CN102680710B - Enzyme-linked immunosorbent assay kit for human oxidized low-density lipoprotein, and using method and application - Google Patents
Enzyme-linked immunosorbent assay kit for human oxidized low-density lipoprotein, and using method and application Download PDFInfo
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Abstract
The invention provides an enzyme-linked immunosorbent assay (ELISA) kit for human oxidized low-density lipoprotein, which comprises the following ingredients: (1) an ELISA plate coated with an anti-human oxidized low-density lipoprotein antibody; (2) serial standard solution of human oxidized low-density lipoprotein; (3) an enzyme-labeled antibody; (4) diluting solution; (5) washing solution; (6) substrate solution; and (7) stopping solution. The invention further provides a method for using the kit and application of the kit. Compared with the traditional detection means for coronary heart disease, the kit has the characteristics of no wound, good simplicity, convenience and rapidness, high sensitivity and low cost.
Description
Technical field
The present invention relates to a kind of detection kit, be specifically related to detect the kit of OxLDL content in sample to be checked.
Background technology
Coronary heart disease, coronary atherosclerotic heart disease, the main basis of its diagnosis is patient's typical history, clinical symptoms, and carries out in conjunction with instrument and biochemical analysis.Conventional detection methods has routine electrocardiogram, electrocardiogram stress test, echocardiogram, spiral CT and UFCT Ultra-fast Computed Tomograph, coronary artery ultrasonoscopy, intravascular ultrasound, Nuclear Cardiac Imaging and selective coronary arteriography at present, and laboratory examination comprises Myocardial Enzymologic inspection, troponin, c reactive protein, blood fat and lipoprotein inspection etc.In above detection methods, great majority are that the electrocardiosignal detecting after myocardial ischemia or myocardial infarction changes (various cardiogram) and ventricular wall motion metamorphosis (echocardiogram, cardiac radionuclide measurement, spiral CT and UFCT Ultra-fast Computed Tomograph, magnetic resonance imaging) for carrying out the diagnosis of coronary heart disease in basis, if degree of stenosis not yet causes myocardial ischemia, above detection means often can not contribute to diagnosis of coronary heart disease.
Selective coronary arteriography can be directly acquainted with the degree of coronary artery stenosis, be considered at present the goldstandard of diagnosis of coronary heart disease, but it belongs to traumatic inspection, there is certain danger, need to be in hospital, and expensive, for need to row surgical intervention or the patient of interventional therapy be essential inspection, and have the patient of coronary heart disease for light disease patients with coronary heart disease or suspection, and be often difficult to accept, need to find traumatic little detection method.
Research shows, OxLDL ELISA OxLDL is relevant with the generation development of coronary heart disease, it has been generally acknowledged that and can, by detecting the content of OxLDL ELISA OxLDL in sample serum to be checked, assess the risk of suffering from coronary heart disease.In prior art, some relevant detection methods are disclosed, as: number of patent application: 03109935.1, denomination of invention: with the quantitative measurement of anti-phosphocholine antibody to OxLDL ELISA and the application in diagnosing atherosclerotic thereof.The quantitative detecting method of this disclosure of the invention comprises the following steps: that (a) contacts the antibody of anti-phosphocholine with the sample that contains OxLDL; (b) described antibody is combined with OxLDL; (c) measure the binding capacity of described antibody and OxLDL; (d) according to the typical curve recording taking phosphocholine as standard items, quantitatively OxLDL content.Because of antigen-antibody reaction very complicated, relevant with several factors, although the epiope of phosphocholine is identical with the epiope that low-density lipoprotein presents through oxidative modification rear surface, but the degrees of specificity of the antigen-antibody reaction of anti-phosphocholine antibody and OxLDL ELISA is difficult to prediction, not high enough with the accuracy of anti-phosphocholine antibody test OxLDL ELISA, be difficult to be applied to clinical detection.Application number is: 200710202084.7, denomination of invention: a kind of detecting trace quantity oxygenize low density lipoprotein by indirect competition method colloidal gold strip, disclose a kind of half-quantitative detection trace OxLDL ELISA and replaced gold test paper strip, comprise basic supporting pieces, this test strips comprises basic supporting pieces, and the adsorptive pads of once arranging at basic supporting pieces, chromatographic film, gold pad and absorption of sample pad, it is characterized in that fixing can only be in conjunction with the golden labeling antibody of approximately 600 μ g concentration on gold pad, adopt indirect competitive, it is the OxLDL ELISA competition colour developing in OxLDL ELISA and the sample to be checked being fixed in chromatographic film.This detection method is half-quantitative detection, is difficult to accurately detect OxLDL ELISA content, is more difficult to the possibility that Accurate Prediction sample to be checked is suffered from coronary heart disease.
Therefore, also do not have clinically at present a kind of easy, quick, without wound, the diagnosis of coronary heart disease product that cheap and accuracy is high, develop new coronary heart disease detection methods very necessary.
Summary of the invention
Technical scheme of the present invention has been to provide a kind of enzyme-linked immunologic detecting kit of people's OxLDL ELISA, in order to further diagnosis coronary atherosclerotic heart disease.Another technical scheme of the present invention provides using method and the purposes of this kit.
An enzyme-linked immunologic detecting kit for people's OxLDL ELISA, it comprises following composition:
(1) be coated with the ELISA Plate of anti-human OxLDL ELISA antibody;
(2) people's OxLDL ELISA series standard product:
Standard items 1: every liter of standard items 1 contain 20g bovine serum albumin(BSA), 50g sucrose;
Standard items 2: every liter of standard items 2 contain 20g bovine serum albumin(BSA), 50g sucrose and 1U OxLDL ELISA;
Standard items 3: every liter of standard items 3 contain 20g bovine serum albumin(BSA), 50g sucrose and 2U OxLDL ELISA;
Standard items 4: every liter of standard items 4 contain 20g bovine serum albumin(BSA), 50g sucrose and 4U OxLDL ELISA;
Standard items 5: every liter of standard items 5 contain 20g bovine serum albumin(BSA), 50g sucrose and 8U OxLDL ELISA;
Standard items 6: every liter of standard items 6 contain 20g bovine serum albumin(BSA), 50g sucrose and 16U OxLDL ELISA;
(3) enzymic-labelled antibody: be the goat-anti human apolipoprotein b antibody of peroxidase or horseradish peroxidase mark;
(4) dilution;
(5) cleansing solution;
(6) substrate solution;
(7) stop buffer.
Wherein, described kit also comprises Quality Control thing:
High Quality Control thing: every rising Quality Control thing contains 20g bovine serum albumin(BSA), 50g sucrose and 13U OxLDL ELISA;
Low Quality Control thing: every liter low Quality Control thing contains 20g bovine serum albumin(BSA), 50g sucrose and 1.5U OxLDL ELISA.
Wherein, the described ELISA Plate that is coated with anti-human OxLDL ELISA antibody of step (1) is prepared as follows:
A, antibody dilution: anti-human OxLDL ELISA monoclonal antibody is diluted to 2.5 ~ 20 μ g/ml by the Tris-HCl buffer solution of the 50mM that is 8.0 with pH, obtains coating buffer;
B, coated: get microwell plate, with cleansing solution washing 3 times, add the above-mentioned coating buffer containing anti-human OxLDL ELISA monoclonal antibody, 100 μ l/ holes, every hole, hatch 12 hours for 4 DEG C;
C, sealing: the coating buffer that inclines, be placed on thieving paper and pat several times, remove raffinate, the Tris-HCl confining liquid that to add containing the concentration of 1% bovine serum albumin(BSA) and 5% sucrose be 50mmol/L, its pH is 8.0,300 μ l/ holes,, room temperature, 1 hour;
D, vacuum drying, seal, and must be coated with the ELISA Plate of anti-human OxLDL ELISA antibody.
Preferably, described anti-human OxLDL ELISA monoclonal antibody be by preserving number be CCTCCNO:C 200304 hybridoma cell line produce.
Wherein, the preparation method of described OxLDL ELISA is as follows:
I, get low-density lipoprotein, be dissolved in 10mM phosphate buffer, wherein, the concentration of low-density lipoprotein is 5mg/ml;
II, get the phosphate buffer of step I, add copper sulphate, making its final concentration is 2 μ mol/L, 37 DEG C of reactions 2 hours;
III, add ethylenediamine tetraacetic acid again, making its final concentration is 0.01%(w/v), cessation reaction, dialysis, obtains OxLDL ELISA.
Wherein, described dilution be every liter containing NaCl 40g, NaH
2pO
42H
2o 1.184g, Na
2hPO
412H
2o 11.6g, the aqueous solution of bovine serum albumin(BSA) 50g;
Described cleansing solution is every liter and contains NaCl 68g, NaH
2pO
42H
2o 6.22g, Na
2hPO
412H
2o61g, the aqueous solution of Tween-2021ml;
Described substrate solution comprises substrate solution A and substrate solution B, and substrate solution A is the aqueous solution that contains TMB, and substrate solution B contains H
2o
2aqueous solution;
Described stop buffer is every liter of aqueous solution that contains concentrated sulphuric acid 108ml.
The present invention also provides the using method of aforesaid kit, and it comprises the following step:
I, sample pre-treatments;
II, detect with aforesaid kit;
III, result treatment and analysis.
Preferably, it comprises the following step:
1. Sample Dilution: with dilution by 4 times of testing sample dilutions;
2. solution preparation:
Get standard items and quality-control product, redissolve;
Enzymic-labelled antibody working fluid: with dilution by 125 ~ 1000 times of enzymic-labelled antibody dilutions;
3. get the ELISA Plate that is coated with anti-human OxLDL ELISA antibody, with cleansing solution washing 3 times, for subsequent use;
4. application of sample: get standard items, Quality Control thing and testing sample and add respectively microwell plate, every hole 100 μ l; At 37 DEG C, hatch 2 hours; Wash plate 5 times with cleansing solution;
5. add enzyme labelled antibody: with the direction vertical with anti-human OxLDL ELISA monoclonal antibody, add the enzyme labelled antibody after dilution, 100 μ l/ holes, hatch 1 hour at 37 DEG C, wash plate 5 times with cleansing solution;
6. add substrate solution: get isopyknic substrate solution A and substrate solution B, totally 100 μ l/ holes, 37 DEG C of lucifuges are hatched 15 minutes;
7. add stop buffer, 50 μ l/ holes;
8. measure absorbance value: start microplate reader, reading in 30 minutes after cessation reaction, measures wavelength 450nm.
Wherein, 2. 125 times of described enzymic-labelled antibody dilutions of step.
The present invention also provides the application of this kit in the diagnostic reagent of preparation coronary atherosclerotic heart disease.
It is good in-people's OxLDL ELISA series standard product that kit of the present invention provides, provide possibility for accurately detecting OxLDL ELISA in sample to be checked, and anti-oxidation low-density lipoprotein antibody and other solution have all been done preferably, improve kit accuracy in detection.Kit provided by the invention can reach the good range of linearity (related coefficient (R) is not less than 0.99) and well accuracy (this product analyze in accuracy: CV% not higher than 15%; Accuracy between analysis: CV% is not higher than 20%), illustrate that its accuracy is high, and clinical detection can be accurately by patients with coronary heart disease and normal subjects separately, there is good market application foreground.
Obviously,, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area, not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make amendment, replacement or the change of other various ways.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.
Embodiment
Composition and the using method thereof of embodiment 1 detection kit of the present invention
1, the composition of kit
(1) be coated with the ELISA Plate of anti-human OxLDL ELISA antibody;
(2) people's OxLDL ELISA series standard product:
Standard items 1: every liter of standard items 1 contain 20g bovine serum albumin(BSA), 50g sucrose;
Standard items 2: every liter of standard items 2 contain 20g bovine serum albumin(BSA), 50g sucrose and 1U OxLDL ELISA;
Standard items 3: every liter of standard items 3 contain 20g bovine serum albumin(BSA), 50g sucrose and 2U OxLDL ELISA;
Standard items 4: every liter of standard items 4 contain 20g bovine serum albumin(BSA), 50g sucrose and 4U OxLDL ELISA;
Standard items 5: every liter of standard items 5 contain 20g bovine serum albumin(BSA), 50g sucrose and 8U OxLDL ELISA;
Standard items 6: every liter of standard items 6 contain 20g bovine serum albumin(BSA), 50g sucrose and 16U OxLDL ELISA;
(3) enzymic-labelled antibody: be the goat-anti human apolipoprotein b antibody of peroxidase or horseradish peroxidase mark;
(4) dilution: every liter containing NaCl 40g, NaH
2pO
42H
2o1.184g, Na
2hPO
412H
2o11.6g, the aqueous solution of bovine serum albumin(BSA) 50g;
(5) cleansing solution: every liter containing NaCl 68g, NaH
2pO
42H
2o 6.22g, Na
2hPO
412H
2o61g, the aqueous solution of Tween-2021ml;
(6) substrate solution: comprise substrate solution A and substrate solution B, substrate solution A is the aqueous solution that contains TMB, and substrate solution B contains H
2o
2aqueous solution;
(7) stop buffer: every liter of aqueous solution that contains concentrated sulphuric acid 108ml;
(8) Quality Control thing:
High Quality Control thing: every rising Quality Control thing contains 20g bovine serum albumin(BSA), 50g sucrose and 13U OxLDL ELISA;
Low Quality Control thing: every liter low Quality Control thing contains 20g bovine serum albumin(BSA), 50g sucrose and 1.5U OxLDL ELISA.
The composition of commercial kit is in Table 1(96 person-portion/box)
Table 1 kit chief component of the present invention composition
2, the preparation method of the each component of kit of the present invention
(1) OxLDL standard items
Adopt the LDL of disposable density－gradient centrifuga－tion method separation and purification from human plasma, with external acellular metal Cu
2+oxLDL is made in ion method oxidation.Specifically comprise the following steps:
A, the preparation of LDL: separate human plasma lipoprotein by disposable density－gradient centrifuga－tion method, collection density is the LDL component of 1.030 ~ 1.050g/ml, identify that by two-way immunity diffusion, immunoelectrophoresis it is the LDL of purifying, lipoprotein agarose gel electrophoresis is identified its purity > 99%;
Disposable density－gradient centrifuga－tion method:
1. in 11ml centrifuge tube, add 4ml 1.400 blood plasma density fluids, it is 1.200 density gradient liquid that upper strata spreads 3ml density successively; 4ml density is 1.006 density gradient liquid, handles with care, covers gland bonnet, then puts into rotor.
2. ultracentrifugation, 10 DEG C, centrifugal 5 hours of 50000rpm.
3. collect second layer low-density lipoprotein component, 2-8 DEG C of preservation.
4. by the low-density lipoprotein component of collecting to containing 0.01%(w/v) the 10mmol/L phosphate buffer desalination dialysis of ethylenediamine tetraacetic acid 12 hours.
B, the preparation of OxLDL: external acellular metal Cu
2+ionic oxide formation legal system is for OxLDL:
1. OxLDL ELISA is dialysed 12 hours to 10mmol/L phosphate buffer (pH7.4), 2 ~ 8 DEG C of preservations, detect protein content;
2. with 10mmol/L phosphate buffer, the low-density lipoprotein of having dialysed is diluted to 5mg/ml according to institute's Reichl's test content, adding therein final concentration is the copper-bath of 2 μ mol/L, is placed in 37 DEG C of water-baths 2 hours;
3. adding final concentration is 0.01%(w/v) ethylenediamine tetraacetic acid cessation reaction;
4. by the low-density lipoprotein after oxidation at 2 ~ 8 DEG C to containing 0.01%(w/v) the phosphate buffer dialysis of ethylenediamine tetraacetic acid;
5. suck the OxLDL ELISA after dialysis with disposable syringe, put 0.22 μ m disposable aspiration needle filter and filter, in 2 ~ 8 DEG C of refrigerators of filtrate, preserve.
C, the mensuration of OxLDL content: the OxLDL of aforementioned preparation, by the step of OxLDL enzyme linked immunosorbent detection method, records after its OxLDL content, for the preparation of OxLDL kit standard items taking OxLDL enterprise normative reference product as contrast; Content assaying method is as follows:
1. will resist OxLDL monoclonal antibody to be diluted to suitable concentration with coated damping fluid, add microwell plate, 100 μ l/ holes, 2 ~ 8 DEG C are spent the night;
2. by concentration and dilution liquid (5 ×) with purified water be diluted to 1 ×, concentrated cleaning solution (21 ×) with purified water be diluted to 1 ×, substrate solution A and substrate solution B, in the preparation of 1:1 ratio, fully mix rear use;
3. wash plate 3 times with coated damping fluid, with confining liquid closed porosity plate, 300 μ l/ holes, place under room temperature 1 hour;
4. with dilution, enterprise's normative reference product series is diluted to 16,8,4,2,1U/ml; Get appropriate OxLDL, add dilution to do suitably dilution, make its content in enterprise's normative reference product series scope;
5. wash plate 3 times, serial dilution is become to enterprise's normative reference product of 16,8,4,2,1U/ml and the testing sample having diluted, add respectively microwell plate, 100 μ l/ holes, all do multiple hole, and blank hole adds 100 μ l dilutions;
6. wash plate 5 times, add enzymic-labelled antibody working fluid, 100 μ l/ holes, incubation 1 hour at 37 DEG C;
7. wash plate 5 times, add freshly prepared substrate solution, 100 μ l/ holes, incubation 15 minutes at 37 DEG C of dark places;
8. add 50 μ l/ hole stop buffer cessation reactions;
9. reading in 30 minutes after cessation reaction, mensuration wavelength is 450nm.
D, the preparation of OxLDL standard items: with containing 2%(w/v) bovine serum albumin(BSA), 5%(w/v) phosphate buffer of sucrose by the OxLDL serial dilution of aforementioned preparation become 16,8,4,2,1U/ml, taking the dilution that do not add OxLDL as 0U/ml, after packing, freeze drying, gland packing, generate a reagent box standard items.
(2) be coated with the ELISA Plate of anti-human OxLDL ELISA antibody
Synthesizing of anti-OxLDL antibody
A, anti-OxLDL monoclonal antibody is expressed supernatant and is cultivated in vitro by roller bottle training method the hybridoma preparation that preserving number is CCTCC NO:C 200304.Inoculating cell number 2 × 10
5/ ml; Nutrient solution consists of 97%DMEM/F12:RPMI1640=1:1,3%FBS; Roll fast 0.5rpm; Changed liquid once every 96 hours totally, carry out altogether changing liquid twice, gather in the crops three times.
B, culture supernatant is filtered clarification through 0.45 μ m, adopts the labscale small-sized ultrafiltration system of Millipore company, 500ml sample cup, 50ml/min flow velocity.
Streamline 25 expands column receipts peak and is further purified with QXL medium on XK16/20 fixed bed column, antibody yield >60%, and antibody purity is analyzed >90% with HPLC.
The preparation of ELISA Plate
(1) coated: will to resist OxLDL monoclonal antibody to be diluted to working concentration with being coated with damping fluid (every liter of aqueous solution containing 6.06g Tris, its pH is 8.0), and add microwell plate by 100 μ l/ holes, and be placed in 4 DEG C, 12 hours;
(2) sealing: the coating buffer that inclines, be placed on thieving paper and pat several times, remove raffinate, add confining liquid, the Tris-HCl confining liquid that confining liquid is is 50mmol/L containing the concentration of 1% bovine serum albumin(BSA) and 5% sucrose, its pH is 8.0,300 μ l/ holes, room temperature, 1 hour;
(3) dry: the deblocking liquid that inclines, be placed on thieving paper and pat several times, remove raffinate, microwell plate is put into vacuum drying chamber dry, 40 DEG C, 2 hours.Vacuum ranges: ﹣ 0.085～﹣ 0.1Mpa;
(4) after dry, take out microwell plate, with the sealing of shrouding gummed paper, put into aluminium foil bag, with capper sealing, be placed in 4 DEG C of preservations;
(3) enzymic-labelled antibody
The anti-apoB antibody (goat-anti human apolipoprotein b antibody) of HRP mark adopts conventional sodium periodate legal system standby, specifically comprises the following steps:
A, is dissolved in 1ml distilled water by 10mg HRP and adds freshly prepared 0.06mol/LNaIO
41ml, mixes in 4 DEG C and places 30 minutes;
B, adds 0.16mol/L glycol water 1ml, and room temperature is placed 30 minutes;
C, adds the aqueous solution 2ml containing the anti-apoB antibody of 5mg, then at 4 DEG C, to 0.05mol/L carbonic acid buffer (pH9.6) dialysed overnight;
D, solution in sucking-off bag filter, adds 0.5ml NaBH
4, place 2 hours at 4 DEG C;
E, drips equal-volume saturated (NH4)
2sO
4solution, places 30 minutes at 4 DEG C;
F, centrifugal 10 minutes of above-mentioned solution 2500rpm, removes supernatant, the PBS dissolving of a little 0.02mol/LpH7.4 for precipitation, and at 4 DEG C to this damping fluid dialysed overnight;
G, solution in sucking-off bag filter, the centrifugal insolubles of removing, supernatant is crossed Superose 6 chromatographic columns, with the PBS wash-out of 0.02mol/L pH7.4, collects first peak, is the enzymic-labelled antibody of purifying;
H, by the enzymic-labelled antibody aseptic filtration of collecting, adds equal-volume sterile glycerol, and-20 DEG C save backup.
(4) dilution, cleansing solution, substrate solution, stop buffer, according to the solution preparation method preparation of this area routine.
3, the using method of kit of the present invention
(1) reagent preparation:
(1) measure first 10 minutes and from refrigerator, take out kit, balance is to room temperature;
(2) by 10ml dilution (5 ×) with 40ml pure water be diluted to 1 ×;
(3) by 30ml cleansing solution (21 ×) with 600ml pure water be diluted to 1 ×;
(4) redissolution of standard items: add respectively 1ml purified water to redissolve in OxLDL standard items (0,1,2,4,8,16U/ml) and high and low Quality Control thing, leave standstill 3 minutes, shake up;
(5) enzymic-labelled antibody working fluid: get appropriate enzymic-labelled antibody, do 125 times of dilutions with dilution, as get 80 μ l enzymic-labelled antibodies, add 9920 μ l dilutions, mix;
(6) substrate solution: get isopyknic substrate solution A and substrate solution B, fully mix, preparation before use, as get 5ml substrate solution A and add 5ml substrate solution B.
(2) measure:
(7) dilute sample: with dilution by 4 times of Sample Dilutions;
(8) prepare microwell plate: tear sealing bag, take out microwell plate, remove shrouding gummed paper, with cleansing solution washing 3 times, for subsequent use;
Washing methods:
Automatic washing plate: require every hole to inject cleansing solution 350 μ l, injection and sucking-off interval 15 ~ 30 seconds;
Or wash plate by hand: liquid in the hole of inclining, every hole adds cleansing solution 350 μ l, and cleansing solution is removed in hypsokinesis in static 30 seconds, on thieving paper, pats dry.
(9) application of sample: standard items, Quality Control thing and testing sample are added to microwell plate, and 100 μ l/ holes, hatch 2 hours at 37 DEG C.
Carry out application of sample according to the order of application of sample shown in table 2.
Table 2 application of sample order example
(10) wash plate 5 times;
(11) add enzymic-labelled antibody working fluid, 100 μ l/ holes, hatch 1 hour at 37 DEG C;
(12) wash plate 5 times;
(13) add substrate solution, 100 μ l/ holes, lucifuge is hatched 15 minutes at 37 DEG C;
(14) add stop buffer, 50 μ l/ holes;
(15) measure absorbance value: start microplate reader, reading in 30 minutes after cessation reaction, measures wavelength 450nm.
(3) calculating of measurement result
(16) calculate the average light absorption value of each standard items, Quality Control thing and sample;
(17) taking the standard items of 0U/ml as blank, the absorbance value of standard items, Quality Control thing and sample should deduct the absorbance value of blank;
(18) use log-linear drawing, taking the logarithm of standard items concentration as transverse axis, the logarithm of absorbance value is the longitudinal axis, draws OxLDL concentration-absorbance value double-log dose-response curve;
(19) find the OxLDL concentration of dilute sample according to the testing sample absorbance value of dilution from dose-response curve;
(20) concentration that in sample, the ultimate density of OxLDL equals dilute sample is multiplied by extension rate;
(21) if the absorbance value of sample, higher than 16U/ml or lower than the absorbance value of 1U/ml standard items, can calculate by proper extension curve, extrapolation factor (Extrapolation Factor) is no more than 2;
(22) using BioTek EL800 type microplate reader supporting KC4(Version#2.0, Rev#17) data analysis system analyzes test findings.
The clinical practice of embodiment 2 kits of the present invention
Adopt kit of the present invention to detect 508 routine normal subjectses and 176 routine patients with coronary heart disease, result shows, normal subjects's blood plasma OxLDL content is 20.18 ± 15.02U/ml, Patients Plasma with Coronary Heart Disease OxLDL content is 68.90 ± 18.42U/ml, the two has significant difference (<0.0001), can fast, accurately detect coronary heart disease.
To sum up, enzyme linked immunological kit provided by the invention is highly sensitive, high specificity, availablely clinically fast, accurately detects coronary heart disease, and without wound, cheap, application prospect is good.
Claims (7)
1. an enzyme-linked immunologic detecting kit for people's OxLDL ELISA, is characterized in that: it comprises following composition:
(1) be coated with the ELISA Plate of anti-human OxLDL ELISA antibody;
(2) people's OxLDL ELISA series standard product:
Standard items 1: every liter of standard items 1 contain 20g bovine serum albumin(BSA), 50g sucrose;
Standard items 2: every liter of standard items 2 contain 20g bovine serum albumin(BSA), 50g sucrose and 1U OxLDL ELISA;
Standard items 3: every liter of standard items 3 contain 20g bovine serum albumin(BSA), 50g sucrose and 2U OxLDL ELISA;
Standard items 4: every liter of standard items 4 contain 20g bovine serum albumin(BSA), 50g sucrose and 4U OxLDL ELISA;
Standard items 5: every liter of standard items 5 contain 20g bovine serum albumin(BSA), 50g sucrose and 8U OxLDL ELISA;
Standard items 6: every liter of standard items 6 contain 20g bovine serum albumin(BSA), 50g sucrose and 16U OxLDL ELISA;
(3) enzymic-labelled antibody: be the goat-anti human apolipoprotein b antibody of peroxidase or horseradish peroxidase mark;
(4) dilution;
(5) cleansing solution;
(6) substrate solution;
(7) stop buffer;
The described ELISA Plate that is coated with anti-human OxLDL ELISA antibody of composition (1) is prepared as follows:
A, antibody dilution: anti-human OxLDL ELISA monoclonal antibody is diluted to 2.5 ~ 20 μ g/ml by the Tris-HCl buffer solution of the 50mM that is 8.0 with pH, obtains coating buffer;
B, coated: get microwell plate, with cleansing solution washing 3 times, add the above-mentioned coating buffer containing anti-human OxLDL ELISA monoclonal antibody, 100 μ l/ holes, every hole, hatch 12 hours for 4 DEG C;
C, sealing: the coating buffer that inclines, be placed on thieving paper and pat several times, remove raffinate, the Tris-HCl confining liquid that to add containing the concentration of 1% bovine serum albumin(BSA) and 5% sucrose be 50mmol/L, its pH is 8.0,300 μ l/ holes, room temperature, 1 hour;
D, vacuum drying, seal, and must be coated with the ELISA Plate of anti-human OxLDL ELISA antibody;
Described monoclonal antibody is that the hybridoma cell line that is CCTCC NO:C200304 by preserving number produces; Described dilution is every liter and contains NaCl 40g, NaH
2pO
42H
2o 1.184g, Na
2hPO
412H
2o 11.6g, the aqueous solution of bovine serum albumin(BSA) 50g; Described cleansing solution is every liter and contains NaCl 68g, NaH
2pO
42H
2o 6.22g, Na
2hPO
412H
2o 61g, the aqueous solution of Tween-20 21ml; Described substrate solution comprises substrate solution A and substrate solution B, and substrate solution A is the aqueous solution that contains TMB, and substrate solution B contains H
2o
2aqueous solution; Described stop buffer is every liter of aqueous solution that contains concentrated sulphuric acid 108ml.
2. kit according to claim 1, is characterized in that: described kit also comprises Quality Control thing:
High Quality Control thing: every rising Quality Control thing contains 20g bovine serum albumin(BSA), 50g sucrose and 13U OxLDL ELISA;
Low Quality Control thing: every liter low Quality Control thing contains 20g bovine serum albumin(BSA), 50g sucrose and 1.5U OxLDL ELISA.
3. according to the kit described in claim 1 or 2, it is characterized in that: the preparation method of described OxLDL ELISA is as follows:
I, get low-density lipoprotein, be dissolved in 10mM phosphate buffer, wherein, the concentration of low-density lipoprotein is 5mg/ml;
II, get the phosphate buffer of step I, add copper sulphate, making its final concentration is 2 μ mol/L, 37 DEG C of reactions 2 hours;
III, add ethylenediamine tetraacetic acid again, making its final concentration is 0.01%(w/v), cessation reaction, dialysis, obtains OxLDL ELISA.
4. the using method of the kit described in claim 1-3 any one, is characterized in that: it comprises the following step:
I, sample pre-treatments;
II, detect with the kit described in claim 1-3 any one;
III, result treatment and analysis.
5. using method according to claim 4, is characterized in that: it comprises the following step:
1. Sample Dilution: with dilution by 4 times of testing sample dilutions;
2. solution preparation:
Get standard items and quality-control product, redissolve;
Enzymic-labelled antibody working fluid: with dilution by 125 ~ 1000 times of enzymic-labelled antibody dilutions;
3. get the ELISA Plate that is coated with anti-human OxLDL ELISA antibody, with cleansing solution washing 3 times, for subsequent use;
4. application of sample: get standard items, Quality Control thing and testing sample and add respectively microwell plate, every hole 100 μ l; At 37 DEG C, hatch 2 hours; Wash plate 5 times with cleansing solution;
5. add enzyme labelled antibody: with the direction vertical with anti-human OxLDL ELISA monoclonal antibody, add the enzyme labelled antibody after dilution, 100 μ l/ holes, hatch 1 hour at 37 DEG C, wash plate 5 times with cleansing solution;
6. add substrate solution: get isopyknic substrate solution A and substrate solution B, totally 100 μ l/ holes, 37 DEG C of lucifuges are hatched 15 minutes;
7. add stop buffer, 50 μ l/ holes;
8. measure absorbance value: start microplate reader, reading in 30 minutes after cessation reaction, measures wavelength 450nm.
6. using method according to claim 5, is characterized in that: step 2. described enzymic-labelled antibody is diluted 125 times.
7. the application of the kit described in claim 1-3 any one in preparation coronary atherosclerotic heart disease diagnostic reagent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210160797.2A CN102680710B (en) | 2012-05-22 | 2012-05-22 | Enzyme-linked immunosorbent assay kit for human oxidized low-density lipoprotein, and using method and application |
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| CN103630695A (en) * | 2013-11-20 | 2014-03-12 | 中国人民解放军南京军区南京总医院 | Chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoprotein (a) and oxidized low-density lipoprotein of human serum |
| 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 |
| CN104914255A (en) * | 2015-05-21 | 2015-09-16 | 北京协和洛克生物技术有限责任公司 | Kit for detecting concentration of oxidized low-density lipoprotein in sample and preparation method thereof |
| CN105158486B (en) * | 2015-08-21 | 2016-07-06 | 陈立国 | For detecting the enzyme linked immunological kit of people's OxLDL ELISA |
| CN105974143A (en) * | 2016-04-08 | 2016-09-28 | 刘庆平 | Enzyme-linked immunosorbent assay kit for detection of human oxidized low-density lipoprotein and preparation method thereof |
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| 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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