CN111965357B - Fluorescent immunochromatography detection method, test paper and application thereof - Google Patents
Fluorescent immunochromatography detection method, test paper and application thereof Download PDFInfo
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- CN111965357B CN111965357B CN201911188167.4A CN201911188167A CN111965357B CN 111965357 B CN111965357 B CN 111965357B CN 201911188167 A CN201911188167 A CN 201911188167A CN 111965357 B CN111965357 B CN 111965357B
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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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
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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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Abstract
A fluorescent immunochromatography detection method comprises the steps of adding a sample liquid to be detected and a sample diluent into a sample pad and a sample diluent respectively, carrying out capillary action on the sample liquid to be detected, and combining a to-be-detected object with a T line. Under the action of liquid diffusion, after the sample liquid dissolves the instant membrane between the sample pad and the fluorescent pad, fluorescent microspheres carried by the diluent face the T line chromatography together and are combined with a sample at the T line, and finally, a fluorescent value at the T line is obtained and is used as a basis for sample detection. Proved by verification, the result obtained in the detection of the antibody on the surface of hepatitis B by adopting the detection method and the test paper shows that the variation Coefficient (CV) of T/C is obviously reduced, the interference factor is controlled, and the accuracy of fluorescence chromatography detection is obviously improved.
Description
Technical Field
The application relates to a composition, in particular to a composition for fluorescent detection reagent, which is used for improving the precision and accuracy of the reagent on antibody detection.
Background
Fluorescent immunochromatography is a novel membrane detection technology based on antigen-antibody specific immune reaction. The technology uses strip fiber chromatographic materials fixed with detection lines (coated antibodies or coated antigens) and quality control lines (anti-antibodies) as stationary phases, test liquid as mobile phases, fluorescent labeled antibodies or antigens are fixed on a connecting pad, and analytes are enabled to move on chromatographic strips through capillary action. For macromolecular antigens (proteins, viruses, pathogenic bacteria and the like) with multiple antigenic determinants, a sandwich-type double-antibody sandwich immunochromatography method is generally adopted, namely, an object to be detected is firstly combined with a fluorescent labeled antibody under the action of a mobile phase, and then is combined with a coated antibody to form a sandwich-type double-antibody sandwich when reaching a detection line. For small molecule antigens (agricultural veterinary drugs, illicit drugs and the like) with only a single epitope, after the small molecule antigen to be detected is combined with a fluorescent labeled antibody, the small molecule antigen is difficult to be combined with a coated antibody on a detection line due to steric hindrance. Therefore, small molecule analytes with a single epitope are often detected by competitive immunochromatography.
The structure of the common test strip is as follows from one end to the other end: after the sample is added into the sample pad, the sample is driven by capillary phenomenon to move towards the direction of the water absorbing paper to generate chromatography, then the sample is combined with the marked microsphere attached to the fluorescent pad, and the chromatography is continued, and reacts with a detection line (also called T line) and a quality control line (also called C line) which are fixed on the NC film in sequence. When a substance to be detected exists in a sample, the substance to be detected combined with the fluorescent microsphere is fixed on a T line, and the qualitative or quantitative detection of the substance to be detected is realized by detecting the fluorescent signal and the intensity thereof. Such one-way chromatography with one sample (i.e., the "one-step method") has a large amount of interference to detection, and particularly in a one-step reaction performed by an indirect method, similar detection antibodies (e.g., igG antibodies) carried by a sample (e.g., blood) may bind to the microspheres and interfere with the results.
Patent CN200980117868.7 discloses a time-of-accumulation resolved emission two-dimensional gel electrophoresis that uses the difference in fluorescence lifetime imaging to distinguish between fluorescence from specific protein markers and non-specific background fluorescence, resulting in significant improvements in both sensitivity and dynamic range over the prior art. The fluorescent dye bound to the protein is excited with a pulsed laser scanner to provide an in-gel detection and quantification of the fluorescent lifetime imaging assay for the protein on a pixel-by-pixel level, and a multi-exponential fit of the fluorescent decay curve is used to separate the fluorescence from the fluorescent dye-labeled protein species, from the gel matrix itself, from the source of the solution or particles present in the gel, or from the scattering effect for subsequent background subtraction.
Patent CN201810206451.9 discloses an immunofluorescence chromatography test paper, which has high sensitivity and good repeatability, and comprises a sample pad, a marking pad, a chromatography strip and absorbent paper which are sequentially lapped on a PVC bottom plate. Wherein, the chromatographic strip takes the inverse opal structure nitrocellulose photonic crystal film as a solid phase carrier. According to the immunofluorescence chromatography test paper, the traditional nitrocellulose membrane is replaced by the inverse opal structure nitrocellulose photonic crystal film, so that the detection sensitivity of the immunofluorescence chromatography test paper is improved, the nonspecific combination of false positive, false negative and the like can be reduced, the background signal interference is effectively reduced, the signal to noise ratio and the resolution are improved, the reaction time is shortened, and the coating concentration of the capture antibody can be greatly reduced.
Disclosure of Invention
The application aims to provide a fluorescence immunochromatography detection method which is used for fluorescence chromatography, so that interference of similar antibodies in a sample on a detection result is avoided, and the detection accuracy is improved.
The application also aims to provide the fluorescent immunochromatography test paper, which avoids the interference of similar antibodies in a sample on a detection result, so as to be beneficial to improving the detection accuracy.
The application also aims to provide an application of the fluorescent immunochromatography detection test paper in preparing a hepatitis B surface antibody detection reagent.
A fluorescent immunochromatography detection method comprises the steps of adding a sample liquid to be detected and a sample diluent into a sample pad and a sample diluent respectively, carrying out capillary action on the sample liquid to be detected, and combining a to-be-detected object with a T line. Under the action of liquid diffusion, after the sample liquid and the diluent dissolve the instant membrane between the sample pad and the fluorescent pad, the fluorescent microspheres carried by the diluent face the T line chromatography together and are combined with the to-be-detected object at the T line, and finally, the fluorescent value at the T line is obtained and used as the basis of sample detection.
According to the fluorescent immunochromatography detection method, after the sample liquid and the diluent are added, most of the sample liquid faces the T line chromatography, the object to be detected is fixed at the T line, and under the action of liquid diffusion, a small part of the sample liquid and the diluent wets and dissolves the instant membrane, so that the diluent drives fluorescent microspheres in the fluorescent pad to face the T line chromatography and is combined with the object to be detected fixed at the T line. And forming a detection form that the sample liquid is contacted with the T line firstly, and then contacted with the T line after carrying the diluent of the fluorescent microsphere. The object to be detected is combined with the T line, a large amount of irrelevant components can be removed (namely substances which are not fixed at the T line are continuously chromatographed and move towards the absorbent paper), and interference factors are further reduced because the fluorescent microspheres are subjected to the cleaning action of the diluent in the chromatography process towards the T line, so that the interference substances are greatly reduced when the substances are combined at the T line.
In order to implement the fluorescence immunochromatography detection method, the application also provides a fluorescence immunochromatography detection test paper which comprises a dilution pad and a water absorption paper, wherein the dilution pad and the water absorption paper are respectively arranged at two ends of the detection test paper, a fluorescence pad, an instant membrane, a sample pad and a nitrocellulose membrane (also called NC membrane) are sequentially arranged in the direction from the dilution pad to the water absorption paper, and a T line and a C line are arranged on the NC membrane.
An instant membrane is used between the sample pad and the dilution pad, and the dilution liquid and the sample liquid are isolated by utilizing the physical isolation function of the membrane, so that most of the sample liquid faces the T-line chromatography before the dilution liquid. After the instant membrane is dissolved, the diluent is opened to the channel of the T line chromatography and is combined with the object to be detected on the T line after the diluent is in the sample liquid.
An instant membrane implementation mode for fluorescent immunochromatography detection test paper adopts polyethylene glycol (PEG), the molecular weight of which is 2,000 Da-6,000 Da, and the instant membrane implementation mode is preferably prepared by mixing PEG with the molecular weight of 2,000Da and PEG with the molecular weight of 4,000Da according to the weight ratio of 1.7:1, so that the instant membrane can be properly dissolved, namely, the instant membrane can be dissolved by sample liquid and diluent, and the fluorescent microsphere can be driven by the diluent to face T-line chromatography.
Another embodiment of the instant membrane for use in a fluorescent immunochromatographic test strip employs polyvinyl alcohol (PVA) or soluble starch.
The fluorescent immunochromatography test paper provided by the application adopts glass fiber as a sample pad, and the unit weight of the sample pad is 75g/m 2 0.43mm in thickness, chromatography speed 5s/2cm, and water storage capacity 79mg/cm 2 。
The fluorescent immunochromatography test paper provided by the application adopts glass fiber as a diluting pad, and the unit weight of the diluting pad is 75g/m 2 0.43mm in thickness, chromatography speed 5s/2cm, and water storage capacity 79mg/cm 2 。
The fluorescent immunochromatography test paper provided by the application adopts glass fiber as a fluorescent pad, and the unit weight of the fluorescent pad is 75g/m 2 The thickness is 0.38mm, the chromatography speed is 3s/2cm, and the water storage capacity is 63mg/cm 2 。
By verification, the detection method and the test paper of the application show that the variation Coefficient (CV) of T/C is obviously reduced, the interference factor is controlled, the accuracy of fluorescence chromatography detection is obviously improved, and the detection method is consistent with the use method of a commercially available test paper strip.
A fluorescent immunochromatography kit comprises various detection test papers provided by the application.
Drawings
FIG. 1 is a schematic diagram of an embodiment of a test strip for fluorescence immunochromatography according to the present application.
Detailed Description
The technical scheme of the present application is described in detail below with reference to the accompanying drawings. The embodiments of the present application are only for illustrating the technical scheme of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical scheme of the present application, which is intended to be covered by the scope of the claims of the present application.
Example 1
Fig. 1 is a fluorescent immunochromatographic test strip, which includes a dilution pad 600 and a water-absorbing paper 100, which are respectively disposed at both ends of the test strip, a fluorescent pad 500, an instant membrane 400, a sample pad 300 and an NC membrane 200 are sequentially disposed in a direction from the dilution pad 600 to the water-absorbing paper 100, and T-lines 210 and C-lines 220 are disposed on the NC membrane 200, and fluorescent microspheres (not shown) are disposed on the fluorescent pad 500.
In use, sample fluid is applied to sample pad 300 and diluent is applied to diluent pad 600. After the sample solution is added, on one hand, the sample solution is directed to the T line 210, and the substance to be tested is fixed at the T line 210. On the other hand, under the action of the liquid diffusion, a small part of sample liquid and diluent wets and dissolves the instant membrane 400, so that the diluent drives the fluorescent microspheres in the fluorescent pad 500 to chromatograph towards the T line 210 and combine with the substance to be detected fixed at the T line.
Through practice, PEG, PVA and soluble starch can be used for preparing instant membrane, and the molecular weight of PEG is 2,000 Da-6,000 Da. The instant membrane is prepared by mixing PEG with molecular weight of 2,000Da and PEG with molecular weight of 4,000Da according to the weight ratio of 1.7:1, which is beneficial to the dissolution of sample liquid to the instant membrane and the driving of fluorescent microspheres to the T-line chromatography by diluent.
In order to adjust the chromatographic speed of the diluent carrying the fluorescent microspheres in the dissolving process of the instant membrane, the dissolving of the sample pad to the instant membrane is facilitated. The sample pad and the dilution pad each had a basis weight of 75g/m 2 0.43mm in thickness, chromatography speed 5s/2cm, and water storage capacity 79mg/cm 2 Is a glass fiber of (a). The fluorescent pad adopts 75g/m of unit weight 2 The thickness is 0.38mm, the chromatography speed is 3s/2cm, and the water storage capacity is 63mg/cm 2 Is a glass fiber of (a).
Example 2
The commercial test strip and the test strip of the embodiment are respectively used for detecting a low-concentration hepatitis B surface antibody positive serum sample with the concentration of 20mIU, and the specific detection results are shown in Table 1.
TABLE 1
Example 3
The commercial test strip and the test strip of this example were used to detect a low concentration hepatitis B surface antibody positive serum sample at a concentration of 50mIU, respectively, and the specific detection results are shown in Table 2.
TABLE 2
Claims (1)
1. A fluorescent immunochromatography test paper for detecting positive serum of antibody on hepatitis B surface is characterized by comprising a dilution pad and water absorption paper which are respectively arranged at two ends of the test paper, wherein a fluorescent pad, an instant membrane, a sample pad and an NC membrane are sequentially arranged from the dilution pad to the water absorption paper, and T lines and C lines are arranged on the NC membrane, wherein the instant membrane is prepared by mixing PEG with molecular weight of 2,000Da and PEG with molecular weight of 4,000Da according to the weight ratio of 1.7:1, the sample pad adopts glass fiber, and the unit weight of the sample pad is 75g/m 2 Thickness of (thickness)0.43mm, chromatography speed 5s/2cm, and water storage capacity 79mg/cm 2 The dilution pad is made of glass fiber, and the unit weight of the dilution pad is 75g/m 2 0.43mm in thickness, chromatography speed 5s/2cm, and water storage capacity 79mg/cm 2 The fluorescent pad is made of glass fiber, and the unit weight of the fluorescent pad is 75g/m 2 The thickness is 0.38mm, the chromatography speed is 3s/2cm, and the water storage capacity is 63mg/cm 2 The detection method comprises the steps of respectively adding sample liquid to be detected and sample diluent into a sample pad and a diluent pad, carrying out capillary action on the sample liquid, carrying out T-line chromatography on the sample liquid, combining a to-be-detected object with the T-line, carrying out liquid diffusion on an instant membrane between the sample pad and a fluorescent pad by the sample liquid and the diluent, enabling fluorescent microspheres carried by the diluent to jointly face the T-line chromatography, combining the fluorescent microspheres with the to-be-detected object at the T-line, and finally obtaining a fluorescent value at the T-line as a basis of sample detection.
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| CN201911188167.4A CN111965357B (en) | 2019-11-28 | 2019-11-28 | Fluorescent immunochromatography detection method, test paper and application thereof |
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Citations (4)
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| CN101726596A (en) * | 2009-11-04 | 2010-06-09 | 无锡中德伯尔生物技术有限公司 | Fluorescent microsphere immunochromatographic testing card for testing five indexes of hepatitis b and method for preparing same |
| CN103743900A (en) * | 2014-01-24 | 2014-04-23 | 厦门为正生物科技有限公司 | Immunochromatography detection device and method by adopting two-step method |
| CN108318690A (en) * | 2018-03-13 | 2018-07-24 | 深圳市第二人民医院 | A kind of immunofluorescence chromatographic test paper and its preparation method and application |
| CN110308275A (en) * | 2019-08-08 | 2019-10-08 | 深圳市易瑞生物技术股份有限公司 | A kind of diplopore detection card and kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110195525A1 (en) * | 2010-02-11 | 2011-08-11 | Aquilino Arnold J | Method of use and Apparatus for an Enhanced Lateral Flow Rapid Test Device |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101726596A (en) * | 2009-11-04 | 2010-06-09 | 无锡中德伯尔生物技术有限公司 | Fluorescent microsphere immunochromatographic testing card for testing five indexes of hepatitis b and method for preparing same |
| CN103743900A (en) * | 2014-01-24 | 2014-04-23 | 厦门为正生物科技有限公司 | Immunochromatography detection device and method by adopting two-step method |
| CN108318690A (en) * | 2018-03-13 | 2018-07-24 | 深圳市第二人民医院 | A kind of immunofluorescence chromatographic test paper and its preparation method and application |
| CN110308275A (en) * | 2019-08-08 | 2019-10-08 | 深圳市易瑞生物技术股份有限公司 | A kind of diplopore detection card and kit |
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| 王兵等.苯甲酸利扎曲普坦速溶膜剂的研制及体内外评价.《中国医药工业杂志》.2016,第47卷(第11期),第1398-1403页. * |
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