CN106370844A - Dual colloidal gold test strip for detecting human bone metabolic biomarker and detection device applying same - Google Patents
Dual colloidal gold test strip for detecting human bone metabolic biomarker and detection device applying same Download PDFInfo
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- G—PHYSICS
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- 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
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- 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
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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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Abstract
The invention belongs to the field of clinical laboratory techniques and in particular relates to a colloidal gold test strip for quantitatively detecting human bone metabolic biomarker and a detection device applying same. The test strip provided by the invention comprises a sample pad, a glass fiber mat coated with gold-labeled streptavidin, a glass fiber mat coated with a gold-labeled antibody, a nitrocellulose membrane and an absorbent pad which are sequentially adhered with one another on a bottom plate in a staggered manner. A quality control line pre-enveloped with a goat anti-rat IgG antibody is formed in the nitrocellulose membrane; a detection line parallel to the quality control line is formed in the nitrocellulose membrane; an antibody capable of being specifically bound with a to-be-detected antigen human bone metabolic biomarker is coated on the detection line; the gold-labeled antibody is an antibody capable of being specifically bound with the to-be-detected antigen human bone metabolic biomarker. According to the test strip provided by the invention, the human bone metabolic biomarker can be quantitatively detected, the requirement on instruments is low, the time of clinically detecting the human bone metabolic biomarker is shortened, and the detection complexity and cost can be reduced.
Description
Technical field
The invention belongs to clinical medical inspection field is and in particular to a kind of be based on double colloidal gold probe detection by quantitative people's bone generations
The colloidal gold strip thanking to mark and the detection means applying it.
Technical background
World Health Organization (WHO) is that bone mineral content is relatively low to the definition of osteoporosises (osteoporosis, op), and skeleton is micro-
Structural change, easily destroyed, skeleton toughness reduces, and is susceptible to fracture.Whole world osteoporosis and relative
Complication and sequela patient reach more than 200,000,000 people.Osteoporotic sickness rate growth rate is accelerated year by year.According to nearest system
Meter, its global incidence comes the 7th of all chronic diseases.
Clinically diagnosis of osteoporosis most common means are bone density (bmd) detections, and most popular method is that dual energy x-ray is inhaled
Receive instrument (dexa) detection.But, the specific standards of bmd yet suffer from larger dispute, and ethnic group, region, sex are to the standard of bmd all
There is certain impact.Additionally, bmd detection is typically all body local detection it is difficult to provide human body comprehensive bone strength information.
Another defect of bmd detection is not good to the Function of Evaluation of osteoporosis therapy effect.The interval of the adjacent detection of bmd twice is
Need 3 months less, therefore cannot react the effect of osteoporosis therapy in time.
Multiple biochemical substances can produce in bone resorption and bone restructuring procedure, and enters into blood, urine or other body fluid.
These materials are referred to as Bone markers.Deepen continuously and many new diagnosing osteoporosis with study to osteoporosises
With the appearance for the treatment of meanss, the degree of concern more and more higher to Bone markers for the people.It is now recognized that Bone markers can
To assess patient bone change over condition, the risk of prediction postmenopausal osteoporosis patients with fractures and assessment patient with bone disease medicine
Curative effect after thing treatment, being one has certain dependency with bmd, but the bone strength phase independent of bmd
Close mark.
Bone markers can be divided into two big class, and bone formation mark, including serum osteocalcin (s-oc), serum bone alkali
Acid phosphatase (s-balp);Serum t type procollagen n end propetide (s-pinp);Serum i type procollagen c end propetide (s-picp);Bone
Absorption marker thing, including Pyridinoline (u-ptd);Urine i Collagen Type VI crosslinking n terminal peptide (u-ntx);Urine i Collagen Type VI crosslinking c end
Peptide (u-ctx);Serum i Collagen Type VI crosslinking n- terminal peptide (s-ntx);Urinary DPD (u-dpd);The anti-tartaic acid of serum
Phosphatase 5b (s-tracp-5b);Serum i Collagen Type VI crosslinking c terminal peptide (s-ctx).
Clinically generally use electro-chemistry immunity method and elisa method detection Bone markers, the detection of both approaches
Overlong time, typically more than 45 minutes, the detection time of elisa method is typically at 1 hour for the detection time of electro-chemistry immunity method
More than 30 minutes.And, the operation of both the above method is complex, need the reviewer of specialty could operate.
Content of the invention
Present invention aims to the deficiencies in the prior art, based on colloidal gold-labeled method and Streptavidin-life
A kind of thing element amplification system, there is provided double gold colloidals of quantitative, quick, easy, highly sensitive detection people's Bone markers
Test strips and the detection means applying it, time-consuming, complex operation, instrument and equipment have high demands can to solve existing detection technique
Problem.This test strips can complete the Bone markers detection by quantitative in human serum sample in 10 minutes, sensitive quick, one-tenth
This is low, miniaturization instrument, specimen in use amount are few, has broad application prospects in clinical medical inspection field.
The purpose of the present invention can be achieved by employing the following technical solutions:
The invention provides a kind of people's Bone markers detection is with double colloidal gold strips, including test strips end liner, sample
Product pad, the first pad, the second pad, nitrocellulose filter and absorption pad are it is characterised in that suitable on described test strips end liner
Sample pad that secondary mutual overlap joint is pasted, the first pad, the second pad, nitrocellulose filter and absorption pad, described nitric acid
Cellulose membrane is provided with a nature controlling line being coated sheep anti mouse igg antibody in advance, nitrocellulose filter is additionally provided with and above-mentioned matter
The parallel detection line of control line, detection line is coated with the antibody being combined with people's Bone markers antigenic specificity to be measured, institute
The first pad stated is coated with gold mark Streptavidin, and described gold mark Streptavidin can be with gold mark biotinylated antibody
Specific bond, the second described pad is coated with gold mark biotinylated antibody, and described gold mark biotinylated antibody is can be with
The antibody that people's Bone markers antigen-specific to be measured combines.
Further, the described antibody being combined with people's Bone markers antigenic specificity to be measured is can be with people's bone to be measured
The monoclonal antibody that metabolic markers antigenic specificity combines.
Further, described detection line is to be coated anti-human Bone markers monoclonal antibody.
Further, described gold mark biotinylated antibody is the biotinylated mAb of colloid gold label.
Further, described gold mark Streptavidin preparation method is: takes colloidal gold solution, is adjusted using solution of potassium carbonate
Section ph value;Add Streptavidin;Peg-20000 is added after incubation 15min under room temperature;Centrifugation, resuspended, 4 DEG C of holdings after mixing
Standby.
Further, described gold mark biotinylated antibody preparation method is: takes colloidal gold solution, using solution of potassium carbonate
Adjust ph value;Add anti-human Bone markers monoclonal antibody;It is slowly stirred;4 DEG C of incubations;Add 3 ' end Biotinylated single strand
Nucleotide;4 DEG C of incubations;Add peg-20000;After mixing centrifugation, resuspended, 4 DEG C of holdings are standby.
Present invention also offers a kind of gold colloidal detection means of detection by quantitative people's Bone markers, described detection means
The jam used double colloidal gold strips and wrap up described test strips including above-mentioned people's Bone markers detection, described jam
Further include panel and base plate, described panel is provided with observation groove and adding mouth, described adding mouth is opened on described sample
Pad top, and exposed portion or whole described sample pad, described observation channel opening is on the upside of described nitrocellulose filter, and exposes
All described detection line and control lines.
Further, the panel of described jam and base plate are made using high-density polyethylene material.
This test strips can be used to detect people's Bone markers fluid sample, during operation, whole blood, blood plasma or serum is pressed
A certain amount Deca, in the sample pad of this test strips, test strips is put in the golden scalar quantity reading apparatus supporting with it, reaction
After 10 minutes, draw testing result by golden scalar quantity reading apparatus.
Present invention beneficial effect compared to existing technology is:
Present invention employs most popular colloidal gold technique in modern poct detection, by gold mark Streptavidin and gold mark
Biotinylated antibody amplifies the signal that gold colloidal produces, thus reaching the purpose improving detection sensitivity.The detection of this test strips
Result is accurately, quickly, easy and simple to handle, can be widely applied to the evaluation of diagnosing osteoporosis and medication effect.
Brief description
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.
The cross-sectional configuration schematic diagram of double colloidal gold strips of Fig. 1 present invention;
Fig. 2 is the structural representation of the jam of double colloid gold test paper bar detection devices of the present invention;
Wherein: 1, end liner;2nd, sample pad;3rd, the first pad;4th, the second pad;5th, nitrocellulose filter;6th, absorb water
Pad;7th, it is coated the detection line of anti-human Bone markers antibody;8th, it is coated the nature controlling line of sheep anti mouse igg antibody;201st, jam;
202nd, panel;203rd, base plate;204th, observe groove;205th, adding mouth.
Specific embodiment
Hereinafter explanation is only the illustration to claimed technical scheme, not to these technical schemes
Any restriction.The content that protection scope of the present invention is recorded by appended claims is defined.
Taking one of people's Bone markers mark human serum Bone Gla protein (oc) as a example.It is specifically described the present invention.
The previously prepared monoclonal antibody anti-osteocalcin antibody 1 for human serum Bone Gla protein different loci
(anti-oc mab 1), anti-osteocalcin antibody 2 (anti-oc mab 2).Prepared by 41nm gold colloidal: by 100ml matter
Amount fraction 0.01% chlorauric acid solution is heated to 95 DEG C, is rapidly added 1ml mass fraction 1% citric acid three sodium solution, and fully
Stirring.After mixed solution becomes redness, continue heating 15min.It is cooled to room temperature, 0.22 μm of filtration, using double after stopping heating
Steam water and supply nano-Au solution to 100ml, add 700 μ l 0.1mol/l solution of potassium carbonate.
Prepared by 13nm gold colloidal: 100ml mass fraction 0.01% chlorauric acid solution is heated to 95 DEG C, is rapidly added
3.5ml mass fraction 1% citric acid three sodium solution, and be sufficiently stirred for.After mixed solution becomes redness, continue heating 15min.Stop
It is cooled to room temperature after only heating, 0.22 μm of filtration, supply nano-Au solution to 100ml using distilled water, add 500 μ l
0.1mol/l solution of potassium carbonate.
Described gold mark biotinylated antibody preparation method is: using pbs buffer (ph=8.0,10mmol/l) dilution
Anti- Bone Gla protein monoclonal antibody to concentration is 0.036mg/ml.Take 0.036mg/ml anti-Bone Gla protein monoclonal antibody ab13418
(anti-oc mab 1) 100 μ l, adds to 1ml aunp solution (13nm), is slowly stirred 15min using magnetic stirring apparatuss.Incubate for 4 DEG C
Educate 1h.It is subsequently adding 3 ' end Biotinylated single strand nucleotide solution 10 μ l, mass fraction is 3.3mg/ml.4 DEG C of incubation 12h.?
Afterwards, add 100 μ l 1%peg-20000, under room temperature, stir 15min.4 DEG C of centrifugations of mixed liquor, 10000g × 50min, abandon supernatant,
Add 1ml gold labeling antibody storing liquid, mix.It is repeated 3 times.Last centrifugation, after abandoning supernatant, adds the storage of 0.2ml gold labeling antibody
Liquid storage, 4 DEG C save backup.
Described gold mark Streptavidin preparation method is: dilutes chain using pbs buffer (ph=8.5,10mmol/l)
Mould Avidin mass fraction is 0.15mg/ml.Take 0.15mg/ml solution of streptavidin 100 μ l, add to 1ml aunp solution
(particle diameter is 41nm), is slowly stirred using magnetic stirring apparatuss.100 μ l 1%peg-20000 are added after incubation 15min under room temperature.
Continue stirring 15min.4 DEG C of centrifugations of mixed liquor, 6000g × 30min, abandon supernatant, add 1ml gold mark Streptavidin storing liquid,
Mix.It is repeated 3 times.Last centrifugation, after removing supernatant, adds 0.2ml gold mark Streptavidin storing liquid, 4 DEG C save backup.
As shown in Figures 1 and 2, people's Bone markers () detection pair colloidal gold strips, including reagent paper taking oc as a example
The sample pad 2 pasted sequentially mutually is overlapped on bar end liner 1, and test strips end liner, is coated with the first of gold mark Streptavidin
Pad 3, the second pad 4 being coated with gold mark biotinylated antibody, nitrocellulose filter 5 and absorption pad 6.Celluloid
Film 5 is provided with a nature controlling line 8 being coated sheep anti mouse igg antibody in advance, nitrocellulose filter 5 is additionally provided with and above-mentioned nature controlling line
Parallel antibody (the anti-oc mab 2) detection line 7 being coated with determined antigen oc specific binding.
There is the Streptavidin of colloid gold particle labelling in first pad 3 of coating gold mark Streptavidin.
There is the biotinylation anti-oc Dan Ke of colloid gold particle labelling in second pad 4 of coating gold mark biotinylated antibody
Grand antibody.
The gold colloidal detection means of detection by quantitative people's Bone Gla protein, described detection means includes the double colloids of people's Bone Gla protein detection
Gold test paper strip and the jam 201 wrapping up described test strips, described jam 201 further includes panel 202 and base plate 203, institute
State and observation groove 204 and adding mouth 205 are provided with panel;Described adding mouth 205 is opened on described sample pad 2 top, and exposes
Partly or entirely described sample pad 2, described observation groove 204 is opened on the upside of described nitrocellulose filter, all described to expose
Detection line 7 and nature controlling line 8.
Further, the panel 202 of described jam 201 and base plate 203 are made using high-density polyethylene material.
The present embodiment provides the detection method of test strips.
The Deca 120 μ l blood serum sample in the sample pad 2 of the double colloidal gold strip of people's Bone markers () taking oc as a example.
Test strips are put in golden scalar quantity reading apparatus, after reacting 10 minutes, detection line 7 and Quality Control in reading apparatus automatic detection test strips
The color intensity of line 8, and show testing result.
The present embodiment provides the properties of test strips, taking detect human serum Bone Gla protein as a example.
Sensitivity for analysis is defined as: 20 zero standard product is measured, takes its 2 times of average deviation, in its standard curve
Upper corresponding concentration is sensitivity for analysis.
Sensitivity for analysis is: 0.50ng/ml
Precision: variation within batch coefficient≤10%, interassay coefficient of variation≤12%
Linear coefficient: r >=0.9900
The range of linearity: 0.50~180ng/ml.
Specificity: measure the Bone Gla protein enzymolysis fragment of 1000 μ g/l, degree of disturbance≤1%.
Present invention merely illustrates some claimed specific embodiments, one of or more skills
In art scheme, described technical characteristic can be combined with arbitrarily one or more technical schemes, and these are combined and obtain
Technical scheme also in the application protection domain, just as obtained from these are combined, technical scheme is open in the present invention
In content, concrete record is the same.
Claims (8)
1. people's Bone markers detection pair colloidal gold strips, including test strips end liner (1), sample pad (2), the first combination
Pad (3), the second pad (4), nitrocellulose filter (5) and absorption pad (6) are it is characterised in that on described test strips end liner (1)
Sample pad (2), the first pad (3), the second pad (4), nitrocellulose filter (5) and absorption that sequentially mutually overlap joint is pasted
Pad (6), described nitrocellulose filter (5) is provided with a nature controlling line (8) being coated sheep anti mouse igg antibody in advance, and nitric acid is fine
It is additionally provided with the detection line (7) parallel with above-mentioned nature controlling line (8), detection line (7) is coated with can be with people's bone to be measured on the plain film (5) of dimension
The antibody that metabolic markers antigenic specificity combines, described the first pad (3) is coated with gold mark Streptavidin, described
Gold mark Streptavidin can be with gold mark biotinylated antibody specific bond, and described the second pad (4) is coated with gold mark life
Thing elementization antibody, described gold mark biotinylated antibody is the antibody being combined with people's Bone markers antigen-specific to be measured.
2. people's Bone markers according to claim 1 detection with double colloidal gold strips it is characterised in that described energy
The antibody being combined with people's Bone markers antigenic specificity to be measured is to tie with people's Bone markers antigenic specificity to be measured
The monoclonal antibody closed.
3. people's Bone markers according to claim 2 detection with double colloidal gold strips it is characterised in that described
Detection line (7) is to be coated anti-human Bone markers monoclonal antibody.
4. people's Bone markers according to claim 2 detection with double colloidal gold strips it is characterised in that described
Gold mark biotinylated antibody is the biotinylated mAb of colloid gold label.
5. people's Bone markers according to claim 1 detection with double colloidal gold strips it is characterised in that described gold
Mark Streptavidin preparation method is: takes colloidal gold solution, adjusts ph value using solution of potassium carbonate;Add Streptavidin;Room
Temperature is lower to be incubated addition peg-20000 after 15min;After mixing centrifugation, resuspended, 4 DEG C of holdings are standby.
6. people's Bone markers according to claim 1 detection with double colloidal gold strips it is characterised in that described
Gold mark biotinylated antibody preparation method is: takes colloidal gold solution, adjusts ph value using solution of potassium carbonate;Add anti-human bone metabolism
Mark monoclonal antibody;It is slowly stirred;4 DEG C of incubations;Add 3 ' end Biotinylated single strand nucleotide;4 DEG C of incubations;Add peg-
20000;After mixing centrifugation, resuspended, 4 DEG C of holdings are standby.
7. the detection of double colloidal gold strips of the people's Bone markers detection described in a kind of application any one of claim 1-6
Device is it is characterised in that described detection means includes the double colloidal gold strips of people's Bone markers detection and parcel institute
State the jam (201) of test strips, described jam (201) further includes panel (202) and base plate (203), described panel (202)
On be provided with observation groove (204) and adding mouth (205), described adding mouth (205) is opened on described sample pad (2) top, and reveals
Go out partly or entirely described sample pad (2), described observation groove (204) is opened on the upside of described nitrocellulose filter, and exposes complete
Detection line (7) described in portion and nature controlling line (8).
8. detection means according to claim 7 is it is characterised in that the panel (202) of described jam (201) and base plate
(203) made using high-density polyethylene material.
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CN108279309A (en) * | 2018-01-30 | 2018-07-13 | 深圳市伯劳特生物制品有限公司 | A kind of test strip and detection method of PLA2R antibody |
CN108414748A (en) * | 2018-01-30 | 2018-08-17 | 深圳市伯劳特生物制品有限公司 | A kind of test strip and detection method of THSD7A antibody |
CN109900909A (en) * | 2019-02-28 | 2019-06-18 | 中国科学院广州生物医药与健康研究院 | A kind of nano gold mark sidestream immune chromatograph test strip detecting osteopontin |
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CN110873791A (en) * | 2018-08-29 | 2020-03-10 | 中国农业大学 | Indirect background fluorescent colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof |
CN109900909A (en) * | 2019-02-28 | 2019-06-18 | 中国科学院广州生物医药与健康研究院 | A kind of nano gold mark sidestream immune chromatograph test strip detecting osteopontin |
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