CN118067980A - Quadruple detection time-resolved fluorescence microsphere immunochromatography test strip and preparation method thereof - Google Patents
Quadruple detection time-resolved fluorescence microsphere immunochromatography test strip and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 7
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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
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
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- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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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
- 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
- G01N33/577—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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Abstract
The invention provides a quadruple detection time-resolved fluorescence microsphere immunochromatography test strip and a preparation method thereof, the test strip comprises a bottom plate, a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad are sequentially stuck on the bottom plate, 1-9 mu L of fluorescent microsphere markers of hepatitis B surface antigen monoclonal antibodies, hepatitis C antigens, syphilis antigens and AIDS antigens are sprayed on each 1cm of conjugate release pad, and a quality control line, a hepatitis B surface antigen detection line, a hepatitis C antibody detection line, a treponema pallidum antibody detection line and an AIDS antibody detection line are arranged on the reaction membrane. The invention adopts a four-in-one detection technology, and four detection targets are tested on a single test paper for detection together, so that the detection time and the production cost are reduced; compared with the traditional colloidal gold test paper, the sensitivity of the invention is improved by one order of magnitude, the probability of false negative is reduced, and the infection risk is reduced.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a quadruple detection time-resolved fluorescence microsphere immunochromatography test strip and a preparation method thereof.
Background
The immunochromatography technology is an analysis method combining the immunochromatography technology and the chromatographic technology developed at the end of the 20 th century, has the characteristics of specificity, simplicity in operation, rapidness and the like, and is widely applied to important fields such as clinical diagnosis, environmental monitoring, food safety and the like. The traditional immunochromatography technology uses colloidal gold as a marker, and the target is qualitatively detected or semi-quantitatively analyzed through band color development. The method is simple and quick, but has poor sensitivity and is difficult to accurately quantify. As a novel immunochromatography technology, the fluorescence immunochromatography technology not only maintains the on-site rapid detection advantage of the traditional colloidal gold test strip, but also adds the high sensitivity characteristic of the fluorescence detection technology, and becomes one of main ways for improving the detection performance of the immunochromatography method.
The existing hepatitis B surface antigen, hepatitis C antibody, treponema pallidum antibody and human immunodeficiency virus (I+II) antibody quadruple detection card is as follows:
1. the single-phase card arranging detection is complex in operation and high in cost;
2. quantitative analysis of the detection target object cannot be realized, and reasonable detection opinion is given;
3. The production cost is high, the economic pressure of the user is high, and the daily detection cannot be realized;
4. By adopting the traditional colloidal gold immunochromatography technology, false negative exists due to lower sensitivity, the anti-interference capability of the colloidal gold is poor, and various false positive risks are easy to occur.
Disclosure of Invention
In order to make up for the defects existing in the prior art, the invention provides a quadruple detection time-resolved fluorescence microsphere immunochromatography test strip, which adopts the following technical scheme:
The quadruple detection time-resolved fluorescence microsphere immunochromatography test strip comprises a bottom plate, wherein a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad are sequentially adhered to the bottom plate, 1-9 mu L of fluorescent microsphere markers of hepatitis B surface antigen monoclonal antibodies, hepatitis C antigens, syphilis antigens and AIDS antigens are sprayed on each 1cm of conjugate release pad, and a quality control line, a hepatitis B surface antigen detection line, a hepatitis C antibody detection line, a treponema pallidum antibody detection line and an AIDS antibody detection line are arranged on the reaction membrane.
Furthermore, the coating amount of the hepatitis B surface antigen detection line, the hepatitis C antibody detection line, the treponema pallidum antibody detection line and the AIDS antibody detection line is 0.5-3 mu L/cm.
Further, the coating amount of the quality control line is 0.5-3 mu L/cm.
The invention also provides a preparation method of the quadruple detection time-resolved fluorescence microsphere immunochromatography test strip, which comprises the following steps:
S1, preparing fluorescent microsphere markers:
Taking 1mg of fluorescent microspheres, centrifuging, collecting the precipitate, re-suspending the precipitate with 1mL of coupling buffer solution, adding EDC (EDC) according to a molar ratio of 1:2 and NHS according to a molar ratio of 1:20, incubating at room temperature after vortex oscillation, centrifuging, collecting the precipitate, adding the coupling buffer solution to re-suspend the microspheres, adding 40-150 mu g of antibody or antigen into the solution, fully mixing, stirring at room temperature for reaction, centrifuging, removing the supernatant, adding 1mL of blocking buffer solution, mixing, reacting at room temperature, centrifuging and washing for a plurality of times with the blocking buffer solution, and re-suspending the precipitate with PBS (phosphate buffer solution) to obtain fluorescent microsphere markers;
s2, preparing a conjugate release pad:
Soaking the conjugate release pad for a period of time by using a Tris-HCl buffer solution containing 0.1-0.5% of bovine serum albumin and 0.01-0.05M of bovine serum albumin, drying, spraying the fluorescent microsphere marker prepared in the step S1 on the dried conjugate release pad, and drying to obtain a finished product;
S3, preparing a reaction film:
respectively coating hepatitis B surface antigen, hepatitis C antibody, treponema pallidum spiral antibody and AIDS antibody on NC membrane to form corresponding detection lines, coating quality control protein antibody on NC membrane to form quality control lines, and drying to obtain the final product;
s4, preparing a sample absorption pad:
Soaking the sample absorption pad in a phosphate buffer solution containing 0.2% Tween20, 0.5% PVP K30 and 0.5% bovine serum albumin for a period of time, and drying to obtain a finished product;
S5, assembling a test strip:
the sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are adhered on the bottom plate in sequence.
Further, in step S1, the concentration of PBS is 0.02M, pH to 7.4, which contains 0.1 to 1% by mass of BSA and 0.1 to 5% by mass of trehalose.
Further, in the step S2, the spraying amount of the fluorescent microsphere marker is 1-9 mu L of the fluorescent microsphere marker sprayed on each 1cm of the conjugate release pad, and the Tris-HCl contains 0.1-5% of trehalose and 0.01-1% of Tween-20 by mass fraction.
In step S3, the coating amount of the detection line is 0.5-3 mu L/cm, and the coating amount of the quality control line is 0.5-3 mu L/cm.
Compared with the prior art, the invention has the following beneficial technical effects:
1. the quadruple detection time-resolved fluorescence microsphere immunochromatography test strip provided by the invention has the advantages of longer Stokes displacement and low background fluorescence signal, effectively enhances the chromatographic detection sensitivity and the stronger fluorescence quantum yield, and is beneficial to reducing the detection lower limit and realizing easy and convenient detection result reading and quantification.
2. The invention adopts polystyrene nanometer microsphere to wrap rare earth fluorescent ions, and improves the fluorescence intensity of single fluorescent ions through fluorescence pre-enhancement technology. Through optimizing and improving the coating technology, after the fluorescent ions are coated, dextran modification is carried out on the surfaces of the microspheres, so that the stability of the microspheres and the anti-interference performance on the reversible environment are greatly improved.
3. Compared with the traditional colloidal gold test paper, the sensitivity of the invention is improved by one order of magnitude (10 times), the probability of false negative is reduced, and the infection risk is reduced.
4. The time-resolved fluorescence microsphere quadruple detection test paper can realize quantitative detection of a detection target object, the quadruple detection test paper quickly adopts a highly specific antibody antigen reaction and immunochromatography analysis technology, the time-resolved fluorescence microsphere labeled antibody and antigen are fixed on a conjugate release pad, and an object to be detected in a sample is combined with the time-resolved fluorescence microsphere labeled antibody and antigen on the conjugate release pad in the flowing process to form an antibody-antigen-antibody-time-resolved fluorescence microsphere label or an antigen-antibody-antigen-time-resolved fluorescence microsphere label. And combining an object to be detected in the sample with an antibody or an antigen on a reaction membrane detection line, and determining the concentration of cannabidiol according to the fluorescence ratio of the detection line to a quality control line.
5. The invention adopts a four-in-one detection technology, and four detection targets are tested on a single test paper to jointly detect, so that the detection time and the production cost are reduced.
Drawings
FIG. 1 is a front view of a four-way detection time-resolved fluorescence microsphere immunochromatographic test strip of the present invention.
FIG. 2 is a cross-sectional view of a four-way detection time-resolved fluorescence microsphere immunochromatographic test strip of the present invention.
Wherein: 1. a bottom plate; 2. a sample absorbing pad; 3. a conjugate release pad; 4. a reaction membrane; 5. a water absorbing pad; 6. a quality control line; 7. hepatitis B surface antigen detection line; 8. a hepatitis C antibody detection line; 9. treponema pallidum antibody detection line; 10. an AIDS antibody detection line; 11. a face shell; 12. a detection window; 13. sample wells.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly practiced by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.
Example 1
As shown in figures 1 and 2, the quadruple detection time-resolved fluorescence microsphere immunochromatography test strip comprises a bottom plate 1, wherein a sample absorption pad 2, a conjugate release pad 3, a reaction membrane 4 and a water absorption pad 5 are sequentially adhered on the bottom plate 1. The reaction membrane 4 is provided with a quality control line 6, a hepatitis B surface antigen detection line 7, a hepatitis C antibody detection line 8, a treponema pallidum antibody detection line 9 and an AIDS (I+II) antibody detection line 10. The bottom plate 1 is buckled with a surface shell 11, and a detection window 12 and a sample hole 13 are arranged on the surface shell 11.
1-9 Mu L of time-resolved fluorescence microsphere marked hepatitis B surface antigen monoclonal antibody, hepatitis C antigen, syphilis antigen, AIDS antigen and quality control protein with pigment indicator are sprayed on the conjugate release pad 3. The particle size of the time-resolved fluorescence microsphere is 100-400 nm, and the loading capacity of fluorescence signals can be improved. The coating amount of the hepatitis B surface antigen detection line, the hepatitis C antibody detection line, the treponema pallidum antibody detection line and the AIDS antibody detection line is 0.5-3 mu L/cm. The coating amount of the quality control line is 0.5-3 mu L/cm.
The preparation method of the quadruple detection time-resolved fluorescence microsphere immunochromatography test strip comprises the following steps:
S1, preparing fluorescent microsphere markers:
1mg of fluorescent microspheres were centrifuged at 15000rpm for 10min, the pellet was collected and resuspended with 1mL of coupling buffer. EDC in a molar ratio of 1:2 and NHS in a molar ratio of 1:20 are then added, after vortexing, incubated at room temperature for 20-30min, centrifuged at 15000rpm for 10min, and the precipitate is collected. Then adding coupling buffer solution to resuspend microballoons, adding 40-150 mug antibody or antigen (hepatitis B surface antigen monoclonal antibody, hepatitis C antigen, syphilis antigen, AIDS antigen) into the solution, fully mixing uniformly, stirring at room temperature to react for 2-4 h, centrifuging at 10000rpm for 10min to remove supernatant, adding 1mL of sealing buffer solution, mixing uniformly, reacting at room temperature for 1-2 h, centrifuging with sealing buffer solution to wash for 3 times, then resuspension precipitation with PBS (containing 0.02M, pH7.4, 0.1-1% BSA and 0.1-5% trehalose), thus obtaining the time-resolved fluorescent microballoon marker, and standing at 4 ℃ for standby.
S2, preparing a conjugate release pad:
The conjugate release pad is soaked in 0.01-0.05M Tris-HCl buffer solution (containing 0.1-5% trehalose and 0.01-1% Tween-20) with the mass fraction of 0.1-0.5% bovine serum albumin and the pH value of 7.4 for 2 hours, and is dried at 37 ℃ for 2 hours for standby. And uniformly spraying the prepared fluorescent microsphere markers on a conjugate release pad by using a film spraying instrument, spraying 1-9uL of fluorescent microsphere markers on each 1cm of conjugate release pad, drying at 37 ℃ for 1-2h, and placing in a drying environment for standby.
S3, preparing a reaction film:
The concentration of antigen or antibody (hepatitis B surface antigen, hepatitis C antibody, treponema pallidum antibody, AIDS antibody) is regulated to 0.1-2 mg/mL by PBS with pH of 0.02M and pH of 7.4, and corresponding detection lines are formed on NC film by coating, wherein the coating amount is 0.5-3 mu L/cm. The quality control protein antibody is regulated to 0.02-1.0 mg/mL by PBS with the concentration of 0.01M and the pH of 7.4, and a quality control line is formed by coating the NC film, wherein the coating amount is 0.5-3 mu L/cm. And (3) drying the coated reaction film at 37 ℃ for 1-2 hours, and placing the reaction film in a drying environment for standby.
S4, preparing a sample absorption pad:
the sample absorption pad is soaked in phosphate buffer solution containing 0.2% of Tween20, 0.5% of PVP K30 and 0.5% of bovine serum albumin with the pH of 7.4 and 0.02mol/L for 2 hours, dried for 24 hours at 37 ℃, and placed in a dry environment for storage for standby.
S5, assembling a test strip:
the sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially stuck on the bottom plate, finally cut into small strips with the width of 4mm, put into a drying agent, put into an aluminum foil bag for sealing, and can be stored for 12 months at the temperature of 4-30 ℃.
Although the present invention has been described in detail with reference to the embodiments, it should be understood that the invention is not limited to the preferred embodiments, but is capable of modification and equivalents to some of the features described in the foregoing embodiments, but is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (7)
1. The utility model provides a quadruple detects time resolution fluorescence microballon immunochromatography test strip, includes the bottom plate, paste in proper order on the bottom plate and have sample absorption pad, conjugate release pad, reaction membrane, absorbent pad, its characterized in that: each 1cm of the conjugate release pad is sprayed with 1-9 mu L of fluorescent microsphere markers of hepatitis B surface antigen monoclonal antibodies, hepatitis C antigens, syphilis antigens and AIDS antigens, and the reaction membrane is provided with a quality control line, a hepatitis B surface antigen detection line, a hepatitis C antibody detection line, a treponema pallidum antibody detection line and an AIDS antibody detection line.
2. The quadruple detection time-resolved fluorescence microsphere immunochromatographic strip according to claim 1, wherein: the coating amount of the hepatitis B surface antigen detection line, the hepatitis C antibody detection line, the treponema pallidum antibody detection line and the AIDS antibody detection line is 0.5-3 mu L/cm.
3. The quadruple detection time-resolved fluorescence microsphere immunochromatographic strip according to claim 2, wherein: the coating amount of the quality control line is 0.5-3 mu L/cm.
4. The preparation method of the quadruple detection time-resolved fluorescence microsphere immunochromatography test strip is characterized by comprising the following steps of:
S1, preparing fluorescent microsphere markers:
Taking 1mg of fluorescent microspheres, centrifuging, collecting the precipitate, re-suspending the precipitate with 1mL of coupling buffer solution, adding EDC (EDC) according to a molar ratio of 1:2 and NHS according to a molar ratio of 1:20, incubating at room temperature after vortex oscillation, centrifuging, collecting the precipitate, adding the coupling buffer solution to re-suspend the microspheres, adding 40-150 mu g of antibody or antigen into the solution, fully mixing, stirring at room temperature for reaction, centrifuging, removing the supernatant, adding 1mL of blocking buffer solution, mixing, reacting at room temperature, centrifuging and washing for a plurality of times with the blocking buffer solution, and re-suspending the precipitate with PBS (phosphate buffer solution) to obtain fluorescent microsphere markers;
s2, preparing a conjugate release pad:
Soaking the conjugate release pad for a period of time by using a Tris-HCl buffer solution containing 0.1-0.5% of bovine serum albumin and 0.01-0.05M of bovine serum albumin, drying, spraying the fluorescent microsphere marker prepared in the step S1 on the dried conjugate release pad, and drying to obtain a finished product;
S3, preparing a reaction film:
respectively coating hepatitis B surface antigen, hepatitis C antibody, treponema pallidum spiral antibody and AIDS antibody on NC membrane to form corresponding detection lines, coating quality control protein antibody on NC membrane to form quality control lines, and drying to obtain the final product;
s4, preparing a sample absorption pad:
Soaking the sample absorption pad in a phosphate buffer solution containing 0.2% Tween20, 0.5% PVP K30 and 0.5% bovine serum albumin for a period of time, and drying to obtain a finished product;
S5, assembling a test strip:
the sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are adhered on the bottom plate in sequence.
5. The method for preparing the quadruple detection time-resolved fluorescence microsphere immunochromatographic test strip according to claim 4, which is characterized in that: in step S1, PBS was used at a concentration of 0.02M, pH to 7.4, and contained BSA at a mass fraction of 0.1 to 1% and trehalose at a mass fraction of 0.1 to 5%.
6. The method for preparing the quadruple detection time-resolved fluorescence microsphere immunochromatographic test strip according to claim 5, which is characterized in that: in the step S2, the spraying amount of the fluorescent microsphere marker is 1-9 mu L of the fluorescent microsphere marker sprayed on a conjugate release pad per 1cm, and the Tris-HCl contains 0.1-5% of trehalose and 0.01-1% of Tween-20 by mass percent.
7. The method for preparing the quadruple detection time-resolved fluorescence microsphere immunochromatographic test strip according to claim 6, which is characterized in that: in the step S3, the coating amount of the detection line is 0.5-3 mu L/cm, and the coating amount of the quality control line is 0.5-3 mu L/cm.
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| CN102023216A (en) * | 2009-09-16 | 2011-04-20 | 中华人民共和国陕西出入境检验检疫局 | Suspension chip detection method for simultaneously detecting hepatitis B virus, hepatitis C virus, treponema pallidum and human immunodeficiency virus pathogens |
| CN104345148A (en) * | 2013-07-28 | 2015-02-11 | 嘉兴朝云帆生物科技有限公司 | Test strip and method for detecting protein by using immunochromatography |
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| CN214794831U (en) * | 2021-05-17 | 2021-11-19 | 山西瑞豪生物科技有限公司 | Quantum dot fluorescence immunochromatography test strip for multi-project rapid joint detection of blood infectious diseases |
| CN115327126A (en) * | 2022-08-10 | 2022-11-11 | 郑州左安检测科技有限公司 | Time-resolved fluorescent microsphere immunochromatography test strip for detecting cannabidiol and preparation method and application thereof |
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| CN102023216A (en) * | 2009-09-16 | 2011-04-20 | 中华人民共和国陕西出入境检验检疫局 | Suspension chip detection method for simultaneously detecting hepatitis B virus, hepatitis C virus, treponema pallidum and human immunodeficiency virus pathogens |
| CN104345148A (en) * | 2013-07-28 | 2015-02-11 | 嘉兴朝云帆生物科技有限公司 | Test strip and method for detecting protein by using immunochromatography |
| CN106290829A (en) * | 2016-07-19 | 2017-01-04 | 杭州博拓生物科技股份有限公司 | A kind of four-in-one joint-detection paper box and preparation technology |
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