CN104764877A - Immunity chromatography test method and test paper - Google Patents
Immunity chromatography test method and test paper Download PDFInfo
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- CN104764877A CN104764877A CN201410244152.6A CN201410244152A CN104764877A CN 104764877 A CN104764877 A CN 104764877A CN 201410244152 A CN201410244152 A CN 201410244152A CN 104764877 A CN104764877 A CN 104764877A
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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
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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
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
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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
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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Abstract
The invention discloses an immunity chromatography test method and a test paper. One or more specific antibodies or antigens in two or more round dot shape, multilateral shape and other figure shape or a combination of different figures, or a combination of strip shape and other figure are fixed on a reaction film; a binder release pad adsorbs marked specific antibodies or antigens; when a sample to be tested drops on the sample pad, the sample to be tested under the action of capillary moves forward, dissolves the markers on the binder release pad and mutually reacts to form a binder; when the binder moves to the test area T and a quality control area C, the binder combines with antibodies or antigens on the test points and quality control points and is trapped and gathered at the test points and quality control points. By naked eye observation or different coloration results on the test points and quality control points by scanning instrument, the qualitative, semi-quantitative or quantitative detection on the sample to be tested can be realized. The invention has the advantages of simple and rapid operation, and can be widely used in the field of in vitro diagnostic reagents.
Description
Technical field
The invention belongs to external diagnosis reagent field, be specifically related to a kind of immunochromatography detection method and the test paper used and paper box.
Background technology
Immunochromatographic method is a kind of quick diagnosis technology based on immune colloidal gold technique that the nineties is risen, its principle is first fixed on reaction film 2 by specific antibody, after the sample pad 4 of test paper immerses sample to be checked, due to capillarity, sample to be checked will move forward along this film, when moving to the region being fixed with antibody, in sample to be checked corresponding antigen namely with this antibody generation specific binding, if this region can be made to show certain color with immune colloid gold, thus realize specific immunodiagnosis.
Take colloid gold test paper as the various aspects that the immune chromatography test paper technology of main representative has now been widely used in immune detection, specific antibody or antigen are mainly fixed on reaction film 2 with ribbon by this technology, carry out qualitative analysis by the colour developing result of the banded detection line 8 of detection zone T mono-rule, the colour developing result of the banded detection line 8 of many rules carries out half-quantitative detection.
Existing immunochromatography detection method and test paper, the every bar detection line 8 on test paper only provides a kind of Detection Information, and in certain area, increase more detection line 8 quantity can cause information confusion even interpretation mistake to identification.
Application number be 201210200364.5 patent of invention disclose a kind of colloidal gold immunochromatographimethod quantitative testing test paper and detection method thereof, the method detection line 8 has three at least, and feature comprises 3,4,5,6 detection lines 8.The test paper of detection line 8 more than 7 cannot be applicable to.
At present, detection zone T adopts the combination of round point shape, polygon-shaped, other diagram shape, different graphic shape, or the figure that histogram shape and other diagram shape combine is as check point 6, and the setting of check point 6 can be same antibody or the antigen of same concentration or variable concentrations, also can be Multiple Antibodies or the antigen of same concentration or variable concentrations.Carry out by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 the immunochromatography detection method that characteristic signal that instrument that qualitative, half-quantitative detection or employing have signal testing function gathers check point 6 and Quality Control point 7 on test paper carries out quantitatively detecting never to report.
Immunochromatography technique includes but not limited to double antibody sandwich method, indirect method, A competitive inhibition method.
Double antibody sandwich method, belongs to non-competing combination and measures, be applicable to the polyvalent antigen in detection molecules with at least two antigenic determinants.Its basic functional principle is: the antibody that utilization is connected on reaction film 2 and labelled antibody are detected two antigenic determinants on antigen molecule respectively and are combined in sample to be checked, form solid matrix antibody-antigen-labelled antibody immune complex.
Indirect method, measures the method that antibody is the most frequently used, belongs to non-competing combination and measures.Its principle is by antigen immobilization in reaction film 2, and in sample to be checked, first test antibodies is combined with labeled molecule, form compound, then the antigen on reaction film 2 is combined, and forms labeled molecule-Antibody-antigen complex to be checked.
Competition law can be used for antigen and TPPA.To measure antigen, its ultimate principle is that the envelope antigen competition by inspection antigen and solid phase in sample to be checked is combined with labelled antibody, and when the amount by inspection antigen in sample to be checked is greater than the threshold value of reagent, labelled antibody then can not be combined with the envelope antigen of solid phase; If without being subject to inspection antigen in sample to be checked, labelled antibody is directly combined with the envelope antigen of solid phase.
The quantivative approach of the semi-quantitative method that the assay method of immunochromatography detection method comprises the quilitative method of detection line 8 color determining detection zone T that detects by an unaided eye, multiple detection lines 8 of the detection zone T that detects by an unaided eye develop the color situation and detection line 8 color intensity by specialized instrument and equipment mensuration detection zone T.
Summary of the invention
The object of the invention is to, for prior art, provide a kind of immunochromatography detection method and test paper, can realize treating sample this qualitative, sxemiquantitative or quantitatively detect.
A kind of immunochromatography detection method, adopts following steps:
(1) reaction film 2 wraps quilt: according to the kind of the object determination coated antibody to be checked in sample to be checked or antigen, the concentration of setting check point 6 and Quality Control point 7 and quantity;
(2) bond release pad 5 is prepared according to the object to be checked in sample to be checked;
(3) prepared by sample pad 4;
(4) test paper is assembled: test paper by base plate 1, and is successively set on sample pad 4 on base plate 1, bond release pad 5, reaction film 2, absorption pad 3 are formed;
(5) treat sample originally to detect: in test paper sample pad 4, add sample to be checked, can treat sample by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 and originally carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point 6 and Quality Control point 7 on test paper quantitatively can detect.Or the object to be checked in sample to be checked is first determined according to the colour developing situation of multiple check point 6, again by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 can treat sample in this different objects to be checked carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point 6 and Quality Control point 7 on test paper can quantitatively detect.
A kind of immunity chromatography detection test paper, for realize treating sample this qualitative, sxemiquantitative or quantitatively detect; Test paper by base plate 1, and is successively set on sample pad 4 on base plate 1, bond release pad 5, reaction film 2, absorption pad 3 are formed.
Described reaction film 2 is divided into two regions, and one is discharge the adjacent detection zone T of pad 5 with bond, and one is the quality control region C adjacent with absorption pad 3.
Described detection zone T is provided with 2 to 30 check points 6, and check point 6 does not comprise only histogram shape, and check point 6 is the combination of round point shape, polygon-shaped, other diagram shape or different graphic shape, also can be the combination of histogram shape and other diagram shape.The setting of check point 6 can be same antibody or the antigen of same concentration or variable concentrations, can treat sample by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 and originally carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point 6 and Quality Control point 7 on test paper quantitatively can detect.Or check point 6 is coated with different antibody or the antigen of same concentration or variable concentrations, first the object to be checked in sample to be checked is determined according to the colour developing situation of multiple check point 6, again by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 can treat sample in this different objects to be checked carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point 6 and Quality Control point 7 on test paper can quantitatively detect.
Described quality control region C is provided with 1 to 30 Quality Control point 7, and Quality Control point 7 is the combination of round point shape, polygon-shaped, histogram shape, other diagram shape or different graphic shape; Quality Control point 7 is coated with and can discharges with bond the antibody or antigen that on pad 5, bond is combined.
Described bond release pad 5 is marked with the relevant specific antibody of single object to be checked or antigen, or is marked with the relevant multiple specific antibody of each object to be checked or antigen.
Described immunochromatography detection method, its labeling method includes but not limited to quantum dot-labeled method, Collaurum marking, colloidal selenium marked method, coloured or fluorescent latex labelling method, magnetic particle labels method, time resolution chromatography, chemoluminescence method.
Described sample to be checked is from the blood of clinical or non-clinical, body fluid, urine, saliva, genital discharge liquid or other liquid samples or thick sample.
Described base plate 1 material includes but not limited to PVC (Polyvinylchloride) plate, reaction film 2 material includes but not limited to nitrocellulose filter and cellulose acetate membrane, the material of absorption pad 3 includes but not limited to filter paper, sample pad 4 material includes but not limited to glass fibre and nonwoven fabrics, and bond release pad 5 includes but not limited to glass fibre or nonwoven fabrics.
A kind of immunochromatography detection method and test paper, comprise the immune chromatography test paper of any one of claim 1-8.
technique effect
The present invention is a kind of immunochromatography detection method and test paper, and the test paper that the method uses is simple to operate, fast, easy to use, do not need one of skill in the art to operate, the needs of different levels personnel can be met, easily spread to hospital of grass-roots community and average family.And detection zone T adopts the combination of round point shape, polygon-shaped, other diagram shape, different graphic shape, or the figure that histogram shape and other diagram shape combine is as check point 6, the instrument having a signal testing function by the colour developing situation of the multiple check point 6 of visual inspection and Quality Control point 7 or employing gathers the characteristic signal of check point 6 and Quality Control point 7 on test paper, can realize treating sample this qualitative, sxemiquantitative or quantitatively detect.Simple to operate, quick, be widely used in external diagnosis reagent field.
A kind of immunochromatography detection method of the present invention and test paper is different from existing method and test paper part is:
Existing immunochromatography detection method and detection paper district T are one or more ribbon detection line 8, and each detection line 8 only provides an information, and in certain area, increase more detection line 8 quantity can cause information confusion even interpretation mistake to identification.
The detection zone T of a kind of immunochromatography detection method of the present invention and test paper thereof is provided with 2 to 30 check points 6, check point 6 does not comprise only histogram shape, check point 6 is the combination of round point shape, polygon-shaped, other diagram shape or different graphic shape, also can be the combination of histogram shape and other diagram shape.The setting of check point 6 can be same concentrations, also can be same antibody or the antigen of variable concentrations.Check point 6 can also be set as Multiple Antibodies or antigen, and the check point 6 of allo-antibody or antigen can not represent by different diagram shape.
A kind of immunochromatography detection method of the present invention and test paper thereof, can can treat sample by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 and originally carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point 6 and Quality Control point 7 on test paper quantitatively can detect.Or the object to be checked in sample to be checked is first determined according to the colour developing situation of multiple check point 6, again by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 can treat sample in this different objects to be checked carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point 6 and Quality Control point 7 on test paper can quantitatively detect.
Existing method of comparing the invention provides more different check points 6 and the better different graphic information identified.
Accompanying drawing explanation
Accompanying drawing 1 is the schematic diagram of a kind of immunochromatography detection method of the present invention and test paper.
Accompanying drawing 2 is schematic diagram of existing immunochromatography detection method and test paper.
Accompanying drawing 3 test paper for human chorionic gonadotrophin detection (colloidal gold immunity chromatography) schematic diagram.
Accompanying drawing 4 microdose urine protein, urine β
2microglobulin, press down Guang element C tri-joint-detection test paper (colloidal gold immunity chromatography) schematic diagram.
embodiment describes
Implementation step:
(1) reaction film 2 wraps quilt: according to the kind of the object determination coated antibody to be checked in sample to be checked or antigen, the concentration of setting check point 6 and Quality Control point 7 and quantity;
(2) bond release pad 5 is prepared according to the object to be checked in sample to be checked;
(3) prepared by sample pad 4;
(4) test paper is assembled: test paper by base plate 1, and is successively set on sample pad 4 on base plate 1, bond release pad 5, reaction film 2, absorption pad 3 are formed;
(5) treat sample originally to detect: in test paper sample pad 4, add sample to be checked, can treat sample by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 and originally carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point 6 and Quality Control point 7 on test paper quantitatively can detect.Or the object to be checked in sample to be checked is first determined according to the colour developing situation of multiple check point 6, again by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 can treat sample in this different objects to be checked carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point 6 and Quality Control point 7 on test paper can quantitatively detect.
Reaction film 2 is divided into two regions, and one is discharge the adjacent detection zone T of pad 5 with bond, and one is the quality control region C adjacent with absorption pad 3.
Described detection zone T is provided with 2 to 30 check points 6, check point 6 does not comprise only histogram shape, check point 6 is round point shape, polygon-shaped, the combination of other diagram shape or different graphic shape, also can be the combination of histogram shape and other diagram shape, the setting of check point 6 can be same antibody or the antigen of same concentration or variable concentrations, sample can be treated by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 and originally carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point 6 and Quality Control point 7 on test paper can quantitatively detect.Or check point 6 is coated with different antibody or the antigen of same concentration or variable concentrations, first the object to be checked in sample to be checked is determined according to the colour developing situation of multiple check point 6, again by the colour developing situation of the multiple check point of visual inspection 6 and Quality Control point 7 can treat sample in this different objects to be checked carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point 6 and Quality Control point 7 on test paper can quantitatively detect.
Described quality control region C is provided with 1 to 30 Quality Control point 7, and Quality Control point 7 is the combination of round point shape, polygon-shaped, histogram shape, other diagram shape or different graphic shape; Quality Control point 7 is coated with and can discharges with bond the antibody or antigen that on pad 5, bond is combined.
Described bond release pad 5 is marked with the relevant specific antibody of single object to be checked or antigen, or is marked with the relevant multiple specific antibody of each object to be checked or antigen.
In the present invention, labelled reagent normally can be used as the carrier of sessile antibody or antigen in immunochromatography detects.Such as, colloid gold particle, electroselenium particle, coloured or fluorescent latex particles and magnetic-particle, particularly preferably colloid gold particle.
In the present invention, base plate 1 supporting layer is the material do not absorbed water, and avoids propping material to absorb sample to be checked thus affects the movement of horizontal direction.The material of base plate 1 comprises PVC (Polyvinylchloride) plate and other plastic materials, preferred PVC (Polyvinylchloride) plate.
In the present invention, the material of reaction film 2 includes but not limited to nitrocellulose filter, nylon membrane or cellulose acetate membrane.Preferred nitrocellulose filter.
In the present invention, absorption pad 3 refers to absorb the liquid absorbing member spread with Quality control by the sample to be checked of reaction film 2.The material of absorption pad 3 can be but be not limited to filter paper.Absorption pad 3 can use one deck also can use multilayer.
In the present invention, sample pad 4 is the parts receiving sample to be checked, comprises any material and form, as long as liquid and object to be checked can be allowed to pass through.Be suitable for specifically including but not limited to of the material of sample pad 4: glass fibre, acrylic fibre, hydrophilic polyethylene material, dry paper, nonwoven fabrics and fabric.Preferred use glass fibre and nonwoven fabrics.Sample pad 4 can use one deck also can use multilayer.
In the present invention, the material being suitable for bond release pad 5 includes but not limited to paper, cellulose mixtures, cellulose nitrate, dacron, acrylonitrile copolymer, glass fibre and nonwoven fabrics.Preferred use nonwoven fabrics.
In the present invention, except a kind of immunochromatographyassay assay method and test paper, also comprise plastic clip, test paper can be arranged within plastic clip and use.The mode that described plastic clip and the size of test paper, sample add and the position that position, antibody or antigen solidify on reaction film 2 match.
specific implementation method
Following embodiment is provided to be to understand the present invention further better; be not limited to described preferred forms; content of the present invention and protection domain are not construed as limiting; anyone is under enlightenment of the present invention or any and the present invention feature of other prior aries of the present invention being carried out combining and draw is identical or akin product, all belongs within protection scope of the present invention.
Embodiment 1
The preparation embodiment 1 of immunity chromatography detection test paper of the present invention, preparation and detection human chorionic gonadotrophin (HCG) Test paper (colloidal gold immunity chromatography).
Biologically active raw material: α-HCG monoclonal antibody, β-HCG monoclonal antibody, IgG polyclonal antibody are all bought from market.
Reagent: bovine serum albumin(BSA) is bought from market.
Nitrocellulose filter is bought from market.
Test paper preparation procedure is as follows:
Step 1. reaction film 2 wraps quilt
Object determination coated antibody kind to be checked according in sample to be checked: α-HCG monoclonal antibody, IgG polyclonal antibody.
Concentration and the quantity of check point 10 and Quality Control point 11 is set: check point 10 arranges 6 points, 4 variable concentrations according to the object to be checked in sample to be checked; Quality Control point 11 arranges 2 points, 1 concentration.
Be coated on nitrocellulose filter by the α-HCG monoclonal antibody of 15.42mg/ml, the IgG polyclonal antibody of 8.02mg/ml with the figure according to accompanying drawing 3, dry 24h, temperature is 37 DEG C, wet degree≤40%RH.
Prepared by step 2. bond release pad 5
Get the colloidal gold solution of 40nm, add the K of 0.2M in 1.2% ratio
2cO
3(sal tartari) regulates pH value, adds β-HCG monoclonal antibody, make label in 15 μ g/ml ratios, places room temperature 5min, adds BSA(bovine serum albumin(BSA) in 0.8% ratio) stabilizing agent.
The centrifugal 30min of 4500r, gets concentrating and precipitating thing; Supernatant is continued the centrifugal 45min of 6500r, get concentrating and precipitating thing; Supernatant is continued the centrifugal 60min of 9000r, get concentrating and precipitating thing; Three centrifugal concentrating sediments are mixed.
Diluted by centrifugal good concentrate, be laid on the nonwoven fabrics of 90cm × 30cm, liquid volume added 10-11ml/ opens, dry 24h, temperature 37 DEG C, Shi Du≤40%RH.
The preparation of step 3. sample pad 4
To the glass fibre of 83.5cm × 30cm and nonwoven fabrics be cut into comprising 0.5%NaCl(potassium chloride), 0.5% sucrose, 0.1%BSA(bovine serum albumin(BSA)) and Tris-HCl damping fluid (trishydroxymethylaminomethane-hydrochloric acid) (pH7.4) working fluid soak.At 37 DEG C by dry for described pad 24h, obtain sample pad 4.
Step 4. assembles test paper
Base plate 1 is lain on operator's console, sticks double faced adhesive tape.
Bag is attached on base plate 1 by good reaction film 2.
Press the 2mm of reaction film 2 to stick absorption pad 3, the other end aligns with base plate 1.
The 2mm place of reaction film 2 layer of non-woven fabric is pressed to stick.
Bond is discharged pad 5 to be pressed on together above nonwoven fabrics.
Glass fibre is pressed on bond release pad 5 half position, the other end aligns with base plate 1.
Again layer of non-woven fabric one end and bond are discharged pad 5 upper end to align, the other end aligns with base plate 1.
Step 5. Product checking
The sample urine to be checked of getting containing HCG is added drop-wise in sample pad 4, sentence read result during 5min, T
1colour developing, other T point, without colour developing, represents that containing HCG concentration in sample urine to be checked is 25-125mIU/ml; T
1and T
2colour developing, other T, without colour developing, represents that containing HCG concentration in sample urine to be checked is 125-500mIU/ml; T
1, T
2, T
3colour developing, T
4without colour developing, represent that containing HCG concentration in sample urine to be checked is 500-2500mIU/ml; T all develops the color, then represent in sample urine to be checked that containing HCG concentration is higher than 2500mIU/ml.
Embodiment 2
The preparation embodiment 2 of immunity chromatography detection test paper of the present invention, preparation and detection microdose urine protein, urine β
2microglobulin, press down Guang element C tri-joint-detection test paper (colloidal gold immunity chromatography).
Biologically active raw material: human albumin monoclonal antibody, mouse-anti human albumin monoclonal antibody, Cys-C(press down Guang element C) monoclonal antibody, mouse-anti people Cys-C(press down Guang element C) antibody, β
2microglobulin monoclonal antibody, mouse-anti people β
2microglobulin antibody, IgG polyclonal antibody are all bought from market.
Reagent: bovine serum albumin(BSA) is bought from market.
Nitrocellulose filter is bought from market.
Test paper preparation procedure is as follows:
Step 1. reaction film 2 wraps quilt
Object determination coated antibody kind to be checked according in sample to be checked: human albumin monoclonal antibody, Cys-C(press down Guang element C) monoclonal antibody, β
2microglobulin monoclonal antibody, IgG polyclonal antibody.
Set concentration and the quantity of check point 12 and Quality Control point 13 according to the object to be checked in sample to be checked: check point 12 arranges 3 points, each point is a kind of detection antibody; Quality Control point 13 arranges 2 points, 1 concentration.
By the human albumin monoclonal antibody of 2.5mg/ml, the β of 2.26mg/ml
2the Cys-C(of microglobulin monoclonal antibody, 2.42mg/ml presses down Guang element C) monoclonal antibody, 8.02mg/ml IgG polyclonal antibody with reference to the accompanying drawings 4 figure be coated on nitrocellulose filter, dry 24h, temperature is 37 DEG C, wet degree≤40%RH.
Prepared by step 2. bond release pad 5
The preparation of microdose urine protein gold mark bond release pad
Get the colloidal gold solution of 40nm, add the K of 0.2M in 1.2% ratio
2cO
3(sal tartari) regulates pH value, adds mouse-anti human albumin monoclonal antibody, make label in 12 μ g/ml ratios, places room temperature 5min, adds BSA(bovine serum albumin(BSA) in 0.8% ratio) stabilizing agent.
The centrifugal 30min of 4500r, gets concentrating and precipitating thing; Supernatant is continued the centrifugal 45min of 6500r, get concentrating and precipitating thing; Supernatant is continued the centrifugal 60min of 9000r, get concentrating and precipitating thing; Three centrifugal concentrating sediments are mixed.
Diluted by centrifugal good concentrate, be laid on the nonwoven fabrics of 90cm × 30cm, liquid volume added 10-11ml/ opens, dry 24h, temperature 37 DEG C, Shi Du≤40%RH.
Urine β
2the preparation of microglobulin gold mark bond release pad
Get the colloidal gold solution of 40nm, add the K of 0.2M in 1.2% ratio
2cO
3(sal tartari) regulates pH value, adds mouse-anti people β in 10 μ g/ml ratios
2microglobulin antibody, makes label, places room temperature 5min, adds BSA(bovine serum albumin(BSA) in 0.8% ratio) stabilizing agent.
The centrifugal 30min of 4500r, gets concentrating and precipitating thing; Supernatant is continued the centrifugal 45min of 6500r, get concentrating and precipitating thing; Supernatant is continued the centrifugal 60min of 9000r, get concentrating and precipitating thing; Three centrifugal concentrating sediments are mixed.
Diluted by normal concentration by centrifugal good concentrate, be laid on the nonwoven fabrics of 90cm × 30cm, liquid volume added 10-11ml/ opens, dry 24h, temperature 37 DEG C, Shi Du≤40%RH.
Press down the preparation of Guang element C gold mark bond release pad
Get the colloidal gold solution of 40nm, add the K of 0.2M in 1.2% ratio
2cO
3(sal tartari) regulates pH value, adds mouse-anti people Cys-C(press down Guang element C in 11 μ g/ml ratios) antibody, make label, place room temperature 5min, add BSA(bovine serum albumin(BSA) in 0.8% ratio) stabilizing agent.
The centrifugal 30min of 4500r, gets concentrating and precipitating thing; Supernatant is continued the centrifugal 45min of 6500r, get concentrating and precipitating thing; Supernatant is continued the centrifugal 60min of 9000r, get concentrating and precipitating thing; Three centrifugal concentrating sediments are mixed.
Diluted by normal concentration by centrifugal good concentrate, be laid on the nonwoven fabrics of 90cm × 30cm, liquid volume added 10-11ml/ opens, dry 24h, temperature 37 DEG C, Shi Du≤40%RH.
The preparation of step 3. sample pad 4
To the glass fibre of 83.5cm × 30cm and nonwoven fabrics be cut into comprising 0.5%NaCl(potassium chloride), 0.5% sucrose, 0.1%BSA(bovine serum albumin(BSA)) and Tris-HCl damping fluid (trishydroxymethylaminomethane-hydrochloric acid) (pH7.4) working fluid soak.At 37 DEG C by dry for described pad 24h, obtain sample pad 4.
Step 4. assembles test paper
Base plate 1 is placed on operator's console, sticks double faced adhesive tape.
Bag is attached on base plate 1 by good reaction film 2.
Press the 2mm of reaction film 2 to stick absorption pad 3, the other end aligns with base plate 1.
The 2mm place of reaction film 2 layer of non-woven fabric is pressed to stick.
By microdose urine protein bond release pad, urine β
2the release of microglobulin bond is padded, press down Guang element C bond release pad is pressed on above nonwoven fabrics successively together.
Glass fibre is pressed on bond release pad 5 half position, the other end aligns with base plate 1.
Again layer of non-woven fabric one end and bond are discharged pad 5 upper end to align, the other end aligns with base plate 1.
Step 5. Product checking
Get on sample urine specimen pad 4 to be checked, during 8min, sentence read result, T
1colour developing, represents that in sample urine to be checked, microdose urine protein is for positive; T
2colour developing, represents in sample urine to be checked containing urine β
2microglobulin is positive; T
3colour developing, represents in sample urine to be checked containing pressing down Guang element C for positive.
The foregoing is only embodiments of the invention, do not limit the present invention, all on basis of the present invention, any amendment, improvement etc. done, within the protection domain that all should be included in invention.
Claims (10)
1. an immunochromatography detection method, for realize treating sample this qualitative, sxemiquantitative or quantitatively detect; It is characterized in that: adopt following steps:
(1) reaction film bag quilt: according to the kind of the object determination coated antibody to be checked in sample to be checked or antigen, the concentration of setting check point and Quality Control point and quantity;
(2) bond release pad is prepared according to the object to be checked in sample to be checked;
(3) sample pad is prepared;
(4) test paper is assembled: test paper by base plate, and is successively set on sample pad, bond release pad, reaction film, the absorption pad formation on base plate;
(5) treat sample originally to detect: in test paper sample pad, add sample to be checked, can treat sample by the colour developing situation of the multiple check point of visual inspection and Quality Control point and originally carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point and Quality Control point on test paper quantitatively can detect; Or the object to be checked in sample to be checked is first determined according to the colour developing situation of multiple check point, again by the colour developing situation of the multiple check point of visual inspection and Quality Control point can treat sample in this different objects to be checked carry out qualitative or half-quantitative detection, or the characteristic signal adopting the instrument with signal testing function to gather check point and Quality Control point on test paper can quantitatively detect.
2. immunochromatography detection method according to claim 1, is characterized in that: reaction film is divided into two regions, and one is discharge with bond to pad adjacent detection zone, and one is the quality control region adjacent with absorption pad; Detection zone is provided with 2 to 30 check points, and check point does not comprise only histogram shape, and check point is the combination of round point shape, polygon-shaped, other diagram shape or different graphic shape, also can be the combination of histogram shape and other diagram shape; The setting of check point can be same antibody or the antigen of same concentration or variable concentrations, also can be Multiple Antibodies or the antigen of same concentration or variable concentrations; Described quality control region is provided with 1 to 30 Quality Control point, and Quality Control point is the combination of round point shape, polygon-shaped, histogram shape, other diagram shape or different graphic shape; Quality Control point is coated with to discharge with bond and pads the antibody or antigen that bond is combined.
3. immunochromatography detection method according to claim 1, is characterized in that: described bond release pad is marked with the relevant specific antibody of single object to be checked or antigen, or is marked with the relevant multiple specific antibody of each object to be checked or antigen.
4. immunochromatography detection method according to claim 1, is characterized in that: described labeling method comprises quantum dot-labeled method, Collaurum marking, colloidal selenium marked method, coloured or fluorescent latex labelling method, magnetic particle labels method, time resolution chromatography, chemoluminescence method.
5. immunochromatography detection method according to claim 1, is characterized in that: described method comprises double antibody sandwich method, indirect method, A competitive inhibition method.
6. immunochromatography detection method according to claim 1, is characterized in that: described sample to be checked is from the blood of clinical or non-clinical, body fluid, urine, saliva, genital discharge liquid or other liquid samples or thick sample.
7. immunity chromatography detection test paper according to claim 1, is characterized in that: for realize treating sample this qualitative, sxemiquantitative or quantitatively detect; Test paper by base plate, and is successively set on sample pad, bond release pad, reaction film, the absorption pad formation on base plate.
8. immunity chromatography detection test paper according to claim 1, it is characterized in that: described baseboard material is PVC (Polyvinylchloride) plate, reaction film material is nitrocellulose filter or cellulose acetate membrane, the material of absorption pad is filter paper, sample pad material is glass fibre and nonwoven fabrics, and bond release cushion material is glass fibre or nonwoven fabrics.
9. the immunity chromatography detection test paper according to claim 1-8, is characterized in that: test paper also can be assemblied in plastic clip that position that the mode that adds with the size of test paper, sample and position, antibody or antigen solidifies on reaction film matches becomes paper box.
10. a complete paper box for immunochromatography detection method, is characterized in that: comprise test paper and paper box described in Sample dilution to be checked and the claims 1-9.
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| CN201410244152.6A CN104764877A (en) | 2014-05-14 | 2014-06-04 | Immunity chromatography test method and test paper |
| PCT/CN2015/072273 WO2015184847A1 (en) | 2014-05-14 | 2015-02-05 | Immunochromatographic test method and test paper |
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| CN201410202928 | 2014-05-14 | ||
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| CN201510233016.1A Pending CN104820104A (en) | 2014-05-14 | 2015-05-08 | Human chorionic gonadotropin immunochromatography detection test paper and preparation method thereof |
| CN201510233043.9A Pending CN104820094A (en) | 2014-05-14 | 2015-05-08 | Immune-chromatographic detection test paper and detection method |
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Also Published As
| Publication number | Publication date |
|---|---|
| CN104820094A (en) | 2015-08-05 |
| WO2015172688A1 (en) | 2015-11-19 |
| CN104820104A (en) | 2015-08-05 |
| WO2015172689A1 (en) | 2015-11-19 |
| WO2015184847A1 (en) | 2015-12-10 |
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Application publication date: 20150708 |