CN1773283A - Integrated testing method for protein marker rolated to predicting cardiocerebrovascular diseases accuring - Google Patents
Integrated testing method for protein marker rolated to predicting cardiocerebrovascular diseases accuring Download PDFInfo
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Abstract
The present invention adopts liquid-phase chip method, on the polystyrene fluorescent microbead the correspondent antibody of protein marker related to cardiovascular disease and cerebrovascular disease can be coated, and can be used for capturing correspondent protein marker in the tested liquor, and the detection antibody of every protein marker can be used to determine concentration of every protein marker in the sample. The invented integrated detection can accurately obtain the information of several indexes within a short time, and its sensitivity is above 80%.
Description
Technical field
The present invention relates to the detection technique of protein, be specifically related to take place the detection technique of relevant protein marker with cardiovascular and cerebrovascular diseases.
Background technology
Cardiovascular and cerebrovascular disease is one of topmost factor of harm China's people's health.Along with rolling up of people at highest risk, predict the generation of cardiovascular and cerebrovascular diseases rapidly and accurately and judge that the prognosis situation of cardiovascular and cerebrovascular diseases becomes a difficult problem that presses for solution.Thought in the past and just can assess the onset risk of cardiovascular and cerebrovascular disease, assessed the patient that may miss 50% acute coronary artery syndrome if only rely on traditional hazards by conventional risk factors such as blood pressure, blood sugar, blood fat and smokings.Along with going deep into of research, some protein become the focus of research as new hazards and mark.The progress of modern detecting and the development of proteomics make and set about studying the pathogenetic forecasting tool of cardiovascular and cerebrovascular from protein markers and become possibility.Because existing detection method such as immunoturbidimetry major part can only detect single index, and the accuracy rate of single index is lower, must repeatedly test repeatedly if assess a plurality of indexs, is not easy to clinical the development.High-sensitive C-reactive protein (hs-CRP), homocysteine (Hcy), lipoprotein a (LP (a)), TpP (TpP), D-dimer (D-dimer), fibrinogen (Fig), the von Willebrand factor (vWF) etc. is considered to predict coronary heart disease, the independent hazard factor of cerebral apoplexy, inquire into the clinical value of these protein markers, illustrate its combined action in the cardiovascular and cerebrovascular disease event prediction, study the integrated protein markers detection technique of a kind of height, the detection method of multiple index is unified on the platform, can be easy in the short period of time, fast, obtain the information of a plurality of even up to a hundred indexs accurately, for predicting that accurately the cardiovascular and cerebrovascular incident provides comprehensive foundation, find the high-risk object of cardiovascular and cerebrovascular diseases early, in time effectively intervene, reduce the generation of cardiovascular and cerebrovascular disease incident, be one and have Study on Value problem of crucial importance.
Summary of the invention
The present invention aims to provide the integrated detection technique that relevant protein marker takes place for a kind of and prediction cardiovascular and cerebrovascular diseases, thereby it is easy, analyze the variation of protein marker relevant with the generation of cardiovascular and cerebrovascular incident in the human body quickly, to find the high-risk object of cardiovascular and cerebrovascular diseases early, in time effectively intervene, reduce the generation of cardiovascular and cerebrovascular incident.
Realize that foregoing invention purpose technical scheme is: a kind of integrated testing method of the protein marker relevant with the generation of prediction cardiovascular and cerebrovascular diseases, comprise determining of associated protein mark, the preparation of capture antibodies, detect the preparation of antibody and the mensuration of sample, the hs-CRP relevant in the same sample of one-time detection of the present invention with cardiovascular and cerebrovascular diseases, Hcy, LP (a), TpP, D-dimer, protein marker more than three kinds or three kinds among Fig and the vWF, concrete steps are: the preparation of (1) capture antibodies, the monoclonal antibody of determining of protein marker more than three kinds or three kinds is coated on the polystyrene fluorescence microballon solid matrix, with the bag of sheep anti-mouse igg-determination of biotin antibody by efficient, (2) preparation of detection antibody, in addition will be above-mentioned the monoclonal antibody adding BAC-S-NHS of another different epitopes of protein marker more than three kinds or three kinds, carry out biotin labeling, measure antibody content behind the purifying, calculate biotin labeling efficient, (3) mensuration of sample: wrap by the protein marker microballon suspension of good antibody in the liquid-phase system of 96 hole suction filtration plates [1], [2] add the antigen standard items of blood serum sample or various protein marker variable concentrations in the suction filtration plate respectively, [3] the detection antibody of the biotin labeled protein marker of adding, [4] PE dyeing back liquid phase protein chip reading apparatus detects.
Be described in further detail the present invention below in conjunction with accompanying drawing.
Description of drawings
The relevant integrated detection technique process flow diagram of protein marker takes place with cardiovascular and cerebrovascular diseases in Fig. 1;
Fig. 2 Bio-plex liquid phase protein chip reading apparatus detects bag by the fluorescent value of microballon of monoclonal antibody;
Fig. 3 crosses A280 and the A500 detection that post is collected 10 pipe labelled antibody percolation liquid;
Fig. 4 ultraviolet-visible spectrophotometer detects the 4th, the 5 pipe labelled antibody A280 and the A500 values of collecting;
A280 value (E280) when Fig. 5 UV spectrophotometer measuring IgG antibody concentration is 1mg/ml;
Fig. 6 biotin typical curve.
1, with cardiovascular and cerebrovascular diseases determining of relevant protein markers takes place:
Coronary heart disease (myocardial infarction, angina pectoris) and cerebral apoplexy are main cardiovascular and cerebrovascular diseases, and hs-CRP, Hcy, LP (a), TpP, D-dimer, Fig and vWF are considered to predict the independent hazard factor of coronary heart disease, cerebral apoplexy.Integrated detection technique selects wherein the protein more than 3 kinds or 3 kinds as detecting mark.
2, the design of the integrated detection technique of protein marker:
Adopt the liquid-phase chip system that the protein marker monoclonal antibody more than 3 kinds or 3 kinds is coated on the different color numbering microballon, these microballons are suspended in the liquid-phase system, utilize this system that the protein marker more than 3 kinds or 3 kinds in the same sample is detected simultaneously, it is as follows specifically to detect step:
Liquid phase suspension microballon method detects step:
2.1, the polystyrene fluorescence microballon bag quilt of multiple protein mark monoclonal antibody
Get 300～700 μ l microballons, the centrifugal 4min of 14000g abandons supernatant, add 240～560 μ l microballon lavation buffer solutions, concussion 30s, the ultrasonic 30s of 50Hz, the centrifugal 4min of 14000g, abandon supernatant, add 240～560 μ l microballons activation damping fluid, concussion 30s, the ultrasonic 30s of 50Hz, add 30～70 μ l EDC and 30～70 μ l S-NHS, 1400 change concussion 20min, the centrifugal 4min of 14000g, abandon supernatant, add 500 μ l～1ml PBS washing, concussion 30s, the ultrasonic 30s of 50Hz, the centrifugal 4min of 14000g, abandon supernatant, 300～700 μ l PBS are resuspended in adding, are distributed into 3～7 pipes, every pipe adds each 9 μ g of 3～7 kinds of protein marker monoclonal antibodies respectively, replenish PBS to every pipe 500 μ l final volume, 4 ℃, 1400 change concussion spends the night; The centrifugal 4min of 14000g, abandon supernatant, every pipe adds 500 μ l PBS washing respectively, add 250 μ l sealing damping fluid, 1400 change concussion 20min, and every pipe adds the washing of 500 μ l store buffer liquid respectively, the centrifugal 6min of 16000g, abandon supernatant, it is resuspended that every pipe adds 150 μ l store buffer liquid respectively, detects the bag of antibody by efficient with sheep anti-mouse igg-biotin.
2.2, the biotin labeling of multiple protein mark monoclonal antibody
The monoclonal antibody of 3～7 kinds of another different epitopes of protein marker is diluted to 1mg/ml respectively, add 7.6 μ lBAC-S-NHS (5mg/ml), 1400 change concussion 30min, the antibody of mark biotin is crossed sephadex-G25 post purifying, collect 10 pipe percolation liquid successively, every pipe 1ml, ultraviolet spectrophotometer is measured antibody content (A280 value), A280 value (E280) when mensuration IgG antibody concentration is 1mg/ml, computing formula is: albumen (mg/ml)=A280/E280; Measure the biotin content (A500 value) of antibody labeling; Calculate the ratio (mole molecular proportion) of the mark biotin and the albumen of 3～7 kinds of protein marker monoclonal antibodies, the biotin labeling efficient of expression antibody.
2.3, the typical curve of protein marker antigen standard items is set up and the detection of blood serum sample
Various protein marker antigen standard items press 400,200,100,50,25,10,5, the concentration production standard curve of 0ng/L.Every hole adds 100 μ l bag by good microballon (dilution in 1: 100) in 96 orifice plates, lavation buffer solution washing 20 times, suction filtration adds the various protein marker antigen standard items of 100 each gradient concentration of μ l, make 3 repeating holes, other hole added 100 μ l test serum samples (1: 10
5Dilution), 600 change concussion 1h; Suction filtration, lavation buffer solution washing 3 times adds biotin labeled detection antibody 100 μ l (2 μ g), and 600 change concussion 30min; Suction filtration, lavation buffer solution washing 3 times adds 50 μ l PE (dilution in 1: 25), and 600 change concussion 10min; Suction filtration, lavation buffer solution washing 3 times, it is resuspended to add 125 μ l analysis buffer; Adopt Bio-plex liquid phase protein chip microballon reading apparatus to detect.
Microballon lavation buffer solution (Bead wash buffer); Microballon activation damping fluid (Bead activation buffer); PBS, PH 7.4; Sealing damping fluid (Blocking buffer); Store buffer liquid (Staining buffer); Above-mentioned solution is provided in the kit by the microballon bag of Bio-rad company.
0.1M phosphate buffer (0.1M Sodium Phosphate Buffer); Biotinylation reagent (BiotinylationReagent); Pronase (Pronase); Provide in the antibody biotin labeling kit by Sigma company.
Bio-plex liquid phase protein chip microballon reading apparatus and supporting at random analysis software, the monoclonal antibody of various protein markers and antigen standard items are available from Biodesign company, polystyrene fluorescence microballon, the microballon bag by kit available from Bio-rad company, the biotin labeling kit of antibody is available from Sigma company
The sensitivity of the integrated detection technique diagnosis of coronary heart disease of protein marker of the present invention and specificity obviously are better than the immunoturbidimetry (experimental result sees Table 1) that single index detects.
Table 1 liquid-phase chip method and immunoturbidimetry are relatively to the diagnostic value of CHD
| Mark | Detection method | Sensitivity (%) | Specificity (%) |
| 3 kinds of markers in detecting of lipoprotein a fibrinogen high-sensitive C-reactive protein | Immunoturbidimetry immunoturbidimetry immunoturbidimetry liquid phase protein chip method | 73.9 56.5 63.0 88.7 | 70.3 75.7 86.5 93.6 |
Lipoprotein (a) (Lp (a)), fibrinogen (Fig), high-sensitive C-reactive protein (hs-CRP) joint-detection are higher than detection method Lp (a) (70.3%), Fig (75.7%), the hs-CRP (86.5%) of single index to the specificity (93.6%) of patients with coronary heart disease diagnosis; Joint-detection is higher than detection method Lp (a) (73.9%), Fig (56.5%), the hs-CRP (63.0%) of single index to the sensitivity (88.7%) of patients with coronary heart disease diagnosis; This method has improved the sensitivity of diagnosis of coronary heart disease and specificity, obviously is better than single index detection method.
The present invention has set up the integrated detection technique that relevant protein marker takes place for a kind of and prediction cardiovascular and cerebrovascular diseases, and is not only easy, quick but also significantly improved the accuracy rate predicted is taken place cardiovascular and cerebrovascular diseases.
Embodiment
The integrated testing method of 3 kinds of associated protein marks of diagnosis of coronary heart disease:
1, research object test serum sample collecting:
Be in hospital in the court a year May in October, 2004-2005, have uncomfortable in chest, symptoms such as pectoralgia, doubt and examine patients with coronary heart disease 240 examples, get rid of hepatic and kidney function obstacle, infect, tumour, disease of immune system, coagulation disorders and the major operation history is arranged in the recent period, the acute myocardial infarction AMI acute stage, serum paraoxonase creatine phosphate kinase (CPK) raise greater than 2 times of normal values with the first-class situation that may influence hs-CRP after, have 100 routine conformance with standard and include research in, the male sex's 59 examples wherein, women's 41 examples, 59 ± 11 years old age, the hypertension of record patient simultaneously, diabetes, high fat of blood, the past myocardial infarction medical history.All patients all accept the coronarography inspection.CHD group: coronarography shows to circle round in left trunk, left anterior descending branch, a left side, at least 1 diameter stenosis degree 〉=50%, totally 53 examples in right coronary artery 4 branch vessels; Non-CHD group: coronarography shows that all blood vessel diameter stenosis＜50% are classified as control group, totally 47 examples; The patient takes a blood sample equal early morning on an empty stomach.
2, the selection of protein marker: lipoprotein (a), fibrinogen and high-sensitive C-reactive protein detect mark as 3 kinds of associated protein of the integrated detection technique of diagnosis of coronary heart disease;
3, liquid phase suspension microballon method (liquid phase protein chip method) detection method:
3.1, the polystyrene fluorescence microballon bag quilt of preparation-3 kind of the protein marker monoclonal antibody of capture antibody:
Get 300 μ l microballons, the centrifugal 4min of 14000g abandons supernatant, add 240 μ l microballon lavation buffer solutions, concussion 30s, the ultrasonic 30s of 50Hz, the centrifugal 4min of 14000g abandons supernatant, adds 240 μ l microballons activation damping fluid, concussion 30s, the ultrasonic 30s of 50Hz adds 30 μ l EDC and 30 μ l S-NHS, 1400 rev/mins, concussion 20min, the centrifugal 4min of 14000g abandons supernatant, add 500 μ l PBS washing, concussion 30s, the ultrasonic 30s of 50Hz, the centrifugal 4min of 14000g, abandon supernatant, it is resuspended to add 300 μ l PBS, is distributed into 3 pipes, and every pipe adds lipoprotein (a) respectively, the monoclonal antibody 7 μ g of fibrinogen and high-sensitive C-reactive protein, 12 μ g, 9 μ g, replenish PBS to every pipe 500 μ l final volume, 4 ℃, 1400 rev/mins, concussion is spent the night; The centrifugal 4min of 14000g abandons supernatant, and every pipe adds 500 μ l PBS washing respectively, add 250 μ l sealing damping fluid, 1400 rev/mins, concussion 20min, every pipe add the washing of 500 μ l store buffer liquid respectively, the centrifugal 6min of 16000g, abandon supernatant, it is resuspended that every pipe adds 150 μ l store buffer liquid respectively, detects the bag of antibody by efficient with sheep anti-mouse igg-biotin, the result show the bag detected fluorescent value be 25500-26500FI, considerably beyond 2000FI minimum bag by fluorescent value.See Fig. 2 and table 2.
The highest mean fluorecence value that the per 100 μ l microballons of table 2 wrap 3 kinds of monoclonal antibodies being measured by difference respectively
| The antibody title | The antibody amount (μ g) of per 100 μ l microballon bag quilts | The highest mean fluorecence value (FI) |
| Lipoprotein (a), fibrinogen high-sensitive C-reactive protein | 7 12 9 | 25709 25655 26227 |
3.2, detect the biotin labeling of preparation-3 kind of the protein marker monoclonal antibody of antibody
The monoclonal antibody of 3 kinds of another different epitopes of protein marker is diluted to 1mg/ml respectively, add 7.6 μ lBAC-S-NHS (5mg/ml), 1400 rev/mins, concussion 30min crosses sephadex-G25 post purifying with the antibody of mark biotin, collects 10 pipe percolation liquid successively, every pipe 1ml, the A280 of ultraviolet-visible spectrophotometer detects protein content, and the plain labeling effciency of A500 detection of biological is seen Fig. 3; Wherein antibody purified mainly concentrates on the 4th, 5 pipes, sees Fig. 4; Ultraviolet spectrophotometer is measured antibody content (A280 value), and the A280 value (E280) when mensuration IgG antibody concentration is 1mg/ml is seen Fig. 5; The biotin typical curve is seen Fig. 6; Computing formula is: albumen (mg/ml)=A280/E280; Measure the biotin content (A500 value) of antibody labeling; Calculate the ratio (mole molecular proportion) of the mark biotin and the albumen of 3 kinds of protein marker monoclonal antibodies, the biotin labeling efficient of expression antibody, the result shows that the biotin labeling efficient of every kind of antibody is about 12, and promptly the antibody protein of every mole of molecule has the biotin of 12 moles of molecules to be labeled.
3.3 the typical curve of 3 kinds of protein marker antigen standard items is set up and the detection of blood serum sample
3 kinds of protein marker antigen standard items are by 400,200,100,50,25,10,5, the concentration production standard curve of 0ng/L, every hole adds 100 μ l bag by good microballon (dilution in 1: 100), lavation buffer solution washing 2 times in 96 orifice plates, suction filtration, add 3 kinds of protein marker antigen standard items of 100 each gradient concentration of μ l, make 3 repeating holes, other hole adds the blood serum sample (1: 10 that 100 μ l patients take a blood sample early morning on an empty stomach
5Dilution), 600 rev/mins, concussion 1h; Suction filtration, lavation buffer solution washing 3 times adds biotin labeled detection antibody 100 μ l (2 μ g), and 600 rev/mins, concussion 30min; Suction filtration, lavation buffer solution washing 3 times, the PE fluorescein (dilution in 1: 25) that adds 50 μ l marked by streptavidin dyes, and 600 rev/mins, concussion 10min; Suction filtration, lavation buffer solution washing 3 times, it is resuspended to add 125 μ l analysis buffer; Adopt Bio-plex liquid phase protein chip microballon reading apparatus to detect.
Testing result: the liquid-phase chip method detects lipoprotein a, fibrinogen and high-sensitive C-reactive protein value in the blood plasma
| Numbering | Sex | Age | LP(a)(mg/L) | Fig(g/L) | Hy-CRP (mg/L) | Coronary heart disease whether |
| 1 | M | 65 | 602 | 4.6 | 5.9 | 1 |
| 2 | M | 74 | 805 | 4.3 | 4.5 | 1 |
| 3 | M | 41 | 433 | 3.2 | 3.0 | 0 |
| 4 | M | 52 | 661 | 2.6 | 5.7 | 1 |
| 5 | M | 81 | 231 | 4.5 | 4.6 | 1 |
| 6 | M | 77 | 953 | 4.3 | 9.4 | 1 |
| 7 | M | 81 | 385 | 4.9 | 4.7 | 1 |
| 8 | M | 77 | 361 | 5.3 | 1.4 | 1 |
| 9 | F | 49 | 331 | 6.7 | 4.8 | 1 |
| 10 | F | 50 | 209 | 3.1 | 2.9 | 0 |
| 11 | F | 62 | 164 | 5.1 | 2.2 | 0 |
| 12 | F | 46 | 437 | 5.6 | 1.7 | 1 |
| 13 | M | 76 | 108 | 3.7 | 3.2 | 0 |
| 14 | F | 67 | 283 | 2.6 | 2.9 | 0 |
| 15 | M | 48 | 362 | 5.0 | 5.5 | 1 |
| 16 | M | 65 | 368 | 4.6 | 5.9 | 1 |
| 17 | M | 53 | 203 | 3.1 | 1.2 | 0 |
| 18 | M | 68 | 631 | 4.5 | 4.9 | 0 |
| 19 | M | 57 | 260 | 3.3 | 3.6 | 0 |
| 20 | M | 65 | 164 | 3.3 | 0.9 | 0 |
| 21 | F | 73 | 485 | 4.1 | 4.7 | 1 |
| 22 | F | 61 | 461 | 5.1 | 6.5 | 1 |
| 23 | M | 62 | 123 | 3.2 | 2.2 | 0 |
| 24 | F | 47 | 352 | 3.5 | 2.8 | 0 |
| 25 | F | 61 | 158 | 3.2 | 1.8 | 0 |
| 26 | F | 64 | 229 | 4.1 | 3.3 | 0 |
| 27 | F | 60 | 308 | 3.6 | 3.4 | 0 |
The continuous table of going up:
| 28 | M | 57 | 352 | 5.3 | 5.6 | 1 |
| 29 | M | 69 | 527 | 3.5 | 4.9 | 1 |
| 30 | M | 69 | 203 | 3.2 | 3.1 | 0 |
| 31 | M | 74 | 362 | 5.4 | 5.9 | 1 |
| 32 | M | 58 | 221 | 4.8 | 5.3 | 1 |
| 33 | F | 61 | 291 | 2.6 | 1.7 | 0 |
| 34 | M | 74 | 1232 | 7.7 | 5.6 | 1 |
| 35 | F | 63 | 178 | 2.8 | 2.6 | 0 |
| 36 | M | 59 | 237 | 1.3 | 3.6 | 0 |
| 37 | M | 56 | 497 | 4.6 | 5.1 | 1 |
| 38 | F | 64 | 323 | 5.2 | 6.8 | 1 |
| 39 | F | 66 | 424 | 5.1 | 2.4 | 0 |
| 40 | F | 57 | 350 | 5.1 | 6.8 | 1 |
| 41 | M | 62 | 236 | 3.2 | 4.2 | 0 |
| 42 | M | 67 | 751 | 5.2 | 4.8 | 1 |
| 43 | M | 70 | 318 | 3.2 | 5.5 | 1 |
| 44 | M | 76 | 360 | 4.8 | 5.4 | 1 |
| 45 | M | 65 | 238 | 2.3 | 3.2 | 0 |
| 46 | F | 71 | 336 | 6.2 | 5.3 | 1 |
| 47 | M | 67 | 210 | 4.2 | 3.8 | 0 |
| 48 | M | 67 | 361 | 5.2 | 5.8 | 1 |
| 49 | F | 69 | 559 | 4.6 | 2.8 | 1 |
| 50 | M | 62 | 140 | 2.1 | 5.3 | 1 |
| 51 | M | 38 | 1714 | 4.5 | 5.9 | 1 |
| 52 | M | 62 | 384 | 4.9 | 5.6 | 1 |
| 53 | F | 61 | 313 | 4.3 | 4.7 | 1 |
| 54 | F | 54 | 292 | 3.4 | 2.3 | 0 |
| 55 | M | 48 | 60 | 3.6 | 1.9 | 0 |
| 56 | M | 58 | 319 | 3.2 | 2.3 | 0 |
| 57 | F | 49 | 276 | 3.8 | 1.7 | 0 |
| 58 | M | 46 | 237 | 6.1 | 3.7 | 0 |
| 59 | M | 47 | 490 | 5.9 | 8.2 | 1 |
| 60 | F | 61 | 1193 | 5.3 | 22.9 | 1 |
| 61 | M | 71 | 996 | 6.2 | 5.9 | 1 |
| 62 | F | 40 | 249 | 3.6 | 4.6 | 1 |
| 63 | F | 67 | 745 | 4.3 | 5.2 | 1 |
| 64 | M | 56 | 218 | 3.0 | 2.3 | 0 |
| 65 | M | 69 | 135 | 3.4 | 2.1 | 0 |
| 66 | M | 54 | 345 | 5.3 | 5.2 | 1 |
| 67 | F | 65 | 243 | 5.2 | 6.7 | 1 |
| 68 | F | 61 | 104 | 2.2 | 2.1 | 0 |
| 69 | M | 81 | 132 | 5.9 | 4.5 | 0 |
| 70 | M | 46 | 223 | 3.3 | 1.6 | 0 |
| 71 | F | 61 | 575 | 8.6 | 8.3 | 1 |
| 72 | M | 77 | 328 | 4.9 | 4.5 | 1 |
The continuous table of going up:
| 73 | F | 59 | 369 | 4.3 | 5.6 | 1 |
| 74 | M | 65 | 387 | 5.5 | 3.5 | 1 |
| 75 | M | 58 | 373 | 4.1 | 5.8 | 1 |
| 76 | F | 66 | 325 | 4.6 | 6.2 | 1 |
| 77 | F | 57 | 258 | 2.3 | 1.8 | 0 |
| 78 | F | 50 | 318 | 3.1 | 2.2 | 0 |
| 79 | M | 40 | 180 | 4.1 | 3.8 | 0 |
| 80 | M | 43 | 277 | 3.5 | 1.9 | 0 |
| 81 | M | 73 | 866 | 6.9 | 12.3 | 1 |
| 82 | F | 70 | 372 | 4.3 | 5.1 | 1 |
| 83 | F | 60 | 200 | 3.7 | 2.1 | 0 |
| 84 | M | 70 | 162 | 3.3 | 2.5 | 0 |
| 85 | M | 72 | 76 | 6.2 | 3.6 | 0 |
| 86 | F | 67 | 198 | 3.2 | 2.3 | 0 |
| 87 | F | 58 | 165 | 4.6 | 5.8 | 1 |
| 88 | M | 36 | 568 | 4.4 | 10.5 | 1 |
| 89 | F | 69 | 269 | 3.4 | 1.6 | 0 |
| 90 | F | 70 | 242 | 3.0 | 3.1 | 0 |
| 91 | M | 70 | 389 | 5.3 | 6.3 | 1 |
| 92 | M | 61 | 226 | 1.4 | 2.7 | 0 |
| 93 | M | 67 | 379 | 5.1 | 6.3 | 1 |
| 94 | F | 61 | 188 | 3.9 | 2.3 | 0 |
| 95 | M | 65 | 478 | 3.2 | 1.9 | 1 |
| 96 | M | 55 | 392 | 4.9 | 5.1 | 1 |
| 97 | F | 75 | 203 | 3.0 | 1.8 | 0 |
| 98 | F | 51 | 196 | 2.3 | 2.8 | 0 |
| 99 | F | 56 | 359 | 3.7 | 3.1 | 0 |
| 100 | M | 66 | 262 | 5.6 | 6.1 | 1 |
Annotate: the coronary heart disease criterion is according to the result of coronary angiography; 1: expression is a coronary heart disease; 0: expression is not a coronary heart disease.
The positive boundary of various detection thing parameters is respectively Lp (a) 〉=300mg/L, Fig 〉=4.0g/L, hs-CRP 〉=4.5mg/L;
Result with coronary angiography is a standard, and the sensitivity of the integrated detection technique diagnosis of coronary heart disease of protein marker of the present invention is 88.7%; Specificity is 93.6%.
Claims (1)
1, the integrated testing method of relevant protein marker takes place in a kind of and prediction cardiovascular and cerebrovascular diseases, comprises determining of associated protein mark, the preparation of capture antibodies, and the preparation of detection antibody and the mensuration of sample the invention is characterized in:
(1) the associated protein mark that adopts of test sample is the protein marker more than three kinds or three kinds among hs-CRP, Hcy, lipoprotein (a), TpP, D-dimer, fibrinogen and the vWF relevant with cardiovascular and cerebrovascular diseases;
(2) preparation of capture antibodies is coated on the monoclonal antibody of determining of protein marker more than three kinds or three kinds on the polystyrene fluorescence microballon solid matrix, and the bag of using sheep anti-mouse igg-determination of biotin antibody is by efficient;
(3) detect the preparation of antibody, in addition will be above-mentioned more than three kinds or three kinds the monoclonal antibody of another different epitopes of protein marker add BAC-S-NHS, carry out biotin labeling, measure antibody content behind the purifying, calculating biotin labeling efficient;
(4) mensuration of sample:
[1] wrap by the protein marker microballon suspension of good antibody in the liquid-phase system of 96 hole suction filtration plates,
[2] add the antigen standard items of blood serum sample or protein marker variable concentrations in the suction filtration plate respectively,
[3] the detection antibody of the biotin labeled protein marker of adding,
[4] PE dyeing back liquid phase protein chip reading apparatus detects.
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