CN103529224A - Quick quantitative detecting device and method for simultaneously detecting human brain natriuretic peptides and N-terminal pro-brain natriuretic peptides - Google Patents
Quick quantitative detecting device and method for simultaneously detecting human brain natriuretic peptides and N-terminal pro-brain natriuretic peptides Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
Abstract
A quick quantitative detecting device and method for simultaneously detecting human brain natriuretic peptides and N-terminal pro-brain natriuretic peptides are characterized in that the device is composed of human brain natriuretic peptide (BNP) test paper, N-terminal pro-brain natriuretic peptide (NT-ProBNP) test paper, a double link clamping shell and an immune chromatography result interpretation recorder, the BNP test paper and the NT-ProBNP test paper are mutually independent, and corresponding standard curves and judging methods are arranged in the immune chromatography result interpretation recorder. The immune chromatography result interpretation recorder is an optical detection system and is used for quantitative judging of detection results. The detection range for the BNP and the detection range for the NT-ProBNP are 0.1-10ng/mL and 0.2-20ng/mL respectively. The detection is finished in 15-20 minutes. The quick quantitative detecting device and method have the advantages of detecting in a combined mode, and being simple and convenient to operate, quick in response, accurate in result, suitable for on-site detection and the like, and suitable for requirements of clinic for more accurate, simpler and more convenient detection.
Description
Technical field
Patent of the present invention relates to medical clinic applications technical field, particularly relates to a kind of Quantitative detection device and detection method that detects human brain natriuretic peptide and N terminal brain natriuretic peptide when preparing with immune colloid gold method for quick.
Background technology
Brain natriuretic peptide (BNP) is first to be equaled from pig brain, to separate thereby gain the name for 1988 by Japanese scholars Sudoh, also claims B-typeNatriuretic Peptide.Mankind's brain natriuretic peptide (BNP) is distributed widely in the tissues such as brain, heart, lungs, alimentary canal, urogenital tract, wherein the highest with the level of heart.Cardiac muscle cell directly synthesizes BNP, but first synthetic pro-BNP, and pro-BNP has 108 amino acid, in blood, can decompose, and the fragment of decomposing is 76 amino acid that its N holds, i.e. NT-ProBNP, remaining 32 amino acid, i.e. BNP.
Research discovery, BNP has very great help to the early diagnosis of heart failure, early intervention and prognosis.In ESC's heart failure practice guidelines of calendar year 2001 revision, the instrument using brain natriuretic peptide as kit for diagnosing heart failure.Europe in 2005 and the guide of the U.S., all further affirmed the effect of brain natriuretic peptide in kit for diagnosing heart failure.Because BNP and NT-ProBNP come from same precursor, there is close contact between the two.U.S.'s " clinical chemistry " magazine has been delivered one piece of paper in 2007: " BNP and the NT-proBNP diagnosis accuracy comparison in acute and chronic HF ", draws the following conclusions through a large amount of document statistics: BNP and NT-proBNP chemical examination have higher diagnosis accuracy and have higher correlativity in the diagnosis of acute and chronic HF.
From clinical diagnosis angle analysis, the BNP difference main with NT-proBNP is as follows: 1) removing approach is in vivo different: the removing of BNP is mainly degraded by endocytosis and lysosome then by removing receptors bind with natriuretic peptide, only have a small amount of BNP to remove by kidney, when renal function lacks, neutral endopeptidase (NEP) also can be opened the ring texture of BNP and it is removed; The unique channel that NT-proBNP removes is glomerular filtration, and renal function occurs that disappearance is very big to the metabolic effect of NT-proBNP.2) half life period is different: the half life period of BNP is 22 minutes, and the half life period of NT-proBNP is 120 minutes.From the angle of clinical examination, consider, NT-proBNP is comparatively stable in vitro, to detection, brings convenience, but consider from the angle of clinical practice, the half life period that BNP is shorter more can be reacted in time conditions of patients and be changed, and is beneficial to clinical monitoring result for the treatment of, thereby gives the clinical better using value of bringing.3) the cutoff value of BNP and NT-proBNP has very large difference, and the cutoff value that has diagnostic value that BNP generally acknowledges is 0.1ng/mL, and NT-proBNP is 0.3ng/mL.
BNP and NT-ProBNP detect reagent at present has application clinical, but not yet has the quick detection reagent/device that simultaneously detects these two kinds of indexs to occur.Detect the mutual supplement with each other's advantages can realize two reagent simultaneously, testing result is be sure of more, can get rid of the false negative that the false positive that causes because of some cross reaction or molecule isomery cause; Whether be the false positive of the renal function dysbolism NT-ProBNP that cause, can improve the sensitivity to heart failure fast detecting if can analyze.
Summary of the invention
The object of the invention is to provide a kind of Quantitative detection device and detection method that simultaneously detects human brain natriuretic peptide and N terminal brain natriuretic peptide, is used for detecting BNP and NT-ProBNP in serum, blood plasma and whole blood sample.Have easy and simple to handle, reaction fast, result is accurate, credible, be applicable to the advantages such as Site Detection.Summary of the invention is as follows:
Detect Quantitative detection device and the detection method of human brain natriuretic peptide and N terminal brain natriuretic peptide, it is characterized in that device by separate human brain natriuretic peptide (BNP) and N terminal brain natriuretic peptide (NT-ProBNP) Test paper, duplex gets stuck and the immunochromatography sentence read result registering instrument of built-in respective standard curve and decision method forms.
Described pick-up unit, is characterized in that BNP detects reagent test strip and is comprised of the collaurum pad of the anti-human BNP antibody that contains colloid gold label, the NC film, sample pad, suction sample pad and the plastic bottom board that are coated with pairing BNP antibody (T line) and anti-mouse IgG (C line).NT-ProBNP detects reagent test strip and is comprised of the collaurum pad of the anti-human NT-ProBNP antibody that contains colloid gold label, the NC film, sample pad, suction sample pad and the plastic bottom board that are coated with pairing NT-ProBNP antibody (T line) and anti-mouse IgG (C line).The width of each test paper is 3-5mm, and length is 7-8cm.Sample pad, collaurum pad, NC film and suction sample pad are arranged in order to the other end from plastic bottom board one end.
Described pick-up unit, is characterized in that duplex gets stuck and comprises upper cover and two parts of lower cover, inside can two Test papers of parallel placement.The bottom counter sample pad of upper cover of getting stuck is partly provided with sample well and dilution hole, and for adding sample and dilution, the center section of the upper cover that gets stuck reply test paper NC film middle part is provided with viewing window, for observing C, the T line on NC film, result of determination.
Described pick-up unit, is characterized in that described NC film is a kind of film of the porous structure consisting of cellulose nitrate, and aperture is 8-15um.Dilution fluid cushion is glass fibre membrane or nonwoven fabrics, and inhaling sample pad is absorbent filter.
Described pick-up unit, it is characterized in that the NC film preparation process that is coated with pairing antibody is: with 0.01M pH7.4 phosphate buffer (PBS), the BNP of pairing or NT-ProBNP antibody are mixed with to suitable concentration, in spray film instrument parameter with 1.2-1.4ul/cm on NC film, rule, coated T line is coated with the anti-mouse IgG of 1-2mg/ml as C line simultaneously on NC film top.Dry, standby.
Described pick-up unit, the preparation process that it is characterized in that collaurum pad is: get 100ml collaurum liquid and be placed in beaker, with 0.2M K2C03, be adjusted to pH7.0-8.0, then add BNP or NT-ProBNP antibody for 1-2mg mark, stirring at room 1 hour, sealing, centrifugal 30 minutes of 12000rpm, abandons supernatant, with working fluid, redissolves to 60ml, then with metal spraying machine, be sprayed on equably respectively on collaurum pad drying for standby.
Described pick-up unit, its feature in test card assembly method is: in relative humidity, be less than under 30% condition, get plastic bottom board, the NC film of coated BNP or NT-ProBNP is sticked on to the middle part of base plate, in NC film T line one side, paste collaurum pad, at collaurum pad opposite side, paste sample pad; In NC film C line one side, paste and inhale sample pad; Each is pasted component interface and mutually laminates 1-2mm, and the large plate pasting is cut into the wide test strips of 3-5mm.Then two kinds of test strips are positioned over respectively in the groove of the lower cover that gets stuck, then cover upper cover, compress, complete assembling.
Described pick-up unit, it is characterized in that immunochromatography result interpretation registering instrument is a kind of Systems for optical inspection, built-in typical curve and decision method to BNP and NT-ProBNP test paper, quantitative judgement for to testing result, is respectively 0.1-10ng/mL and 0.2-20ng/mL to BNP and NT-ProBNP sensing range.
Described pick-up unit, it is characterized in that detection method is: during detection, the tested sample of 50ul is joined in sample aperture as serum, blood plasma, whole blood, then in dilution hole, add 50ul sample diluting liquid, in 15-20 minute, with immunochromatography result interpretation registering instrument, testing result is judged.
The invention has the beneficial effects as follows: a kind of Quantitative detection device and detection method that simultaneously detects human brain natriuretic peptide and N terminal brain natriuretic peptide is provided, is used for detecting BNP and NT-ProBNP in serum, blood plasma and whole blood sample.Have easy and simple to handle, reaction fast, result is accurate, credible, be applicable to the advantages such as Site Detection.
Accompanying drawing explanation:
Fig. 1 is Test paper structural drawing
Fig. 2 is dual card shell structure figure
Embodiment
Embodiment: the preparation of Quantitative detection device and the utilization of human brain natriuretic peptide and N terminal brain natriuretic peptide
1 main material
1.1 biological raw material BNP and NT-ProBNP pairing antibody ，Cong Finland HYTEST company buy; Mouse-anti human IgG antibody, sheep anti-mouse igg: self-control; Gold chloride: Sigma company product; NC film: Sartorius company product; Caseinhydrolysate, polyglycol PEG20000:Sigma product.Other common agents is analytical reagent.
1.2 clinical samples are obtained in relevant hospital by company, and totally 120 parts, be wherein diagnosed as 60 parts, patients serum's sample of heart failure, BNP detected value is from 0.2-8ng/mL, and NT-ProBNP detected value is from 0.2-20ng/mL.60 parts, normal human serum sample, BNP and NT-ProBNP value do not detect.
1.3 duplexs get stuck: by our company, designed, associated companies is produced on request, provided.
1.4 immunochromatography result knot interpretation registering instruments: model: NS3001, Newscen Coast Bio-Pharmaceutical Co., Ltd.'s product.
2 methods
2.1BNP and NT-ProBNP antibody colloidal gold mark adopt gold chloride-sodium citrate to prepare the colloidal gold solution that diameter is 40 ± 5nm, get three parts of colloidal gold solutions, with 0.2M K2CO3, solution is transferred to pH7.0 respectively, pH7.5 and pH8.0, then collaurum is slowly stirred, by every ml solution, add 5ug, 10ug, 20ug joins BNP labelled antibody in solution, continue to stir 30 minutes, joining final concentration again and be 0.5% PEG2000 and 0.5% caseinhydrolysate seals, it is centrifugal with 12000g after mark finishes, abandon supernatant, precipitation is redissolved to (pH8.0 in the collaurum working fluid of different proportionings by 60% original volume, containing caseinhydrolysate, sheep blood serum, sucrose and surfactant).Then mark colloidal gold solution is pressed to 1ml solution paving 20cm
2ratio application of sample on nonwoven fabrics, at temperature 20-25 ℃, relative humidity, at the dry 3-5 hour of the drying room of < 30%, is made collaurum pad.NT-ProBNP antibody labeling method is consistent with BNP, and the antibody just adding is NT-ProBNP antibody.
2.2NC film is coated is mixed with 0.8 with 0.01M pH7.4PBS by the BNP antibody of pairing, 1.0 and 1.2mg/mL, sheep anti-mouse igg is diluted to respectively 1mg/ml, 2mg/ml, then with spray film instrument, on NC film, by 1.4ul/cm, rule respectively coated, after being coated with by NC film at temperature 20-25 ℃, relative humidity is at the dry 3-5 hour of drying room of < 30%.NT-ProBNP antibody method for coating is consistent with BNP, and just preparation is NT-ProBNP antibody.
2.3 under relative humidity is less than 30% condition, gets plastic bottom board, the NC film of coated BNP or NT-ProBNP is sticked on to the middle part of base plate, in NC film T line one side, pastes collaurum pad, at collaurum pad opposite side, pastes sample pad; In NC film C line one side, paste and inhale sample pad; Each is pasted component interface and mutually laminates 1-2mm, and the large plate pasting is cut into the wide test strips of 3-5mm.Then two kinds of test strips are positioned over respectively in the groove of the lower cover that gets stuck, then cover upper cover, compress, complete assembling.
2.4 test card technological parameters debugging by concentration not isolabeling, coated reagent combine pairing, prepare sample, utilize quality-control product to test reagent, searching best of breed.
2.5 built-in typical curve curves and parameter setting determine after test paper technological parameter, use respectively 0,0.1,0.5,1.5,4, the BNP standard items of 8ng/mL are measured reagent, and the standard items of variable concentrations demonstrate varying strength colour band, with immunochromatography interpretation registering instrument by after the color band digital of respective strengths, calculate typical curve, and input in immunochromatography result interpretation registering instrument, complete the setting of BNP typical curve parameter; Use respectively 0,0.2,0.5,2,8, the NT-ProBNP standard items of 20ng/mL are measured reagent, the standard items of variable concentrations demonstrate varying strength colour band, with immunochromatography interpretation registering instrument, by after the color band digital of respective strengths, calculate typical curve, and input in immunochromatography result interpretation registering instrument, complete the setting of NT-ProBNP typical curve parameter.The Cutoff value of BNP is made as to 0.1ng/mL, and the Cutoff value of NT-ProBNP is made as 0.3ng/mL, if two kinds of detections are all negative, synthesis result is judged to be feminine gender, if two kinds of result test positive, synthesis result is judged to be the positive.If two kinds of results detect as inconsistent, be judged to be result suspicious, complete the setting of result critical parameter.
2.6 detection methods 1) will detect reagent and sample balance to room temperature, take out test card, keep flat; 2) accurately draw respectively 50 μ l serum, blood plasma or whole blood sample, join in two sample aperture, in the dilution hole of bottom, add respectively immediately 50 μ L sample dilutions (PBS), in 15-20 minute, with immunochromatography result interpretation record, quantitatively judge result; 3) set and test card is put into storehouse after instrument correlation parameter and detect, instrument will show the quantitative measurement result that goes out respectively BNP and NT-ProBNP sample concentration; And the comprehensive detection result to heart failure.
2.7 clinical samples detection reagent detect all clinical samples by detection method after having prepared, and analyzing and testing result.
3 results
3.1 test paper parameters are determined according to the testing result of sample, have determined that BNP and NT-ProBNP antibody labeling pH value are for 7.0-8.0, no significant difference; Optimum mark amount is 5-10ug/ml colloidal gold solution; Best collaurum working fluid is 0.1MTris.Cl damping fluid, and pH7.5, containing 0.2% caseinhydrolysate, 6% sucrose, 0.5%Tween20; The concentration of best bag antigen is that BNP is 0.8mg/mL, and NT-ProBNP is 1.2mg/mL.The optimal decision time of testing result is 15-20 minute.But above parameter when preparing different batches product because biological raw material activity change may need suitable adjustment.
3.2 clinical samples detect 120 parts of clinical samples are detected, and two kinds of detections are all positive is 58 examples, and all negative is 57 examples, compares with clinical, and positive, negative desired value is 100%.Other 5 examples are suspicious result.The concordance rate of synthesis result is 95.8%.Compare with independent detection, concordance rate is suitable, but positive, negative desired value obviously rises.In quantitative context of detection, correlation coefficient r is all greater than 0.95, t check P > 0.05, inconsistent detection outlier < 2%.Quantitatively detect effect better.By above result, can judge the functional of pick-up unit, have easy and simple to handle, reaction fast, result is accurate, credible, be applicable to the advantages such as Site Detection, is applicable to clinical in needs more accurate, easy detection.
Claims (9)
1. detect Quantitative detection device and the detection method of human brain natriuretic peptide and N terminal brain natriuretic peptide simultaneously, it is characterized in that device by separate human brain natriuretic peptide (BNP) and N terminal brain natriuretic peptide (NT-ProBNP) Test paper, duplex gets stuck and the immunochromatography sentence read result registering instrument of built-in respective standard curve and decision method forms.
2. pick-up unit according to claim 1, is characterized in that BNP detects reagent test strip and is comprised of the collaurum pad of the anti-human BNP antibody that contains colloid gold label, the NC film, sample pad, suction sample pad and the plastic bottom board that are coated with pairing BNP antibody (T line) and anti-mouse IgG (C line).NT-ProBNP detects reagent test strip and is comprised of the collaurum pad of the anti-human NT-ProBNP antibody that contains colloid gold label, the NC film, sample pad, suction sample pad and the plastic bottom board that are coated with pairing NT-ProBNP antibody (T line) and anti-mouse IgG (C line).The width of each test paper is 3-5mm, and length is 7-8cm.Sample pad, collaurum pad, NC film and suction sample pad are arranged in order to the other end from plastic bottom board one end.
3. pick-up unit according to claim 1, is characterized in that duplex gets stuck and comprises upper cover and two parts of lower cover, inside can two Test papers of parallel placement.The bottom counter sample pad of upper cover of getting stuck is partly provided with sample well and dilution hole, and for adding sample and dilution, the center section of the upper cover that gets stuck reply test paper NC film middle part is provided with viewing window, for observing C, the T line on NC film, result of determination.
4. according to the pick-up unit described in claim 1 and 2, it is characterized in that described NC film is a kind of film of the porous structure consisting of cellulose nitrate, aperture is 8-15umm.Dilution fluid cushion is glass fibre membrane or nonwoven fabrics, and inhaling sample pad is absorbent filter.
5. pick-up unit according to claim 1, it is characterized in that the NC film preparation process that is coated with pairing antibody is: with 0.01M pH7.4 phosphate buffer (PBS), the BNP of pairing or NT-ProBNP antibody are mixed with to suitable concentration, in spray film instrument parameter with 1.2-1.4ul/cm on NC film, rule, coated T line is coated with the anti-mouse IgG of 1-2mg/ml as C line simultaneously on NC film top.Dry, standby.
6. according to pick-up unit described in claim 1 and 2, the preparation process that it is characterized in that collaurum pad is: get 100ml collaurum liquid and be placed in beaker, with 0.2M K2C03, be adjusted to pH7.0-8.0, then add BNP or NT-ProBNP antibody for 1-2mg mark, stirring at room 1 hour, sealing, centrifugal 30 minutes of 12000rpm, abandons supernatant, with working fluid, redissolves to 60ml, then with metal spraying machine, be sprayed on equably respectively on collaurum pad drying for standby.
7. according to the pick-up unit described in claim 1 and 2, its feature in test card assembly method is: in relative humidity, be less than under 30% condition, get plastic bottom board, the NC film of coated BNP or NT-ProBNP is sticked on to the middle part of base plate, in NC film T line one side, paste collaurum pad, at collaurum pad opposite side, paste sample pad; In NC film C line one side, paste and inhale sample pad; Each is pasted component interface and mutually laminates 1-2mm, and the large plate pasting is cut into the wide test strips of 3-5mmm.Then two kinds of test strips are positioned over respectively in the groove of the lower cover that gets stuck, then cover upper cover, compress, complete assembling.
8. pick-up unit according to claim 1, it is characterized in that immunochromatography result interpretation registering instrument is a kind of Systems for optical inspection, built-in typical curve and decision method to BNP and NT-ProBNP test paper, quantitative judgement for to testing result, is respectively 0.1-10ng/mL and 0.2-20ng/mL to BNP and NT-ProBNP sensing range.
9. pick-up unit according to claim 1, it is characterized in that detection method is: during detection, the tested sample of 50ul is joined in sample aperture as serum, blood plasma, whole blood, then in dilution hole, add 50ul sample diluting liquid, in 15-20 minute, with immunochromatography result interpretation registering instrument, testing result is judged.
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|---|---|
| CN103529224B (en) | 2016-09-07 |
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