CN102680710A - 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 the 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 combines instrument and biochemical analysis to carry out.Inspection means commonly used at present have routine electrocardiogram, electrocardiogram stress test, echocardiogram, spiral CT and UFCT Ultra-fast Computed Tomograph, coronary artery ultrasonoscopy, intravascular ultrasound, nuclear myocardial perfusion imaging video picture and selective coronary arteriography, and laboratory examination comprises myocardium enzyme labeled compound assay, troponin, c reactive protein, blood fat and lipoprotein inspection etc.In the above inspection means; Great majority are to change (various cardiogram) and ventricular wall motion metamorphosis (echocardiogram with the electrocardiosignal after detection myocardial ischemia or the myocardial infarction; Heart radio nuclide examination, spiral CT and UFCT Ultra-fast Computed Tomograph, magnetic resonance imaging) for carrying out coronary heart disease diagnosis in the basis; If degree of stenosis does not cause myocardial ischemia as yet, above detection means often can not help diagnosis of coronary heart disease.
Selective coronary arteriography can be directly acquainted with the degree of coronary artery stenosis, is considered to the goldstandard of diagnosis of coronary heart disease at present, but it belongs to traumatic inspection; Certain danger is arranged, need to be in hospital, and cost an arm and a leg; Patient for capable surgical intervention of needs or internal medicine PCI is essential inspection; And perhaps suspect the patient that coronary heart disease is arranged for light disease patients with coronary heart disease, and often be difficult to accept, need to seek traumatic little detection method.
Research shows that OxLDL ELISA OxLDL is relevant with the incidence and development of coronary heart disease, it has been generally acknowledged that to assess the risk of suffering from coronary heart disease through detecting the content of OxLDL ELISA OxLDL in the sample serum to be checked.In the prior art, some relevant detection methods are disclosed, like number of patent application: 03109935.1, denomination of invention: with anti-phosphocholine antibody to the quantitative measurement of 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) will resist the antibody of phosphocholine to contact with the sample that contains OxLDL; (b) said antibody is combined with OxLDL; (c) binding capacity of said antibody of mensuration and OxLDL; (d) foundation is the typical curve that standard items record with the phosphocholine, quantitatively OxLDL content.Because of antigen-antibody reaction very complicacy; Relevant with several factors; Though the antigen determining family of phosphocholine is identical with the low-density lipoprotein antigen determining family that the surface appears behind oxidative modification; But the degrees of specificity of the antigen-antibody reaction of anti-phosphocholine antibody and OxLDL ELISA is difficult to prediction, and is not high enough with the accuracy of anti-phosphocholine antibody test OxLDL ELISA, is difficult to be applied to Clinical detection.Application number is: 200710202084.7, and denomination of invention: a kind of detecting trace quantity oxygenize low density lipoprotein by indirect competition method colloidal gold strip discloses a kind of half-quantitative detection trace OxLDL ELISA and has 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 the fixing golden labeling antibody that can only combine about 600 μ g concentration on the gold pad, adopts the indirect competition method, is the OxLDL ELISA and the competition of the OxLDL ELISA in the sample to be checked colour developing of being fixed on the chromatographic film.This detection method is a half-quantitative detection, is difficult to accurately detect OxLDL ELISA content, more is difficult to the possibility of accurately prediction sample trouble to be checked coronary heart disease.
Therefore, also do not have at present a kind of easy, quick, no wound, cheap and diagnosis of coronary heart disease product that accuracy is high clinically, develop new coronary heart disease and check very necessity of means.
Summary of the invention
Technical scheme of the present invention has provided 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 the method for application and the purposes of this kit.
A kind of enzyme-linked immunologic detecting kit of people's OxLDL ELISA, it comprises following composition:
(1) is coated with the ELISA Plate of anti-people's OxLDL ELISA antibody;
(2) people's OxLDL ELISA series standard article:
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 HRPO mark;
(4) dilution;
(5) cleansing solution;
(6) substrate solution;
(7) stop buffer.
Wherein, said kit also comprises the 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-people's OxLDL ELISA antibody of step (1) prepares according to following method:
A, antibody dilution: using pH is that the Tris-HCl buffer solution of 8.0 50mM will resist people's OxLDL ELISA monoclonal antibody to be diluted to 2.5 ~ 20 μ g/ml, coating buffer;
B, encapsulate: get microwell plate, with cleansing solution washing 3 times, add the above-mentioned coating buffer that contains anti-people's OxLDL ELISA monoclonal antibody, hatched 12 hours for 4 ℃ in 100 μ l/ holes, every hole;
C, sealing: the coating buffer that inclines, place and pat on the thieving paper several times, remove raffinate, adding contains 1% bovine serum albumin(BSA) and 5% concentration of sucrose is the Tris-HCl confining liquid of 50mmol/L, and its pH is 8.0,300 μ l/ holes,, room temperature, 1 hour;
D, vacuum drying, sealing promptly gets the ELISA Plate that is coated with anti-people's OxLDL ELISA antibody.
Preferably, described anti-people's OxLDL ELISA monoclonal antibody is to be that the hybridoma cell line of CCTCCNO:C 200304 produces by preserving number.
Wherein, the preparation method of described OxLDL ELISA is following:
ⅰ, get low-density lipoprotein, be dissolved in the 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 ℃ of reactions 2 hours;
ⅲ, add ethylenediamine tetraacetic acid again, making its final concentration is 0.01% (w/v), cessation reaction, and dialysis promptly gets OxLDL ELISA.
Wherein, said dilution is every liter and contains NaCl 40g, NaH
2PO
42H
2O 1.184g, Na
2HPO
412H
2O 11.6g, the WS of bovine serum albumin(BSA) 50g;
Said cleansing solution is every liter and contains NaCl 68g, NaH
2PO
42H
2O 6.22g, Na
2HPO
412H
2O61g, the WS of Tween-2021ml;
Said substrate solution comprises substrate solution A and substrate solution B, and substrate solution A is the WS that contains TMB, and substrate solution B contains H
2O
2The WS;
Said stop buffer is every liter of WS that contains concentrated sulphuric acid 108ml.
The present invention also provides the method for application 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. diluted sample: with dilution with 4 times of testing sample dilutions;
2. solution preparation:
Get standard items and quality-control product, redissolve;
Enzymic-labelled antibody working fluid: enzymic-labelled antibody is diluted 125 ~ 1000 times with dilution;
3. get the ELISA Plate that is coated with anti-people's OxLDL ELISA antibody, with cleansing solution washing 3 times, subsequent use;
4. application of sample: get standard items, Quality Control thing and testing sample and add microwell plate respectively, every hole 100 μ l; Hatched under 37 ℃ 2 hours; Wash plate 5 times with cleansing solution;
5. add enzyme labelled antibody: with the vertical direction of anti-people's OxLDL ELISA monoclonal antibody, add the enzyme labelled antibody after the dilution, hatched 1 hour under 37 ℃ in 100 μ l/ holes, washes 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 ℃ of lucifuges were hatched 15 minutes;
7. add stop buffer, 50 μ l/ holes;
8. measure absorbance value: start ELIASA, reading in 30 minutes after cessation reaction is measured wavelength 450nm.
Wherein, the 2. said enzymic-labelled antibody dilution of step is 125 times.
The present invention also provides the application of this kit in the diagnostic reagent of preparation coronary atherosclerotic heart disease.
Kit of the present invention provides good reference-people's OxLDL ELISA series standard article; OxLDL ELISA provides possibility in the sample to be checked in order accurately to detect; And anti-oxidation low-density lipoprotein antibody and other solution all done preferably, improve the 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 (these article are analyzed interior accuracy: CV% and are not higher than 15%; Accuracy between analysis: CV% is not higher than 20%), explain that its accuracy is high, and Clinical detection can accurately separate patients with coronary heart disease and normal subjects, have better market prospect.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
Below, foregoing of the present invention is remake further detailed description through the embodiment of embodiment form.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Embodiment
The composition and the method for application thereof of embodiment 1 detection kit of the present invention
1, the composition of kit
(1) is coated with the ELISA Plate of anti-people's OxLDL ELISA antibody;
(2) people's OxLDL ELISA series standard article:
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 HRPO mark;
(4) dilution: every liter contains NaCl 40g, NaH
2PO
42H
2O1.184g, Na
2HPO
412H
2O11.6g, the WS of bovine serum albumin(BSA) 50g;
(5) cleansing solution: every liter contains NaCl 68g, NaH
2PO
42H
2O 6.22g, Na
2HPO
412H
2O61g, the WS of Tween-2021ml;
(6) substrate solution: comprise substrate solution A and substrate solution B, substrate solution A is the WS that contains TMB, and substrate solution B contains H
2O
2The WS;
(7) stop buffer: every liter of WS 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 seen table 1 (96 person-portions/box)
The main composition of table 1 kit of the present invention
2, the preparation method of each component of kit of the present invention
(1) OxLDL standard items
Adopt the LDL of disposable density separation and purification from human plasma, with external acellular metal Cu
2+OxLDL is processed in the ion method oxidation.Specifically may further comprise the steps:
A; The preparation of LDL: by disposable density separation of human plasma lipoprotein; Collection density is the LDL component of 1.030 ~ 1.050g/ml, uses two-way immunodiffusion, immunoelectrophoresis to identify its LDL as purifying, and the lipoprotein agarose gel electrophoresis is identified its purity＞99%;
Disposable density:
1. in the 11ml centrifuge tube, add 4ml 1.400 blood plasma density fluids, it is 1.200 density gradient liquid that 3ml density is spread on the upper strata successively; 4ml density is 1.006 density gradient liquid, handles with care, covers gland bonnet, puts into rotor again.
2. ultra centrifugal, 10 ℃, centrifugal 5 hours of 50000rpm.
3. collect second layer low-density lipoprotein component, 2-8 ℃ of preservation.
4. the low-density lipoprotein component of collecting was dialysed 12 hours to the 10mmol/L phosphate buffer desalination that contains 0.01% (w/v) ethylenediamine tetraacetic acid.
B, the preparation of OxLDL: external acellular metal Cu
2+The ionic oxide formation legal system is equipped with OxLDL:
1. OxLDL ELISA was dialysed 12 hours to 10mmol/L phosphate buffer (pH7.4), 2 ~ 8 ℃ of preservations detect protein content;
2. the low-density lipoprotein that will dialyse with the 10mmol/L phosphate buffer is diluted to 5mg/ml according to institute's Reichl's test content, adds the copper-bath that final concentration is 2 μ mol/L therein, places 37 ℃ of water-baths 2 hours;
3. adding final concentration is 0.01% (w/v) ethylenediamine tetraacetic acid cessation reaction;
4. the low-density lipoprotein after the oxidation is dialysed to the phosphate buffer that contains 0.01% (w/v) ethylenediamine tetraacetic acid under 2 ~ 8 ℃;
5. suck the OxLDL ELISA after dialysing with disposable syringe, put 0.22 μ m disposable aspiration needle filter and filter, preserve in 2 ~ 8 ℃ of refrigerators of filtrating.
C, the OxLDL Determination on content: the OxLDL of aforementioned preparation, press the step of OxLDL enzyme linked immunosorbent detection method, serve as after contrast records its OxLDL content, to be used for the preparation of OxLDL kit standard items with OxLDL enterprise normative reference article; Content assaying method is following:
1. will resist the OxLDL monoclonal antibody to be diluted to suitable concentration with encapsulating damping fluid, add microwell plate, 100 μ l/ holes, 2 ~ 8 ℃ are spent the night;
2. with 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 fully use behind the mixing in the preparation of 1:1 ratio;
3. wash plate 3 times with encapsulating damping fluid, with confining liquid closed porosity plate, 300 μ l/ holes, room temperature held 1 hour;
4. with dilution enterprise's normative reference article series is diluted to 16,8,4,2,1U/ml; Get an amount of OxLDL, add dilution and do suitably dilution, make its content in the serial scope of enterprise's normative reference article;
5. wash plate 3 times, serial dilution is become 16,8,4,2, the testing sample that the enterprise's normative reference article of 1U/ml and dilution are good adds microwell plate respectively, and 100 μ l/ holes, the hole of all writing in reply, blank hole add 100 μ l dilutions;
6. wash plate 5 times, add the enzymic-labelled antibody working fluid, 100 μ l/ holes, 37 ℃ of following incubations 1 hour;
7. wash plate 5 times, add freshly prepared substrate solution, 100 μ l/ holes, 37 ℃ of following incubations in dark place 15 minutes;
8. add 50 μ l/ hole stop buffer cessation reactions;
9. reading in 30 minutes after cessation reaction, the mensuration wavelength is 450nm.
D; The preparation of OxLDL standard items: with containing 2% (w/v) bovine serum albumin(BSA); The phosphate buffer of 5% (w/v) sucrose with the OxLDL serial dilution of aforementioned preparation become 16,8,4,2,1U/ml, be 0U/ml with the dilution that does not add OxLDL, after packing, the freeze drying; Gland packing, the generate a reagent box is used standard items.
(2) be coated with the ELISA Plate of anti-people's OxLDL ELISA antibody
Synthesizing of anti-OxLDL antibody
A, it is the hybridoma preparation of CCTCC NO:C 200304 at the in vitro culture preserving number through rolling bottle training method that anti-OxLDL monoclonal antibody is expressed supernatant.Inoculating cell several 2 * 10
5/ ml; Nutrient solution consists of 97%DMEM/F12:RPMI1640=1:1,3%FBS; Roll fast 0.5rpm; Whenever changed liquid once totally, carry out altogether changing liquid twice, gather in the crops three times at a distance from 96 hours.
B, culture supernatant is filtered clarification through 0.45 μ m, adopts the small-sized ultrafiltration system of labscale of Millipore company, 500ml sample cup, 50ml/min flow velocity.
Streamline 25 expansion columns are received the peak and on the XK16/20 fixed bed column, are further purified the antibody yield with the QXL medium>60%, antibody purity is analyzed with HPLC>90%.
The preparation of ELISA Plate
(1) encapsulates: will resist the OxLDL monoclonal antibody to be diluted to working concentration with encapsulating damping fluid (every liter of WS that contains 6.06g Tris, its pH is 8.0), and add microwell plate, and place 4 ℃, 12 hours by 100 μ l/ holes;
(2) sealing: the coating buffer that inclines, place and pat on the thieving paper several times, remove raffinate; Add confining liquid, confining liquid is to contain 1% bovine serum albumin(BSA) and 5% concentration of sucrose is the Tris-HCl confining liquid of 50mmol/L, and its pH is 8.0; 300 μ l/ holes, room temperature, 1 hour;
(3) drying: the deblocking liquid that inclines, place and pat on the thieving paper several times, remove raffinate, microwell plate is put into the vacuum drying chamber drying, 40 ℃, 2 hours.Vacuum ranges: ﹣ 0.085～﹣ 0.1Mpa;
(4) after drying finishes, take out microwell plate,, put into aluminium foil bag,, place 4 ℃ of preservations with the capper sealing with the sealing of shrouding gummed paper;
(3) enzymic-labelled antibody
The anti-apoB antibody of HRP mark (goat-anti human apolipoprotein b antibody) adopts conventional sodium periodate method preparation, specifically may further comprise the steps:
A is dissolved in 10mg HRP in the 1ml distilled water and adds freshly prepared 0.06mol/LNaIO
41ml, mixing was placed 30 minutes in 4 ℃;
B adds 0.16mol/L glycol water 1ml, and room temperature was placed 30 minutes;
C adds the WS 2ml that contains the anti-apoB antibody of 5mg, then under 4 ℃, to 0.05mol/L carbonic acid buffer (pH9.6) dialysed overnight;
Solution in the D, sucking-off bag filter adds 0.5ml NaBH
4, 4 ℃ of held 2 hours;
E drips equal-volume saturated (NH4)
2SO
4Solution, 4 ℃ of held 30 minutes;
F, centrifugal 10 minutes of above-mentioned solution 2500rpm removes supernatant, deposition is with the PBS dissolving of a little 0.02mol/LpH7.4, and under 4 ℃ to this damping fluid dialysed overnight;
Solution in the G, sucking-off bag filter, the centrifugal insolubles of removing, supernatant are crossed Superose 6 chromatographic columns, with the PBS wash-out of 0.02mol/L pH7.4, collect first peak, are the enzymic-labelled antibody of purifying;
H, the enzymic-labelled antibody aseptic filtration with collecting adds the equal-volume sterile glycerol, and-20 ℃ of preservations are subsequent use.
(4) dilution, cleansing solution, substrate solution, stop buffer are according to the conventional solution compound method preparation in this area.
3, the method for application of kit of the present invention
(1) reagent preparation:
(1) measure preceding 10 minutes and from refrigerator, take out kit, balance is to room temperature;
(2) with 10ml dilution (5 *) with the 40ml pure water be diluted to 1 *;
(3) with 30ml cleansing solution (21 *) with the 600ml pure water be diluted to 1 *;
(4) redissolution of standard items: in OxLDL standard items (0,1,2,4,8,16U/ml) and high and low Quality Control thing, add the 1ml purified water respectively and redissolve, left standstill 3 minutes, shake up;
(5) enzymic-labelled antibody working fluid: get an amount of enzymic-labelled antibody, do 125 times of dilutions, as get 80 μ l enzymic-labelled antibodies, add 9920 μ l dilutions, mixing with dilution;
(6) substrate solution: get isopyknic substrate solution A and substrate solution B, fully mixing faces and uses preceding preparation, as gets 5ml substrate solution A and add 5ml substrate solution B.
(2) measure:
(7) dilute sample: with dilution with 4 times of diluted samples;
(8) prepare microwell plate: tear sealing bag, take out microwell plate, remove the shrouding gummed paper, with cleansing solution washing 3 times, subsequent use;
Washing methods:
Automatically wash plate: require every hole to inject cleansing solution 350 μ l, inject and sucking-off 15 ~ 30 seconds at interval;
Perhaps wash plate by hand: liquid in the hole of inclining, every hole add cleansing solution 350 μ l, and cleansing solution is removed in hypsokinesis in static 30 seconds, do at the thieving paper arsis.
(9) application of sample: standard items, Quality Control thing and testing sample are added microwell plate, and hatched 2 hours under 37 ℃ in 100 μ l/ holes.
Carry out application of sample in proper order according to application of sample shown in the table 2.
Table 2 application of sample order example
(10) wash plate 5 times;
(11) add the enzymic-labelled antibody working fluid, hatched 1 hour under 37 ℃ in 100 μ l/ holes;
(12) wash plate 5 times;
(13) add substrate solution, 100 μ l/ holes, 37 ℃ of following lucifuges were hatched 15 minutes;
(14) add stop buffer, 50 μ l/ holes;
(15) measure absorbance value: start ELIASA, reading in 30 minutes after cessation reaction is measured wavelength 450nm.
(3) mensuration result's calculating
(16) calculate the average light absorption value of each standard items, Quality Control thing and sample;
(17) standard items with 0U/ml are blank, and the absorbance value of standard items, Quality Control thing and sample should deduct the absorbance value of blank;
(18) using the log-linear drawing, is transverse axis with the logarithm of standard items concentration, and 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 from dose-response curve according to the testing sample absorbance value of dilution;
(20) concentration that the ultimate density of OxLDL equals dilute sample in the sample multiply by extension rate;
(21) if the absorbance value of sample is higher than 16U/ml or is lower than the absorbance value of 1U/ml standard items, but the proper extension curve calculate, extrapolation factor (Extrapolation Factor) is no more than 2;
(22) (Version#2.0, Rev#17) data analysis system is analyzed test findings to use the supporting KC4 of BioTek EL800 type ELIASA.
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; The result shows; Normal subjects's blood plasma OxLDL content is 20.18 ± 15.02U/ml; Patients with coronary heart disease blood plasma OxLDL content is 68.90 ± 18.42U/ml, and the two has significant difference (< 0.0001), can fast, accurately detect coronary heart disease.
To sum up, highly sensitive, the high specificity of enzyme linked immunological kit provided by the invention availablely clinically fast, accurately detects coronary heart disease, and does not have wound, and cheap, application prospect is good.
Claims (10)
1. the enzyme-linked immunologic detecting kit of people's OxLDL ELISA, it is characterized in that: it comprises following composition:
(1) is coated with the ELISA Plate of anti-people's OxLDL ELISA antibody;
(2) people's OxLDL ELISA series standard article:
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 HRPO mark;
(4) dilution;
(5) cleansing solution;
(6) substrate solution;
(7) stop buffer.
2. kit according to claim 1 is characterized in that: said kit also comprises the 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. kit according to claim 1 is characterized in that: the described ELISA Plate that is coated with anti-people's OxLDL ELISA antibody of step (1) prepares according to following method:
A, antibody dilution: using pH is that the Tris-HCl buffer solution of 8.0 50mM will resist people's OxLDL ELISA monoclonal antibody to be diluted to 2.5 ~ 20 μ g/ml, coating buffer;
B, encapsulate: get microwell plate, with cleansing solution washing 3 times, add the above-mentioned coating buffer that contains anti-people's OxLDL ELISA monoclonal antibody, hatched 12 hours for 4 ℃ in 100 μ l/ holes, every hole;
C, sealing: the coating buffer that inclines, place and pat on the thieving paper several times, remove raffinate, adding contains 1% bovine serum albumin(BSA) and 5% concentration of sucrose is the Tris-HCl confining liquid of 50mmol/L, and its pH is 8.0,300 μ l/ holes, room temperature, 1 hour;
D, vacuum drying, sealing promptly gets the ELISA Plate that is coated with anti-people's OxLDL ELISA antibody.
4. kit according to claim 3 is characterized in that: described anti-people's OxLDL ELISA monoclonal antibody is to be that the hybridoma cell line of CCTCC NO:C 200304 produces by preserving number.
5. according to claim 1 or 2 described kits, it is characterized in that: the preparation method of described OxLDL ELISA is following:
ⅰ, get low-density lipoprotein, be dissolved in the 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 ℃ of reactions 2 hours;
ⅲ, add ethylenediamine tetraacetic acid again, making its final concentration is 0.01% (w/v), cessation reaction, and dialysis promptly gets OxLDL ELISA.
6. kit according to claim 1 is characterized in that:
Said dilution is every liter and contains NaCl 40g, NaH
2PO
42H
2O1.184g, Na
2HPO
412H
2O11.6g, the WS of bovine serum albumin(BSA) 50g;
Said cleansing solution is every liter and contains NaCl 68g, NaH
2PO
42H
2O 6.22g, Na
2HPO
412H
2O61g, the WS of Tween-2021ml;
Said substrate solution comprises substrate solution A and substrate solution B, and substrate solution A is the WS that contains TMB, and substrate solution B contains H
2O
2The WS;
Said stop buffer is every liter of WS that contains concentrated sulphuric acid 108ml.
7. the method for application of any described kit of claim 1-6, it is characterized in that: it comprises the following step:
I, sample pre-treatments;
II, detect with each described kit of claim 1-6;
III, result treatment and analysis.
8. method of application according to claim 7, it is characterized in that: it comprises the following step:
1. diluted sample: with dilution with 4 times of testing sample dilutions;
2. solution preparation:
Get standard items and quality-control product, redissolve;
Enzymic-labelled antibody working fluid: enzymic-labelled antibody is diluted 125 ~ 1000 times with dilution;
3. get the ELISA Plate that is coated with anti-people's OxLDL ELISA antibody, with cleansing solution washing 3 times, subsequent use;
4. application of sample: get standard items, Quality Control thing and testing sample and add microwell plate respectively, every hole 100 μ l; Hatched under 37 ℃ 2 hours; Wash plate 5 times with cleansing solution;
5. add enzyme labelled antibody: with the vertical direction of anti-people's OxLDL ELISA monoclonal antibody, add the enzyme labelled antibody after the dilution, hatched 1 hour under 37 ℃ in 100 μ l/ holes, washes 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 ℃ of lucifuges were hatched 15 minutes;
7. add stop buffer, 50 μ l/ holes;
8. measure absorbance value: start ELIASA, reading in 30 minutes after cessation reaction is measured wavelength 450nm.
9. method of application according to claim 8 is characterized in that: 125 times of the 2. said enzymic-labelled antibody dilutions of step.
10. the application of any described kit of claim 1-6 in preparation coronary atherosclerotic heart disease diagnostic reagent.
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| 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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| CN105158486A (en) * | 2015-08-21 | 2015-12-16 | 孙丽华 | Enzyme linked immunosorbent assay kit used for detecting human ox-LDL (oxidized low-density lipoprotein) |
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| 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 |
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