CN113791209B - Immunochromatography test strip without hook effect and preparation method thereof - Google Patents
Immunochromatography test strip without hook effect and preparation method thereof Download PDFInfo
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- CN113791209B CN113791209B CN202110922074.0A CN202110922074A CN113791209B CN 113791209 B CN113791209 B CN 113791209B CN 202110922074 A CN202110922074 A CN 202110922074A CN 113791209 B CN113791209 B CN 113791209B
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- 238000012360 testing method Methods 0.000 title claims abstract description 54
- 230000000694 effects Effects 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 238000003317 immunochromatography Methods 0.000 title description 5
- 239000012528 membrane Substances 0.000 claims abstract description 48
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 47
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 47
- 230000007704 transition Effects 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000010521 absorption reaction Methods 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 21
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 17
- 229930006000 Sucrose Natural products 0.000 claims description 17
- 239000005720 sucrose Substances 0.000 claims description 17
- 238000001035 drying Methods 0.000 claims description 11
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 150000003445 sucroses Chemical class 0.000 claims description 3
- 230000000903 blocking effect Effects 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 238000012993 chemical processing Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 15
- 239000012491 analyte Substances 0.000 abstract description 13
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000000053 physical method Methods 0.000 description 3
- 108010074051 C-Reactive Protein Proteins 0.000 description 2
- 102100032752 C-reactive protein Human genes 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010013654 Drug abuse Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000003028 elevating effect Effects 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 208000011117 substance-related disease Diseases 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Classifications
-
- 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
Abstract
The invention discloses an immunochromatographic test strip without a hook effect and a preparation method thereof, the immunochromatographic test strip comprises a bottom plate, a nitrocellulose membrane, a sample pad and an absorption pad are arranged above the bottom plate, the nitrocellulose membrane is positioned between the sample pad and the absorption pad, a transition pad, a test line and a control line are arranged above the nitrocellulose membrane, the test line is arranged between the transition pad and the control line, a conjugate pad is arranged above the transition pad, and a labeled antibody is arranged above the conjugate pad. Under the condition of no additional manual operation, the hook effect in the traditional sandwich method LFIA test strip is solved at low cost, false negative in the test result is effectively eliminated, and the application limit of the traditional LFIA test strip is broken; the novel immunochromatographic strip exhibits extremely high detection specificity for an analyte.
Description
Technical Field
The invention belongs to the technical field of in-vitro detection, and particularly relates to an immunochromatography test strip without a hook effect and a preparation method thereof.
Background
The immune side flow chromatography (LFIA) technology based on the sandwich method has been widely developed in the field of in vitro detection due to the advantages of low cost, quick effect, simple operation and the like, and can be used for qualitative or quantitative analysis. The LFIA technology for qualitative analysis is mostly based on a colorimetric method, utilizes the surface plasmon resonance phenomenon of gold nanoparticles for detection, and is suitable for application scenes such as pregnancy, drug abuse, infectious disease detection and the like. The LFIA technology part of quantitative analysis is similar to the colorimetric method of qualitative LFIA, and quantitative detection is realized by utilizing the color shade; in recent years, a fluorescence method using quantum dots or fluorescent microspheres as labels has also been developed. Quantitative LFIA can be applied to early diagnosis of major diseases such as heart disease, chronic obstructive pulmonary disease, cancer, etc. On the other hand, whether qualitative or quantitative, all the current immune lateral flow chromatography test strips based on the sandwich method have a hook effect when the concentration of the analyte to be detected is too high, and the test result shows false negative.
The hook effect greatly limits the commercial application of LFIA and no ideal solution exists. The current solutions are: 1. diluting the sample; 2. rinsing; 3. elevating the concentration of the labeled antibody; 4. adding a third test line or applying a plurality of test zones; 5. and (5) dynamically measuring. Dilution of the sample and rinsing method introduced an additional manual step in the LFIA test. The three methods of increasing the concentration of the labeled antibody, adding a third test zone, and applying multiple test zones can significantly increase LFIA test strip processing costs. Dynamic measurement while Bayer's false negatives still limits the detection range at the time of quantification. Therefore, none of these methods completely solve the problems associated with the hook effect.
Disclosure of Invention
The invention provides an immunochromatography test strip without a hook effect and a preparation method thereof for solving the problems.
The technical scheme adopted by the invention is as follows:
the immunochromatographic test strip without the hook effect comprises a bottom plate, a nitrocellulose membrane, a sample pad and an absorption pad are arranged above the bottom plate, the nitrocellulose membrane is positioned between the sample pad and the absorption pad, a transition pad, a test line and a control line are arranged above the nitrocellulose membrane, the test line is arranged between the transition pad and the control line, a conjugate pad is arranged above the transition pad, the nitrocellulose membrane is isolated from the conjugate pad through the transition pad, the effect of delaying the conjugate pad to release a labeled antibody is achieved, and the labeled antibody is arranged above the conjugate pad.
Wherein the transition pad is a film processed by a chemical method or a film processed by a physical method, and the porosity of the transition pad is distributed in a gradient manner; the permeability and the infiltration speed of a solution sample to be measured in the transition pad are regulated by a chemical method or a physical method, so that the transition pad becomes a barrier for resisting the flow of the solution and a switch for releasing the labeled antibody, and the conjugate pad starts to infiltrate and release the labeled antibody at a specific time; delaying the release of the labeled antibody effectively separates the competing reaction between the analyte to be detected and the immobilized and labeled antibodies, thereby eliminating the hook effect at high analyte concentrations.
The invention also provides a preparation method of the immunochromatographic test strip without the hook effect, which comprises the following steps:
step one: fixing a test line and a control line on the nitrocellulose membrane, and drying at 36 ℃ for 2 hours or until the nitrocellulose membrane is completely dried;
step two: blocking the nitrocellulose membrane with a 0.01MPBS buffer solution containing 2% w/v BSA and 0.02% w/v.Tween-20 at room temperature, drying at 36℃for 2h or until the nitrocellulose membrane is completely dry;
step three: adding supersaturated amount of sucrose into deionized water, and stirring for 3-4 days at room temperature to obtain saturated aqueous solution of sucrose; removing sucrose sediment in the solution, and diluting the saturated sucrose solution to an ideal concentration by using deionized water;
step four: cutting the nitrocellulose membrane, and starting to infiltrate the nitrocellulose membrane with the sucrose solution from the edge of the nitrocellulose membrane until the nitrocellulose membrane is completely soaked in the sucrose solution; any bubbles cannot remain on the nitrocellulose membrane; completely drying the nitrocellulose membrane;
step five: immobilization of the pre-conjugated labeled antibody on a conjugate pad and complete drying at 36 ℃;
step six: assembling the LFIA test strip: firstly, a nitrocellulose membrane is stuck on a bottom plate, then a transition pad, an absorption pad and a sample pad are stuck in sequence, and a conjugate pad is stuck on the transition pad.
Wherein, the pH of the 0.01M PBS buffer solution in the second step is 7.2.
Wherein the labeled antibody is a fluorescent labeled antibody or a nano gold labeled antibody.
The invention has the following advantages:
1. under the condition of no additional manual operation, the hook effect in the traditional sandwich method LFIA test strip is solved at low cost, false negative in the test result is effectively eliminated, and the application limit of the traditional LFIA test strip is broken; the novel immunity side-stream chromatography test strip shows extremely high detection specificity for the analyte;
2. in conventional LFIA test strips, the analyte binds both the labeled antibody and the detection antibody. Wherein the analyte generally has the highest affinity for the labeled antibody in solution. When the analyte is in excess (in too high a concentration), most of the labeled antibody will preferentially bind to the analyte and reach reaction equilibrium, forming a labeled antibody-analyte complex. At this time, the complex and the unlabeled detection object can simultaneously form a competition reaction with the detection antibody of the T line, and the residual labeled antibody in the solution sample is insufficient to label the detection object on the T line, so that a false negative result is caused. By introducing the transition pad with adjustable infiltration rate, the release time of the labeled antibody is controlled, and the competition reaction among the analyte, the labeled antibody and the detection antibody in the traditional test strip is effectively avoided. The hook effect is resolved by optimizing the release time, maximizing the binding of unlabeled analyte to the T-line, and then releasing the labeled antibody, labeling the analyte on the T-line. In addition, the method for treating the cellulose membrane by the sucrose solution is simple and easy to implement. The transition pad treated by the chemical method can effectively block the flow of the solution and realize the switching effect. By contrast, under the same optimized condition, the novel immunity side-stream chromatography test strip with the transition pad has a detection range 100 times wider than that of the traditional test strip.
Drawings
FIG. 1 is a schematic diagram of the structure of an immunochromatographic test strip without a hook effect according to an embodiment of the present invention.
FIG. 2 shows test curves (a and b) and test results (C and d) of a novel LFIA test strip with a transition pad according to the embodiment of the present invention, wherein the test curves are used for detecting C-reactive protein;
FIG. 3 is a graph showing the detection of a conventional LFIA test strip for comparison, using the C-reactive protein as an example, according to the present invention;
FIG. 4 is a graph showing the wetting rate test of transition pads treated with sucrose solutions provided in the examples of the present invention.
The reference numerals are explained as follows:
1. a bottom plate; 2. a nitrocellulose membrane; 3. a sample pad; 4. an absorbent pad; 5. a transition pad; 6. conjugate pad.
Detailed Description
The invention is further described below, but is not limited to these.
Examples
The immunochromatography test strip without the hook effect comprises a bottom plate 1, wherein a nitrocellulose membrane 2, a sample pad 3 and an absorption pad 4 are arranged above the bottom plate 1, the nitrocellulose membrane 2 is positioned between the sample pad 3 and the absorption pad 4, a transition pad 5, a test line and a control line are arranged above the nitrocellulose membrane 2, the test line is arranged between the transition pad 5 and the control line, a conjugate pad 6 is arranged above the transition pad 5, and a labeled antibody is arranged above the conjugate pad 6.
Wherein the transition pad 5 is a film treated by a chemical method or a film processed by a physical method and having gradient porosity.
The invention also provides a preparation method of the immunochromatographic test strip without the hook effect, which comprises the following steps:
step one: fixing a test line and a control line on the nitrocellulose membrane 2, and drying at 36 ℃ for 2 hours or until the nitrocellulose membrane 2 is completely dried;
step two: the nitrocellulose membrane 2 was blocked at room temperature with a 0.01MPBS buffer solution pH7.2 containing 2% w/v BSA and 0.02% w/v.Tween-20, dried at 36℃for 2h or until the nitrocellulose membrane 2 was completely dried;
step three: adding supersaturated amount of sucrose into deionized water, and stirring for 3-4 days at room temperature to obtain saturated aqueous solution of sucrose; removing sucrose sediment in the solution, and diluting the saturated sucrose solution to an ideal concentration by using deionized water;
step four: cutting the nitrocellulose membrane 2, and soaking the nitrocellulose membrane 2 in the sucrose solution from the side of the nitrocellulose membrane 2 until the nitrocellulose membrane 2 is completely soaked in the sucrose solution; no bubbles can remain on the nitrocellulose membrane 2; completely drying the nitrocellulose membrane 2;
step five: fixing the pre-coupled fluorescent-labeled antibody or nano-gold-labeled antibody on the conjugate pad 6, and completely drying at 36 ℃;
step six: assembling the LFIA test strip: firstly, a nitrocellulose membrane 2 is stuck on a bottom plate 1, then a transition pad 5, an absorption pad 4 and a sample pad 3 are stuck in sequence, and a conjugate pad 6 is stuck on the transition pad 5.
It is pointed out that several variations and modifications can be made by a person skilled in the art without departing from the inventive concept, which fall within the scope of the invention.
Claims (4)
1. The immunochromatographic test strip without the hook effect comprises a bottom plate (1), and is characterized in that: the novel multifunctional medical device is characterized in that a nitrocellulose membrane (2), a sample pad (3) and an absorption pad (4) are arranged above the bottom plate (1), the nitrocellulose membrane (2) is located between the sample pad (3) and the absorption pad (4), a transition pad (5), a test line and a control line are arranged above the nitrocellulose membrane (2), the test line is arranged between the transition pad (5) and the control line, a conjugate pad (6) is arranged above the transition pad (5), and a labeled antibody is arranged above the conjugate pad (6), wherein the transition pad (5) is a film with gradient porosity distributed through chemical processing or physical processing.
2. The method for preparing the immunochromatographic test strip without hook effect according to claim 1, which is characterized in that: the method comprises the following steps:
step one: fixing a test line and a control line on the nitrocellulose membrane (2), and drying at 36 ℃ for 2 hours or until the nitrocellulose membrane (2) is completely dried;
step two: blocking the nitrocellulose membrane (2) with a 0.01MPBS buffer solution containing 2% w/v BSA and 0.02% w/v.Tween-20 at room temperature, drying at 36℃for 2h or until the nitrocellulose membrane (2) is completely dry;
step three: adding supersaturated amount of sucrose into deionized water, and stirring for 3-4 days at room temperature to obtain saturated aqueous solution of sucrose; removing sucrose sediment in the solution, and diluting the saturated sucrose solution to an ideal concentration by using deionized water;
step four: cutting the nitrocellulose membrane (2), and starting to infiltrate the nitrocellulose membrane (2) from the side of the nitrocellulose membrane (2) with sucrose solution until the nitrocellulose membrane (2) is completely soaked in the sucrose solution; any bubbles cannot remain on the nitrocellulose membrane (2); completely drying the nitrocellulose membrane (2);
step five: immobilization of the pre-conjugated labeled antibodies on the conjugate pad (6) and complete drying at 36 ℃;
step six: assembling the LFIA test strip: firstly, a nitrocellulose membrane (2) is stuck on a bottom plate (1), then a transition pad (5), an absorption pad (4) and a sample pad (3) are stuck in sequence, and a conjugate pad (6) is stuck on the transition pad (5).
3. The method for preparing the immunochromatographic test strip without hook effect according to claim 2, which is characterized in that: the pH of the 0.01MPBS buffer solution in the second step is 7.2.
4. The method for preparing the immunochromatographic test strip without hook effect according to claim 2, which is characterized in that: the labeled antibody is a fluorescent labeled antibody or a nano gold labeled antibody.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110922074.0A CN113791209B (en) | 2021-08-12 | 2021-08-12 | Immunochromatography test strip without hook effect and preparation method thereof |
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| CN202110922074.0A CN113791209B (en) | 2021-08-12 | 2021-08-12 | Immunochromatography test strip without hook effect and preparation method thereof |
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| CN113791209B true CN113791209B (en) | 2024-01-26 |
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Inventor after: Dong Tao Inventor after: He Guozhen Inventor after: Yang Chaochu Inventor before: Dong Tao Inventor before: He Guozhen Inventor before: Yang Chaochu Inventor before: Tulil Leninda Inventor before: Aro Branstad |
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