101140286 B CN101140286 B CN 101140286B CN 200710202086 A CN200710202086 A CN 200710202086A CN 101140286 B CN101140286 B CN 101140286B
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Abstract
The present invention provides a piece of semiquantitative detecting oxide low-density lipoprotein (OX-LDL) colloid selenium reagent paper, which can capture antigen quantity according to OX-LDL monoclonal antibody to create a relation between color and standard OX-LDL and diagnose that content of OX-LDL in human blood plasma is higher than, lower that or equal to standard OX-LDL quantity in human blood plasma, thus realizing early auxiliary diagnosis to ASCVD. The present invention has the advantages of convenience, quickness, sensitivity, specificity and economic efficiency, rejects specified instruments and equipments and is conveniently taken.
Description
One, technical field
The present invention relates to a kind of energy fast, half-quantitative detection goes out the colloid selenium test paper of OxLDL ELISA in plasma sample and content thereof, can be applicable to the early stage auxiliary clinical diagnosis of atherosclerotic cardiovascular disease.
Two, background technology
Much research shows that oxidized low-density lipoprotein (OX-LDL) has atherogenicity (AS) effect.And atherosclerotic is the pathologic basis in early stage that the angiocardiopathies such as coronary heart disease, high fat of blood, angina pectoris, myocardial infarction occur.First the effect of OX-LDL in AS be to promote foam cells to form, and forms the effect of playing in early days similar inflammatory mediator at AS.Next is that OX-LDL has the cytotoxicity of height to vascular endothelial cell (VEC), vascular smooth muscle cell (VSMC), change VEC form and structure and destroy the integrality of endothelium, make the composition such as monocyte and LDL in blood enter subendothelial layer, promote AS to occur.In blood OX-LDL increase can accelerate that coronary atherosclerosis patch be combined with macrophage, foam cells formation and the increase of patch lactone core, cause atheromatous plaque unstable.Many studies show that, in hypertension, high fat of blood and hyperglycemia human plasma, OX-LDL has obviously and increases, and increases gradually with the progress of the state of an illness.These researchs are enough to illustrate that blood plasma OX-LDL increases with incidence and the mortality ratio of atherosclerotic cardiovascular disease (ASCVD) and has substantial connection.
Clinical research finds that in healthy human body, OX-LDL content, between 0.2～0.5 μ g/ml, thinks abnormal when detecting clinically when OX-LDL content is greater than 0.6 μ g/ml.Because OX-LDL is distinctive composition in AS focus, its content is far above normal person; At AS different times OX-LDL content, there is significant difference; pathology is more to late period; the content of OX-LDL is higher, so it is better than conventional blood fat project to the specificity of ASCVD auxiliary diagnosis, therefore the detection of OX-LDL is extremely important to the early stage auxiliary diagnosis of ASCVD.
The method that detects at present OX-LDL is a lot, nuclear magnetic resonance, apoB fluoroscopic examination, enzyme linked immunosorbent assay (ELISA) detection etc., and wherein the most frequently used is the content that ELISA method detects serum OX-LDL.ELISA method is to take competitiveness enzyme-linked reaction as detecting principle, with OX-LDL antibody coated elisa plate, during detection, test sample and enzyme mark bond is added to ELISA Plate simultaneously, surveys OD value carry out result of determination after reaction by colour developing.But the shortcoming that the method exists is: (1) needs special instrument and equipment to be used in conjunction with as microplate reader; (2) detect operating personnel and need to pass through professional training; (3) operating process is relatively complicated, detects required time long; (4) detect required expense higher, can not realize single part of detection.Therefore, have at present many researchers sight to be concentrated on to the colloidal gold strip that detects OxLDL ELISA in human serum, for example patent of invention 200510200394.6, but the preparation method of gold grain is more loaded down with trivial details, need heating, consuming time longer, and the protein content of mark is more.So the present invention has done significant improvement, changed the colloid gold particle of labelled antibody albumen into electroselenium particle.
Electroselenium immunochromatography technique, as a kind of quick clinical diagnosis technology, is characterized in single part of detection, convenient and swift, and except commercially available reagent, without any instrument and equipment, a few minutes get final product visual inspection result, and long shelf-life.The colloid selenium test paper of semi-quantitative determination oxidized low density lipoprotein of the present invention had both had the high sensitivity of ELISA enzyme process, had again sxemiquantitative function, can replace conventional machines and detect quantitative status, significantly reduced testing cost.Content for OxLDL ELISA in external Rapid Determination of Plasma is highly significant.Row is for colloid gold test paper, colloid selenium test paper has following advantage: the preparation of electroselenium particle can be carried out at normal temperatures, do not need heating, reaction time is short, output is high, and electroselenium particle is highly sensitive, labelled protein amount ratio conventional method consumption is low, has reached convenience, saves time, highly sensitive feature.In addition, colloidal selenium marked protein is a physical bond process, in conjunction with firmly, seldom causes that protein active changes, so test paper is more stable, can at room temperature preserve more than 1 year.
Three, summary of the invention
The object of the invention is to overcome the defect that prior art exists, provide a kind of high specificity, highly sensitive, easy and simple to handle, detect and be specifically designed to fast and accurately rapid semi-quantitative and detect immune colloid selenium test paper of OxLDL ELISA (OX-LDL) content in human plasma and preparation method thereof.
Technology path of the present invention is described below:
1) preparation of electroselenium;
2) preparation of colloidal selenium marked thing: electroselenium and OX-LDL monoclonal antibody 1 mix by a certain percentage, makes electroselenium and antibody form monoclonal antibody 1-colloidal selenium marked thing.Select OX-LDL monoclonal antibody 1 as selenium labelled antibody, by little molecule monoclonal antibody distilled water dialysed overnight, adjusting its concentration is 1mg/ml, with 0.22 μ m filtering with microporous membrane, gets selenium sol solution 100ml, and adjusting its pH is 8.2, with 0.22 μ m filtering with microporous membrane.Under electromagnetic agitation, to dropwise adding in selenium sol solution the need of 2.5ml dilution to examine little molecule monoclonal antibody, after 10min, adding 5% bovine serum albumin(BSA) (BSA) to make its final concentration is 1%, continues to stir 10min.The electroselenium that mark is good is dialysed with sucrose, be concentrated into 1/10 of original volume, again with the centrifugal 30min of 1500rpm, supernatant joins during propylene glucosan S-400 gel leans on, with 0.02mol/l pH8.2 TBS, (include 1%BSA, 0.05% Sodium azide) wash-out, the liquid while collecting color peak value, 4 ℃ save backup;
3) application of sample film, glass fibre element film, nitrocellulose filter, thieving paper, PVC backing are cut into prescribed level;
4) the good selenium mark monoclonal antibody 1 of mark is combined on glass fibre element film;
5) on nitrocellulose filter, with sample applicator point sample, become two lines, respectively as detecting band and contrast quality control band;
6) application of sample film, the glass fibre element film, nitrocellulose filter, the thieving paper that are combined with selenium mark monoclonal antibody are attached on PVC plate successively.
Concrete making and assembly method reference embodiment below, its style is referring to Figure of description 1.
Compared with prior art, the present invention has the following advantages:
1) take electroselenium as indicator, itself forms the color mark after immunoassay process, and testing process is quick rapidly, and result is objective, and naked eyes are easy to judgement, can in minutes obtain a result;
2) specificity is good, accurately sensitive, realizes half-quantitative detection, and result is subject to exogenous influence less, good stability;
3) do not need any instrument and equipment, testing cost is low, easy and simple to handle, and detection time is short;
4) with low cost, sample does not need special processing, and required reagent and sample size are few;
5) test strip of the present invention stores conveniently, less demanding to storage temperature.
Four, accompanying drawing explanation
Fig. 1 is structure front elevation (left side) and the side view (right side) of test strip of the present invention.In Fig. 1,1 is PVC plate, and 2 is suction zones, and 3 for detecting band, and 4 is nitrocellulose filter, and 5 is sxemiquantitative quality control band, and 6 is selenium mark pad, and 7 is sample detecting area.
Fig. 2 is that test strip testing result of the present invention is judged schematic diagram.The negative result of A, E in Fig. 2, shows oxygen-freeization low-density lipoprotein in sample; The positive result of B, F, shows that in sample, OxLDL ELISA content is equal to or higher than 0.6 μ g/ml, ill; The positive result of C, G, shows to contain in sample OxLDL ELISA, but content is lower than 0.6 μ g/ml, normal; D is that test strips lost efficacy.
Five, embodiment
Test strips of the present invention is by application of sample film, label pad, and reaction film, thieving paper, PVC backing forms.Test strips of the present invention is divided into 5 functional areas: sample detecting area, selenium mark monoclonal antibody land, sxemiquantitative contrast district, detection zone, suction zones.By the OX-LDL monoclonal antibody 1 of colloidal selenium marked, be coated in selenium mark monoclonal antibody land, OX-LDL monoclonal antibody 2 (or the anti-mouse IGg of rabbit) is coated on detection zone, do to detect band, a certain amount of OxLDL ELISA is coated on sxemiquantitative contrast district, makes quality control band.This test strips is utilized immunochromatography technique principle, judges the semi-quantitative results of detection by the power of detection zone and sxemiquantitative contrast district color contrast, can judge rapidly having or not and content height of determined antigen in sample.Color contrast is stronger, and in sample, OX-LDL concentration is higher, and ill possibility is larger, and lesion degree is darker.Thereby atherosclerotic cardiovascular disease (ASCVD) is carried out to early stage auxiliary diagnosis.
Embodiment mono-: detect the method for making with the colloid selenium test paper of the semi-quantitative determination oxidized low density lipoprotein of upper spraying OX-LDL monoclonal antibody 2:
1, the preparation of electroselenium particle
In reactor, add successively ascorbic acid, selenous acid and ascorbic acid, from reaction, start interval the 4th minute and drip selenous acid and ascorbic acid, then carry out spectral scan, to occur between 500～600nm that maximum absorption band is advisable, add 10% glucosan cessation reaction, collect product standby.
2, the preparation of monoclonal antibody
Use OX-LDL immune balb/c mice, the splenocyte and the small white mouse myeloma cell that get immune mouse are hybridized fusion, prepare hybridoma, screening can stably excreting OX-LDL monoclonal antibody cell line in vitro culture to certain density, then induced ascites is collected in the abdominal cavity that is inoculated into BALB/C mice, by ion-exchange, gel filtration or affinity chromatography method, obtain the OX-LDL monoclonal antibody 1,2 of purifying, and measure tiring of monoclonal antibody, monoclonal antibody 1,2, for antigenic determinants different on antigen, therefore can not be bonded to each other.
3, the preparation of colloidal selenium marked monoclonal antibody 1
Electroselenium and OX-LDL monoclonal antibody 1 mix by a certain percentage, make electroselenium and antibody form monoclonal antibody 1-colloidal selenium marked thing.Select OX-LDL monoclonal antibody 1 as selenium labelled antibody, by little molecule monoclonal antibody distilled water dialysed overnight, adjusting its concentration is 1mg/ml, with 0.22 μ m filtering with microporous membrane, gets selenium sol solution 100ml, and adjusting its pH is 8.2, with 0.22 μ m filtering with microporous membrane.Under electromagnetic agitation, to dropwise adding in selenium sol solution the need of 2.5ml dilution to examine little molecule monoclonal antibody, after 10min, adding 5% bovine serum albumin(BSA) (BSA) to make its final concentration is 1%, continues to stir 10min.The electroselenium that mark is good is dialysed with sucrose, be concentrated into 1/10 of original volume, again with the centrifugal 30min of 1500rpm, supernatant joins during propylene glucosan S-400 gel leans on, with 0.02mol/L pH8.2 TBS, (include 1%BSA, 0.05% Sodium azide) wash-out, the liquid while collecting color peak value, 4 ℃ save backup.
4, selenium mark monoclonal antibody 1 is combined on selenium mark pad
First the glass fibre element film of long 10mm is soaked to 30min with the PBS damping fluid containing 1%BSA and 1%Tween-20, after 37 ℃ of drying boxes are dry, then be soaked in selenium mark monoclonal antibody 1 solution, after the upper selenium mark monoclonal antibody 1 of glass fibre element film absorption, it is natural drying.
5, the preparation of reaction film
Use some film machine, the OX-LDL monoclonal antibody 2 of the OX-LDL of the 0.6 μ g/ml having diluted with PBS and 6 μ g/ml is sprayed into line on nitrocellulose filter, the width of line is 1mm, respectively as quality control band and detection band.Quality control band square nitrocellulose filter lower end 25mm, detect band apart from nitrocellulose filter upper end 15mm, quality control band and detecting between band at a distance of 20mm, select the PBS damping fluid sealing 2h that is 1%BSA containing mass percent concentration for excellent reaction film, with the washing of 0.01mol/LPBS damping fluid, natural drying 2h; The nitrocellulose filter being coated with is cut into 50mm long, the rectangular strip that 5mm is wide.
6, the assembling of test strips
The PVC plastic plate that cutting 80mm is long, is fixed on the nitrocellulose filter of handling well on PVC plate with powerful double faced adhesive tape, and nitrocellulose filter upper limb is apart from PVC plastic plate upper end 20mm, the handle of using during as detection.Then in the suction zones of nitrocellulose filter, paste the long senior absorbent filter of 10mm successively, the 10mm grade absorbent filter that grows tall is pasted in sample detecting area, in selenium mark monoclonal antibody land, paste the glass fibre element film that is combined with selenium mark monoclonal antibody, be finally cut into the test strips that width is 5mm.The whole range request of crossing is avoided being infected with of other albumen.The colloid selenium test paper Vacuum Package of the semi-quantitative determination oxidized low density lipoprotein of making, 4～8 ℃ of kept dry are standby.
Embodiment bis-: detection is with the method for making of the colloid selenium test paper of the semi-quantitative determination oxidized low density lipoprotein of upper spraying rabbit anti-mouse igg.
1, the preparation of electroselenium particle
In reactor, add successively ascorbic acid, selenous acid and ascorbic acid, from reaction, start interval the 4th minute and drip selenous acid and ascorbic acid, then carry out spectral scan, to occur between 500～600nm that maximum absorption band is advisable, add 10% glucosan cessation reaction, collect product standby.
2, the preparation of monoclonal antibody
Use OX-LDL immune balb/c mice, the splenocyte and the small white mouse myeloma cell that get immune mouse are hybridized fusion, prepare hybridoma, the cell line that screening can stably excreting OX-LDL monoclonal antibody is prepared the monoclonal antibody of anti-OXLDL, and measures tiring of monoclonal antibody.
3, the preparation of rabbit anti-mouse igg polyclonal antibody
First the serum of Balb/c mouse is adopted in addition purifying of caprylic acid-ammonium, to obtain purer mouse IgG.Detailed process is: a serum after centrifugal dilutes with 2 parts of 0.01M pH7.4 PBS, adds while stirring 50% saturated ammonium sulfate, clarifies 2 hours for 4 degrees Celsius, and the centrifugal 30min of 13000r/min, abandons precipitation.Supernatant adjust pH to 7.4.Add again 35% saturated ammonium sulphate protein, repeat twice.Sediment dissolves with a small amount of PBS, 4 degrees Celsius of PBS dialysed overnight, during change liquid 3-5 time, then using and obtain purer mouse IgG as antigen, add appropriate adjuvant, the rabbit of immune health, get serum again with caprylic acid-ammonium purifying in addition, make rabbit anti-mouse igg polyclonal antibody.
4, the method for colloidal selenium marked OX-LDL monoclonal antibody
Electroselenium and OX-LDL monoclonal antibody mix by a certain percentage, make electroselenium and antibody form monoclonal antibody colloid selenium label.Select OX-LDL monoclonal antibody as selenium labelled antibody, by OX-LDL monoclonal antibody distilled water dialysed overnight, adjusting its concentration is 1mg/ml, with 0.22 μ m filtering with microporous membrane, gets selenium sol solution 100ml, and adjusting its pH is 8.2, with 0.22 μ m filtering with microporous membrane.Under electromagnetic agitation, to the OXLDL monoclonal antibody that dropwise adds 2.5ml dilution in selenium sol solution, after 10min, adding 5% bovine serum albumin(BSA) (BSA) to make its final concentration is 1%, continues to stir 10min.The electroselenium that mark is good is dialysed with sucrose, be concentrated into 1/10 of original volume, again with the centrifugal 30min of 1500rpm, supernatant joins during propylene glucosan S-400 gel leans on, with 0.02mol/l pH8.2 TBS, (include 1%BSA, 0.05% Sodium azide) wash-out, the liquid while collecting color peak value, 4 ℃ save backup.
5, selenium mark monoclonal antibody is combined on glass fibre element film
Choose the selenium mark OX-LDL monoclonal antibody that mark is good, the amount of this selenium labeling antibody can be combined with 0.6 μ g/ml OX-LDL just; First the glass fibre element film of long 10mm is soaked to 30min with the PBS damping fluid containing 1%BSA and 1%Tween-20, after 37 ℃ of drying boxes are dry, be soaked in again in ready selenium mark monoclonal antibody solution, after the upper selenium mark monoclonal antibody of glass fibre element film absorption, it is natural drying.
6, nitrocellulose filter is coated
With special spraying equipment, by OX-LDL setting-out on nitrocellulose filter of the rabbit that is prepared into accurate concentration after purifying anti-mouse IGg solution and the 0.6 μ g/ml that diluted with PBS, the width of line is 1mm, respectively as detecting band and quality control band.Quality control band square nitrocellulose filter lower end 25mm, detects band apart from nitrocellulose filter upper end 15mm, quality control band and detecting between band at a distance of 20mm.The PBS damping fluid sealing 2h that is 1%BSA containing mass percent concentration for the nitrocellulose filter being coated with, with the washing of 0.01mol/LPBS damping fluid, natural drying 2h; The nitrocellulose filter being coated with is cut into 50mm long, the rectangular strip that 5mm is wide.
7, the assembling of test strips
The PVC plastic plate that cutting 80mm is long, is fixed on the nitrocellulose filter of handling well on PVC plate with powerful double faced adhesive tape, and nitrocellulose filter upper limb is apart from PVC plastic plate upper end 20mm, the handle of using during as detection.Then in the suction zones of nitrocellulose filter, paste the long senior absorbent filter of 10mm successively, the 10mm grade absorbent filter that grows tall is pasted in sample detecting area, in selenium mark monoclonal antibody land, paste the glass fibre element film that is combined with selenium mark monoclonal antibody, finally cut into the test strips that width is 5mm.The whole range request of crossing is avoided being infected with of other albumen.The colloid selenium test paper Vacuum Package of the semi-quantitative determination oxidized low density lipoprotein of making, 4～8 ℃ of kept dry are standby.
Embodiment tri-: the using method of colloid selenium test paper of the present invention
1, collected specimens:
The morning, take out on an empty stomach forearm blood during 7-10, add immediately antioxidant, in case low-density lipoprotein continues oxidation in vitro, get blood plasma after centrifugal and put-30 ℃ of OX-LDL to be checked.
2, detect
Test paper is connected to packing and take out, first at room temperature place about 5 minutes.Take out test paper ，Jiang Tanyang district and be dipped in sample solution, sample liquid level does not surpass Guo Tanyang district.After 3～5 seconds, take out, lie in a horizontal plane on clean operator's console.In 6～10 minutes, can see result by naked eyes.
(1) for detecting, be with the colloid selenium test paper testing result of the semi-quantitative determination oxidized low density lipoprotein that is coated with OX-LDL monoclonal antibody 2 as follows: sxemiquantitative quality control band sees that blue explanation testing result is negative if detection band is not seen blueness, testing sample does not detect the OxLDL ELISA that can suspect sufferer, test strips device quality intact (as shown in A in accompanying drawing 2); If detection is deeper than sxemiquantitative quality control band with color and shows that the OxLDL ELISA existing in sample, higher than 0.6 μ g/ml level, represents ill (as shown in B in accompanying drawing 2); The OxLDL ELISA existing in the little sample of color distortion, may be ill close to the critical level of 0.6 μ g/ml; If detection is with color lower than sxemiquantitative band, the OxLDL ELISA existing in bright sample, lower than 0.6 μ g/ml level, represents normal (as shown in C in accompanying drawing 2); If detection band is not seen blueness, sxemiquantitative quality control band does not see that blue explanation test strips had lost efficacy yet, can not be for detection of (as shown in D in accompanying drawing 2).
(2) for detecting band, to be coated with the colloid selenium test paper testing result of semi-quantitative determination oxidized low density lipoprotein of rabbit anti-mouse igg as follows: if detect that band is not seen blueness, sxemiquantitative quality control band presents blue explanation testing result and is negative, and testing sample does not contain the OxLDL ELISA (as shown in schematic diagram E in accompanying drawing 2) that can suspect sufferer; If sample detection band occurs blue, the colourless proof of sxemiquantitative quality control band detects positive, the OxLDL ELISA existing in interpret sample is equal to or higher than 0.6 μ g/ml level (as shown in schematic diagram F in accompanying drawing 2) ill possibility or ill, should give and payes attention to and watch out for; If detect band and sxemiquantitative quality control band, all occurs blueness, in interpret sample, contain OxLDL ELISA, but content is lower than 0.6 μ g/ml level, be not enough to ill, normally (as shown in schematic diagram G in accompanying drawing 2); If detection band is not seen blueness, sxemiquantitative band is not seen blue explanation test strips failure of apparatus yet, can not be for detection of (as shown in schematic diagram D in accompanying drawing 2).
Claims (2)
1. the colloid selenium test paper of a semi-quantitative determination oxidized low density lipoprotein, it is characterized in that this test strips comprises PVC plate, detects band, nitrocellulose filter, sxemiquantitative quality control band, selenium mark pad, test strips is divided into 5 functional areas, sequentially arrange 5 functional areas: suction zones, detection zone, selenium mark monoclonal antibody land, sxemiquantitative contrast district, sample detecting area; By the OX-LDL monoclonal antibody of colloidal selenium marked, be coated in selenium mark monoclonal antibody land; Rabbit anti-mouse igg is coated on detection zone, makes to detect band; A certain amount of OxLDL ELISA is coated on sxemiquantitative contrast district, makes sxemiquantitative quality control band.
2. according to the colloid selenium test paper of a kind of semi-quantitative determination oxidized low density lipoprotein described in claim 1, the sxemiquantitative quality control band arranging on the nitrocellulose filter described in it is characterized in that is two parallel segments that the spacing that the injection of film machine forms is 20mm with detecting band.
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| CN101504417B (en) * | 2008-10-22 | 2013-08-28 | 李红玉 | Colloid selenium test paper for semi-quantitative determination of urinary trace albumin |
| CN101451993B (en) * | 2009-01-09 | 2013-07-17 | 王贤俊 | Anti-Lp (alpha)antibody preparation and diagnosis kit for detecting Lp (alpha) |
| CN102192980B (en) * | 2010-03-08 | 2014-04-09 | 苏州浩欧博生物医药有限公司 | Nonmetallic colloidal particle immune analysis reagent and method |
| CN104807999A (en) * | 2014-01-28 | 2015-07-29 | 兰州大学 | Electroselenium rapid detection test strip |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5229073A (en) * | 1992-02-27 | 1993-07-20 | Abbott Laboratories | One-step competitive immunoassay for the semiquantitative determination of plasma lipoprotein(a) |
| CN101004419A (en) * | 2007-01-25 | 2007-07-25 | 兰州大学 | Colloid selenium test paper for detecting pulpy kidney, and type of pathogenesis bacteria |
| CN101004421A (en) * | 2007-01-25 | 2007-07-25 | 兰州大学 | Test paper for semi quantitative detecting oxLDL |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5229073A (en) * | 1992-02-27 | 1993-07-20 | Abbott Laboratories | One-step competitive immunoassay for the semiquantitative determination of plasma lipoprotein(a) |
| CN101004419A (en) * | 2007-01-25 | 2007-07-25 | 兰州大学 | Colloid selenium test paper for detecting pulpy kidney, and type of pathogenesis bacteria |
| CN101004421A (en) * | 2007-01-25 | 2007-07-25 | 兰州大学 | Test paper for semi quantitative detecting oxLDL |
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