CN103630695A - Chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoprotein (a) and oxidized low-density lipoprotein of human serum - Google Patents
Chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoprotein (a) and oxidized low-density lipoprotein of human serum Download PDFInfo
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Abstract
The invention discloses a method for chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoproteins (a) and low-density oxidized lipoproteins of human serum. According to an analysis system, a multi-component immunosensor modified by silanization is used as a solid-phase carrier; a solid-phase antibody is prepared by covering the carrier with a capturing antibody; a sample to be measured or a standard series solution is added into the solid-phase antibody; after the solid-phase antibody and the sample to be measured or the standard series solution fully react, an antibody labeled with horse radish peroxidase is added to form a sandwich immune compound; finally a chemiluminescence substrate, namely luminal-hydrogen peroxide, is added for reaction; the luminous intensity is detected through a high-resolution charge coupler, so that the oxidized lipoproteins (a) and the low-density oxidized lipoproteins of the human serum can be measured simultaneously, and the immune reaction condition, the enzyme conjugate dilution degree, the detection time and the like are optimized; furthermore, the indexes such as linear range, precision, accuracy and detection limit of the method are checked. The analysis method has the advantages of simplicity in operation, high sensitivity, narrow linear range, high stability and the like and has a certain clinical application value.
Description
Technical field
The present invention relates to immuno analytical method field, is a kind of chemiluminescence imaging immune analysis method of simultaneously measuring human serum oxidation Lp(a) and OxLDL ELISA specifically.
Background technology
Oxidation lipoprotein is considered to atherosclerotic (As) at present, the key factor developing occurs angiocardiopathy.Oxidation Lp(a) [ox-Lp(a)] and OxLDL ELISA (ox-LDL) all can pass through scavenger receptor approach, cause accumulating of macrophage inner cholesterol ester, cause the formation of foam cells; Also can be directly by picked-up that macrophage is engulfed.Meanwhile, ox-Lp(a), with ox-LDL can by stimulating, monocyte be synthetic, secretion adhesion molecule, promotion monocyte is assembled, is adhered to blood vessel endothelium, and then changes macrophage into.In addition, ox-Lp(a) also can stimulate the generation of oxygen radical, the permeability of injured blood vessel endothelium with ox-LDL.
Ox-LDL, ox-Lp (a) all can measure in serum.Ox-LDL level at various diseases as coronary artery disease CAD), raise in carotid atherosclerosis, transplanting artery sclerosis, the patient who accepts haemodialysis and the diabetes etc. followed.The ox-LDL quantitative test of atherosclerotic lesion shows, at damage location, has a large amount of ox-LDL to accumulate.Level at a kind of different phase ox-LDL of disease also can change, and in the acute stage ox-LDL of acute myocardial infarction AMI, cerebral infarction, PTCA level, can raise.Studying the ox-LDL that shows to circulate is the strong predictor that CAD occurs, and is proportionate with the lesion degree of acute coronary syndrome (ACS).In the rheumatoid patient of Hypertensive Population, ephrosis hemodialysis patients and angiocardiopathy occurred frequently, ox-Lp (a) level all raises.Ox-Lp (a) level obviously raises in CAD patient, and relevant to the lesion degree of As, and ACS patient's variation is particularly remarkable, and prompting is in CAD patient, and the detection of ox-Lp (a) can reflect the abnormal of blood fat more sensitively.The simultaneous determination of above-mentioned explanation ox-LDL, ox-Lp (a) is significant for the prediction of As disease.
At present, the most frequently used method of content of detection circulation ox-LDL and ox-Lp (a) is enzyme linked immunosorbent assay (ELISA).Ox-LDL concentration also can be measured by chemiluminescence enzyme immunoassay.The detection of ox-Lp (a) level has been set up respectively and adopted the specific antibody for oxidation apo (a) and/or apoB is coated antibody, the anti-apo of enzyme mark (a) is for detecting the ELISA method of antibody, the oxidation level in the apo (a) in mensuration Lp (a) and/or oxidation apoB site, to assess the degree of oxidation of Lp (a).Yet above-mentioned detection method only can detect one-component in single analysis process, analysis time is long, sensitivity is low, the range of linearity is narrow, and external ox-LDL kit is expensive, is not easy to clinical expansion.Chemiluminescence immune assay is combined chemical luminescent detecting technology with immunoassay, because it has the advantage such as highly sensitive, the range of linearity is wide, analysis speed is fast, be widely used in the fields such as clinical diagnosis, food security, environmental monitoring.In recent years, chemiluminescence immune assay and imaging technique coupling, form a kind of analytical technology---chemiluminescence imaging immunoassay of renewal.This technology be energy that chemiluminescence immunoassay reaction is discharged by charge-coupled device (CCD), with the form of integration, with high-resolution camera, be filmed.At present, the pattern based on chemiluminescence imaging has obtained in multi-component immunity analytical field paying close attention to widely.
Summary of the invention
The object of the invention is to utilize the principle of chemiluminescence immune assay, a kind of chemiluminescence imaging immune analysis method that simultaneously detects multiple oxidation lipoprotein content in blood serum sample is provided.Relate in particular to and a kind ofly can measure the chemiluminescence imaging immune analysis method that is oxidized Lp(a) and OxLDL ELISA content in blood serum sample simultaneously.
For achieving the above object, the present invention adopts following technical scheme:
A chemiluminescence imaging immune analysis method that simultaneously detects multiple oxidation lipoprotein content in blood serum sample, comprises following steps:
Step 1: on the microslide array of silylation modification, coated capture antibody, incubation, washing, sealing, make insolubilized antibody;
Step 2: add corresponding standard serial solution, incubation, washing in the micropore of the insolubilized antibody of preparing to step 1; The antibody that adds corresponding horseradish peroxidase-labeled, incubation, washing; Then add chemical luminous substrate luminol-hydrogen peroxide, by CCD, carry out the detection of chemiluminescence image; According to concentration of standard solution and corresponding chemiluminescence intensity value drawing standard curve;
Step 3: add corresponding test serum sample in the micropore of the insolubilized antibody of step 1 preparation, incubation, washing, add the antibody of corresponding horseradish peroxidase-labeled, incubation, washing; Then add chemical luminous substrate luminol-hydrogen peroxide, by CCD, carry out the detection of chemiluminescence image; The typical curve of drawing according to step 2, calculates the different concentration that is oxidized lipoprotein in sample.
In specific embodiments of the invention, the capture antibody described in step 1 is the anti-human OxLDL ELISA antibody of rabbit.
As preferably, the concentration of the anti-human OxLDL ELISA antibody of rabbit is 0.33 μ g/mL.
As preferably, described multiple oxidation lipoprotein is oxidation Lp(a) and OxLDL ELISA.
In specific embodiments of the invention, the standard serial solution described in step 2 is fresh mix serum stoste and doubling dilution solution thereof.
In specific embodiments of the invention, in described fresh mix serum stoste, be oxidized the content of Lp(a) and OxLDL ELISA by the Cu of purifying
2+lp(a) and the Cu of oxidation
2+the low-density lipoprotein of oxidation is demarcated.
In specific embodiments of the invention, the antibody of described horseradish peroxidase-labeled is respectively the apolipoprotein B polyclonal antibody of the apolipoprotein of horseradish peroxidase-labeled (a) monoclonal antibody and horseradish peroxidase-labeled.Preferably, the dilutability of the apolipoprotein B polyclonal antibody of the apolipoprotein of horseradish peroxidase-labeled (a) monoclonal antibody and horseradish peroxidase-labeled is 1:100.
As preferably, the confining liquid that in step 1, sealing is used is the pH 7.4 containing 10% hyclone, 0.01M phosphate buffer.
As preferably, in step 1,2,3, wash the cleansing solution using and be pH 7.4,0.01M phosphate buffer.
As preferably, in step 2 and step 3, the time of first step incubation reaction is 1h, and the time of second step incubation reaction is 1.5h.
Simultaneous quantitative of the present invention being detected to the range of linearity, detectability, precision, the accuracy of ox-Lp (a) and ox-LDL chemiluminescence imaging immune analysis method investigates, result demonstration, ox-Lp (a) and ox-LDL detect the range of linearity and are respectively 2.0 * 10
-5～2.0 * 10
-1with 2.4 * 10
-4～2.4U/mL; Detectability (signal to noise ratio (S/N ratio) is 3) is respectively 2.4 * 10
-6with 3.0 * 10
-5u/mL; Ox-Lp (a) and ox-LDL measure average recovery rate and are respectively 99.3% and 99.6%; Ox-Lp (a) and ox-LDL measure average variation within batch (CV) be respectively 6.2% and 5.2%(n=10), between average batch, CV is 8.1%(n=5).
Compare with traditional single component analytical approach, multi-component detection simultaneously has the outstanding advantages such as analysis throughput is high, required time is short, amount of samples is few, analysis cost is low.In the prediction of clinical disease and the assessment of hazard level, the joint-detection of a plurality of indexs can improve its accuracy.
Compared with prior art, the method for the invention tool has the following advantages:
1, can detect the ox-Lp(a of same sample simultaneously), ox-LDL content;
2, highly sensitive;
3, the range of linearity is wide;
4, analysis time is short;
5, the preparation of polycomponent immunosensor is simple, cost is low.
Accompanying drawing explanation
Fig. 1. be chemiluminescence imaging immunoassay schematic diagram;
Fig. 2. be the optimization of coated antibody concentration in the method for the invention;
Fig. 3. be the optimization of enzyme labelled antibody concentration in the method for the invention;
Fig. 4. be the optimization of immunity inculation time in the method for the invention;
Fig. 5. for chemiluminescence imaging immune analysis method of the present invention detects serum ox-Lp(a simultaneously), the typical curve of ox-LDL content;
Fig. 6. be that the method for the invention measures ox-Lp(a), the result of ox-LDL and the correlation analysis of ELISA measurement result.
Embodiment
The invention discloses a kind of chemiluminescence imaging immune analysis method that simultaneously detects ox-Lp (a) and ox-LDL, those skilled in the art can use for reference content herein, suitably improve technological parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications as herein described or suitably change and combination within not departing from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
In order to make those skilled in the art understand better technical scheme of the present invention, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1: the preparation of immune sensing array
First, with Piranha acid solution (H
2sO
4/ 30%H
2o
2, volume ratio is 7:3) and soak microslide, the standing 12h of room temperature, makes slide hydroxylation; With ultrapure water, rinse, after nitrogen dries up, the slide of activation is immersed in 1% (v/v) GPTMS/ toluene solution (volumn concentration that is GPTMS solution in toluene solution is 1%), room temperature (25 ℃) is spent the night, and makes its silanization.Then, with toluene, absolute ethyl alcohol, rinse successively, to remove the silane of surface of glass slide physisorption, nitrogen dries up.Finally, use screen printing to process slide, make its surface form 48 micropores (diameter 2mm, pitch of holes 4mm, 4 row * 12 row) (see figure 1).By the anti-human ox-LDL antibody of rabbit pH9.6,0.05M carbonate coating buffer is diluted to 0.33 μ g/mL, join in the microslide micropore of 48-hole silylation modification, and 1.5 μ L/ holes, 4 ℃ of placements are spent the night.Then use pH7.4,0.01M phosphate buffer (PBS) washing, nitrogen dries up, every hole adds 1.5 μ L confining liquids (containing the pH7.4 of 10% (v/v) hyclone, 0.01M phosphate buffer), room temperature sealing 1h, pH7.4,0.01M phosphate buffer (hereinafter to be referred as PBS) rinses, and nitrogen dries up, and sealing is placed in 4 ℃ of Refrigerator stores.
Embodiment 2: the preparation of standard serial solution
The separation of lipoprotein: the separated 100 routine normal persons of density gradient ultracentrifugation mix fresh plasma LDL (d=1.019-1.063g/mL).Lp (a) adopts the human plasma lipoprotein component of the separated 1.055-1.110g/mL of ultracentrifugation, through SePharose6B column chromatography.
The oxidation of lipoprotein: lipoprotein is through pH 7.4, and 0.01mol/L PBS fully dialyses, adjustment protein concentration is 0.5mg/mL, adds CuSO
4to final concentration 40 μ mol/L, after 37 ℃ of incubation 20h, with containing 1mmol/L EDTA-Na
2, pH7.4,0.01mol/L PBS fully dialyses.Lowry method is measured the protein content of purifying ox-Lp (a), ox-LDL.
Collect the fresh serum of 50 routine Healthy Peoples, after mixing, with purifying ox-Lp (a), the ox-LDL of concentration known, by ELISA method, pooled serum is demarcated.Regulation 1U=1mg oxidation lipoprotein, the concentration of ox-Lp in pooled serum (a) is 2.0 * 10
-1u/mL; The concentration of ox-LDL is 2.4U/mL.Fresh pooled serum is interpreted into standard serial solution with the PBS containing 5% (v/v) hyclone.
The concentration of the standard serial solution of ox-Lp (a) is 2.0 * 10
-5, 2.0 * 10
-4, 2.0 * 10
-3, 2.0 * 10
-2, 2.0 * 10
-1u/mL; The concentration of the standard serial solution of ox-LDL is 2.4 * 10
-4, 2.4 * 10
-3, 2.4 * 10
-2, 2.4 * 10
-1, 2.4U/mL.
Embodiment 3: the optimization of immune response condition
(1) pH 9.6 for the impact of coated antibody concentration, 0.05M carbonate buffer solution dilutes coated antibody strong solution respectively with 1:375,1:750,1:1500,1:3000,1:6000 ratio, to the antibody-solutions 1.5 μ L that add variable concentrations in array micropore, 4 ℃ spend the night after, room temperature sealing 1h.As preferably, the concentration of coated antibody is that 0.33 μ g/mL(is shown in Fig. 2).
(2) impact of enzyme conjugates dilute concentration be take respectively the ratio of 1:80,1:100,1:200,1:400,1:800,1:1000 by enzyme conjugates strong solution and is diluted (dilution is the PBS containing 5% hyclone).As preferably, the dilutability of the apolipoprotein of horseradish peroxidase-labeled (a) [HRP-apo(a)] monoclonal antibody and HRP-apoB polyclonal antibody is 1:100(and sees Fig. 3).
(3) the immune response time affect coated antibody and antigen respectively with 15,30,45,60,90,120min reacts; Enzyme labelled antibody and solid phase bond react respectively with 15,30,45,60,90,120min investigates.As preferably, first step incubation reaction is 1h, and second step incubation reaction is that 1.5h(is shown in Fig. 4).
Embodiment 4: the range of linearity of the method for the invention
Adopt the method for the invention to detect the standard serial solution of ox-Lp (a) and ox-LDL, drawing standard curve.
Concrete steps are as follows:
(1) add standard serial solution.In the respective aperture of the microslide of silylation modification, add the corresponding standard serial solution of 1.5 μ L, incubated at room 1h, PBS washing, nitrogen dries up;
(2) add the antibody of HRP mark.To the antibody-solutions that adds the HRP mark that 1.5 μ L are corresponding in respective aperture, incubated at room 1.5h, PBS washing, nitrogen dries up.
(3) add substrate.Each Kong Jun adds 1.5 μ L Chemoluminescent substrate luminol-hydrogen peroxide.
(4) detect.With CCD, detect immediately, exposure interval 1min, detection of dynamic 10min, collects chemical signal.
(5) foundation of typical curve.Typical curve Log(x) double-log data fitting mode-Log(y) is set up (see figure 5).
Ox-Lp (a) and ox-LDL detect the range of linearity and are respectively 2.0 * 10
-5～2.0 * 10
-1with 2.4 * 10
-4～2.4U/mL; Detectability (signal to noise ratio (S/N ratio) is 3) is respectively 2.4 * 10
-6with 3.0 * 10
-5u/mL.
Embodiment 5: in the time of serum ox-Lp (a) and ox-LDL concentration, measure
(1) add sample.In the respective aperture of the microslide of silylation modification, add 1.5 μ L samples, incubated at room 1h, PBS washing, nitrogen dries up;
(2) add the antibody of HRP mark.To the antibody-solutions that adds the HRP mark that 1.5 μ L are corresponding in respective aperture, incubated at room 1.5h, PBS washing, nitrogen dries up.
(3) add substrate.Each Kong Jun adds 1.5 μ L Chemoluminescent substrate luminol-hydrogen peroxide.
(4) detect.With CCD, detect immediately, exposure interval 1min, detection of dynamic 10min, collects chemical signal.
(5) calculating of sample size.According to typical curve (see figure 5), the content of ox-Lp (a), ox-LDL in difference calculation sample.
Embodiment 6: the accuracy of the method for the invention and precision
(1) accuracy is selected 1 part of human serum, under optimum experimental condition, measure concentration, then respectively to ox-LDL, ox-Lp (a) sterling that adds variable concentrations in serum, ox-Lp (a) and ox-LDL measure average recovery rate and are respectively 99.3% and 99.6%.
(2) precision is used respectively the serum of basic, normal, high 3 kinds of variable concentrations, and 10 hole replicate determinations repeat 5 times, and ox-Lp (a) and ox-LDL measure average variation within batch (CV) and be respectively 6.2% and 5.2%, and between average batch, CV is 8.1%.
Embodiment 7: the methodology of clinical blood serum sample is to when analyzing
Use the method for the invention to measure 46 routine acute coronary syndrome (ACS) patients, 58 routine stable coronary artery disease (SCAD) patients and 61 routine normal healthy controls crowds' serum ox-Lp (a), ox-LDL level, and contrast with the ELISA method measurement result of standard, result shows, ACS group and SCAD group serum ox-Lp (a), ox-LDL level are all higher than Normal group, and ACS organizes ox-Lp (a), ox-LDL level is all significantly higher than SCAD group:
Ox-Lp (a): ACS group (0.49 ± 0.50U/mL); SCAD group (0.26 ± 0.30U/mL); Control group (0.17 ± 0.18U/mL);
Ox-LDL:ACS group (4.67 ± 2.73U/mL); SCAD group (3.10 ± 1.96U/mL); Control group (1.55 ± 1.00U/mL).
The equal height correlation (see figure 6) of ox-Lp (a), ox-LDL result that adopts chemiluminescence imaging immunoassay and ELISA method to measure.
To sum up, the chemiluminescence imaging immune analysis method of foundation is sensitive, accurate, reliable, can detect serum ox-Lp (a), ox-LDL level simultaneously, is suitable for clinical detection.
Claims (10)
1. a chemiluminescence imaging immune analysis method that simultaneously detects multiple oxidation lipoprotein content in blood serum sample, is characterized in that comprising following steps:
Step 1: on the microslide array of silylation modification, coated capture antibody, incubation, washing, sealing, make insolubilized antibody;
Step 2: add corresponding standard serial solution, incubation, washing in the micropore of the insolubilized antibody of preparing to step 1; The antibody that adds corresponding horseradish peroxidase-labeled, incubation, washing; Then add chemical luminous substrate luminol-hydrogen peroxide, by CCD, carry out the detection of chemiluminescence image; According to concentration of standard solution and corresponding chemiluminescence intensity value drawing standard curve;
Step 3: add corresponding test serum sample in the micropore of the insolubilized antibody of step 1 preparation, incubation, washing, add the antibody of corresponding horseradish peroxidase-labeled, incubation, washing; Then add chemical luminous substrate luminol-hydrogen peroxide, by CCD, carry out the detection of chemiluminescence image; The typical curve of drawing according to step 2, calculates the different concentration that is oxidized lipoprotein in sample.
2. chemiluminescence imaging immune analysis method according to claim 1, is characterized in that, the capture antibody described in step 1 is the anti-human OxLDL ELISA antibody of rabbit.
3. chemiluminescence imaging immune analysis method according to claim 2, is characterized in that, the concentration of the anti-human OxLDL ELISA antibody of described rabbit is 0.33 μ g/mL.
4. chemiluminescence imaging immune analysis method according to claim 1, is characterized in that, described multiple oxidation lipoprotein is oxidation Lp(a) and OxLDL ELISA.
5. chemiluminescence imaging immune analysis method according to claim 1, is characterized in that, the standard serial solution described in step 2 is fresh mix serum stoste and doubling dilution solution thereof.
6. chemiluminescence imaging immune analysis method according to claim 5, is characterized in that, in fresh mix serum stoste, is oxidized the content of Lp(a) and OxLDL ELISA by the Cu of purifying
2+lp(a) and the Cu of oxidation
2+the low-density lipoprotein of oxidation is demarcated.
7. chemiluminescence imaging immune analysis method according to claim 1, it is characterized in that, the antibody of the horseradish peroxidase-labeled described in step 2 and 3 is respectively the apolipoprotein B polyclonal antibody of the apolipoprotein of horseradish peroxidase-labeled (a) monoclonal antibody and horseradish peroxidase-labeled.
8. chemiluminescence imaging immune analysis method according to claim 7, it is characterized in that, the dilutability of the apolipoprotein B polyclonal antibody of the apolipoprotein of described horseradish peroxidase-labeled (a) monoclonal antibody and horseradish peroxidase-labeled is 1:100.
9. chemiluminescence imaging immune analysis method according to claim 1, is characterized in that, the confining liquid that in step 1, sealing is used is the 0.01M phosphate buffer containing 10% hyclone, and pH 7.4; The cleansing solution that in step 1,2,3, washing is used is pH 7.4,0.01M phosphate buffer.
10. chemiluminescence imaging immune analysis method according to claim 1, is characterized in that, in step 2 and step 3, the time of first step incubation reaction is 1h, and the time of second step incubation reaction is 1.5h.
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