CN112526137A - Preparation method of immunochromatography detection test strip - Google Patents
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Abstract
The application relates to the technical field of immunoassay, and particularly discloses a preparation method of an immunochromatography detection test strip. The method comprises the following steps: s1, preparing fluorescent microspheres coupled with the antibodies; s2, preparing microsphere diluent; s3, preparing a working solution of the fluorescent microsphere labeled antibody compound; s4, coating antibody; s5, coating a fluorescent labeled antibody complex: coating the fluorescent microsphere labeled antibody compound working solution on a nitrocellulose membrane in a dot spraying manner to form a fluorescent line; s6, assembling a detection test strip: the coated nitrocellulose membrane was adhered to the sample pad and the absorbent pad and fixed to the substrate region and cut to the desired width. The method has the advantage of better linear correlation of the prepared test strip.
Description
Technical Field
The application relates to the technical field of immunoassay, in particular to a preparation method of an immunochromatography test strip.
Background
The fluorescence immunochromatography technology is a novel membrane detection technology based on antigen-antibody specific immunoreaction. The technology takes strip-shaped fiber chromatography materials fixed with a detection line (coated antibody or coated antigen) and a quality control line (anti-antibody) as a stationary phase, a test solution as a mobile phase, a fluorescence labeled antibody or antigen fixed on a connecting pad, and an analyte to be analyzed moves on the chromatography strip through capillary action. For macromolecular antigens (proteins, viruses, pathogenic bacteria and the like) with a plurality of antigenic determinants, a sandwich-type double-antibody sandwich immunochromatography method is generally adopted, namely, an object to be detected is firstly combined with a fluorescence labeling antibody under the action of a mobile phase and then combined with a coating antibody to form a sandwich-type double-antibody sandwich when reaching a detection line, so that the concentration of the object to be detected can be quantitatively detected.
The existing immunochromatography detection test strip basically consists of a polyvinyl chloride base plate, a nitrocellulose membrane, a sample pad and an absorption pad. The luminescent material is generally fixed on a sample pad made of glass fiber by microsphere spraying. When a human or animal body fluid sample is dripped on the sample pad, the body fluid sample is combined with the luminescent substance along with the chromatography to form an antigen-antibody complex, and then the complex is diffused into the detection area and is diffused to the detection area along with the chromatography. The detection area generally consists of a detection line coated with a detection antibody and a quality control line coated with a quality control antibody, and when the complex to be detected flows through the detection line, the antigen-antibody complex to be detected in the sample is captured by the other detection antibody of the detection line. And the content of the target antigen in the sample is measured by quantitatively detecting the fluorescence signal values of the detection line and the quality control line.
At present, the fluorescent microsphere antibody composite is fixed on glass fiber to form a bonding pad in a microsphere spraying mode which is basically adopted in the market, and the microsphere spraying mode cannot reach the upper limit of a specified linear range when aiming at some high-sensitivity projects, so that the performance of a reagent cannot meet the technical requirement.
Disclosure of Invention
In order to avoid the occurrence that the performance of the detection reagent cannot meet the technical requirements because the detection reagent cannot reach the upper limit of the specified linear range, the application provides an immunochromatography detection test strip and a use method thereof.
The application provides a preparation method of a detection test strip for immunochromatography, which adopts the following technical scheme:
a preparation method of an immunochromatographic detection test strip comprises the following steps:
s1, preparing fluorescent microspheres coupled with the antibodies;
s2, preparing microsphere diluent;
s3, preparing a working solution of the fluorescent microsphere labeled antibody complex: mixing the fluorescent microspheres coupled with the antibody with a microsphere diluent according to the ratio of 1: (4-7) mixing the components in a volume ratio to obtain a working solution of the fluorescent microsphere labeled antibody compound;
s4, coating antibody: coating the detection antibody on the nitrocellulose membrane in a dot spraying manner to form a detection line, and coating the quality control antibody on the nitrocellulose membrane in a dot spraying manner to form a quality control line;
s5, coating a fluorescent labeled antibody complex: coating the fluorescent microsphere labeled antibody compound working solution on a nitrocellulose membrane in a dot spraying manner to form a fluorescent line;
s6, assembling a detection test strip: the coated nitrocellulose membrane was adhered to the sample pad and the absorbent pad and fixed to the substrate region and cut to the desired width.
By adopting the technical scheme, compared with the prior art, the preparation process has the advantages that the step of spraying the fluorescent microspheres on the sample pad is reduced, the step of spraying the working solution of the fluorescence-labeled antibody compound on the nitrocellulose membrane is newly added, the time for the fluorescence-labeled antibody compound to pass through the glass fiber is reduced, the compound to be detected can quickly pass through the sample pad and be combined with the fluorescence microsphere antibody compound close to the sample pad, and under the action of capillary, the fluorescence-labeled antibody compound can be released more quickly, so that the performance of the detection test strip is improved.
Preferably, the specific implementation manner of the step S1 is as follows:
and (3) mixing the antibody and the fluorescent microspheres according to the following ratio of (0.3-1.5): 1 for 60-180min in a coupling buffer at pH 5-8.
By adopting the technical scheme, in the coupling buffer solution, the antibody and the fluorescent microspheres are mixed by adopting the mass-to-volume ratio, so that the antibody can be uniformly dispersed on the fluorescent microspheres, and the utilization rate of the antibody is improved.
Preferably, the fluorescent microspheres are polystyrene-embedded lanthanide microspheres, polyethylene-embedded quantum dot microspheres or polystyrene-embedded organic fluorescent dye microspheres.
Preferably, the coupling buffer is 0.02M phosphate buffer.
Preferably, the particle size of the fluorescent microsphere is 200-300 nm.
By adopting the technical scheme, firstly, the particle size of the fluorescent microspheres is controlled, and the dispersibility of the fluorescent microspheres in the coupling buffer solution can be ensured; then, by controlling the particle size of the fluorescent microspheres, the antibody can be uniformly marked on the surfaces of the fluorescent microspheres in a coupling buffer solution.
Preferably, the microsphere diluent in the step S2 is prepared from the following raw materials in parts by weight:
by adopting the technical scheme, the interference of the Tris-HCL buffer solution on the biochemical process is small, the most appropriate chemical reaction environment can be provided for the fluorescent microspheres coupled with the antibody, and the buffer solution with the pH value in a wide range from acidity to alkalinity can be prepared only by using the buffer system because the alkalinity of the Tris alkali is strong.
Preferably, the polyethylene glycol is PEG4000 purchased from Sigma.
Preferably, the preservative is proclin300 purchased from Sigma.
Preferably, the specific implementation manner of the step S2 is as follows:
s1, preparing raw materials comprising Tween, bovine serum albumin, polyethylene glycol, sucrose, a preservative and a Tris-HCL buffer solution in parts by weight;
s2, sequentially adding Tween, bovine serum albumin, polyethylene glycol, sucrose and a preservative into the Tris-HCL buffer solution, and uniformly stirring to obtain the microsphere diluent.
By adopting the technical scheme, the components are uniformly mixed, so that the purpose is to uniformly disperse the Tween, the bovine serum albumin, the polyethylene glycol, the sucrose and the preservative in the Tris-HCL buffer solution, and the microsphere diluent can fully play a role.
Preferably, the stirring speed in the step S3 is 40-60r/min, and the temperature is 25-30 ℃.
By adopting the technical scheme, the stirring speed in the step S3 is limited to be 40-60r/min, the temperature is limited to be 25-30 ℃, and all components in the microsphere diluent can be uniformly dispersed.
Preferably, the specific implementation manner of the step S4 is as follows:
the quality control line is obtained by spraying the quality control antibody on a nitrocellulose membrane at a spraying speed of 0.8-1.5 mu L/cm through a membrane scratching and gold spraying integrated machine and drying; the detection line is obtained by spraying the detection antibody on a nitrocellulose membrane at a spraying speed of 0.8-1.5 muL/cm through a membrane-scratching and gold-spraying integrated machine and drying.
By adopting the technical scheme, the film-scratching and gold-spraying integrated machine is adopted to prepare the detection line and the quality control line, the efficiency is high, the rejection rate is low, and the obtained lines of the detection line and the quality control line are uniform, so that the detection result is guaranteed.
Preferably, the specific implementation manner of the step S5 is as follows:
the fluorescent line is obtained by spraying the working solution of the fluorescence labeling antibody compound on a nitrocellulose membrane at a spraying speed of 0.8-1.5 mu L/cm through a membrane-scribing and gold-spraying integrated machine and drying.
By adopting the technical scheme, the film-scribing and gold-spraying integrated machine is used for spraying on the nitrocellulose membrane at the spraying speed of 0.8-1.5 mu L/cm, so that a fluorescent line with a certain width is obtained, and the fluorescent line can be ensured to react with a compound to be detected.
Drawings
FIG. 1 is a schematic diagram of the test strip for immunochromatography in this embodiment.
Reference numerals: 1. a substrate region; 2. a sample area; 3. a reaction zone; 31. a fluorescent line; 32. detecting lines; 33. a quality control line; 34. a nitrocellulose membrane; 4. absorption zone
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples.
The source of the raw materials used in the following examples are shown in table 1 below:
TABLE 1 sources of raw materials
| Raw materials | Goods number | Obstetric apparatus |
| Fluorescent microspheres | IF8827 | Bangs |
| Nitrocellulose membrane | B10600004 | Sadoris sp |
| Tris-HCl buffer | T6455 | Sigma |
| Tween-20 | 93773 | Sigma |
| Tween-80 | / | Sigma |
| Bovine serum albumin | A1933 | Sigma |
| Polyethylene glycol | PEG4000 | Sigma |
| Sucrose | / | Is commercially available |
| proclin300 | 48912 | Sigma |
| Phosphate buffer | 17202 | Sigma |
Examples
The application document discloses a test strip for immunochromatography, which refers to fig. 1, and comprises a substrate region 1 and an immunodetection region positioned on the upper surface of the substrate region 1, wherein the substrate region 1 is a polyethylene bottom plate, and in other embodiments, the substrate region 1 is one of a polyvinyl chloride bottom plate, a polypropylene bottom plate or a polystyrene bottom plate;
the immunodetection zone comprises:
(1) sample area 2: a sample pad is arranged on the sample area 2, and the sample pad is made of a glass fiber film or non-woven fabric in the embodiment;
(2) reaction zone 3: the reaction zone 3 is a nitrocellulose membrane 34 having a fluorescence line 31, a detection line 32 and a quality control line 33, and the nitrocellulose membrane 34 may be replaced with a commonly used chromatography medium in other embodiments;
(3) the absorption zone 4: the absorption area 4 is provided with an absorption pad which is formed by cutting filter paper.
The distance of the immunodetection zones was 25 mm. The fluorescence line 31, the detection line 32 and the quality control line 33 are sequentially arranged according to the permeation direction of the complex to be detected on the nitrocellulose membrane 34, the distance between the fluorescence line 31 and the end of the sample pad is 2mm, the distance between the detection line 32 and the fluorescence line 31 is 8mm, and the distance between the detection line 32 and the quality control line 33 is 4 mm.
In another embodiment, the distance between the immunodetection zones is 25mm, the distance between the fluorescent line 31 and the end of the sample pad is 2mm, the distance between the detection line 32 and the fluorescent line 31 is 9mm, and the distance between the detection line 32 and the quality control line 33 is 5 mm.
The application document also discloses a preparation method and a use method of the immunochromatography detection test strip, and the following technical scheme is adopted:
s1, preparing fluorescent microspheres coupled with the antibodies;
s2, preparing a microsphere diluent:
s21, preparing raw materials comprising Tween, bovine serum albumin, polyethylene glycol, sucrose, a preservative and a Tris-HCL buffer solution in parts by weight;
s22, sequentially adding Tween, bovine serum albumin, polyethylene glycol, sucrose and a preservative into the Tris-HCL buffer solution, and uniformly stirring to obtain the microsphere diluent.
S3, preparing a microsphere compound working solution: uniformly mixing the fluorescent microspheres coupled with the antibody with a microsphere diluent to obtain a microsphere compound working solution;
s4, coating antibody: coating the detection antibody on the nitrocellulose membrane 34 by a dot spraying mode to form a detection line 32, and coating the quality control antibody on the nitrocellulose membrane 34 by a dot spraying mode to form a quality control line 33;
s5, coating a fluorescent labeled antibody complex: coating the fluorescence labeling antibody compound working solution on the nitrocellulose membrane 34 by a dot spraying mode to form a fluorescence line 31;
s6, assembling a detection test strip: the coated nitrocellulose membrane 34 is adhered to the sample pad and the absorbent pad and fixed on the substrate region 1, and cut into test strips of a desired width.
S7, uniformly mixing the compound to be detected and the sample diluent, dripping the mixture on a sample pad, and flatly placing and standing;
s8, reading signals through a fluorescence instrument
Examples 1-3 of the preparation and method of use are: human C-reactive protein (CRP) assay.
Example 1: the embodiment provides a preparation method and a using method of a detection test strip for immunochromatography, which adopt the following technical scheme:
s1, preparing fluorescent microspheres coupled with antibodies: the CRP-labeled antibody (CRP monoclonal antibody-1) and the fluorescent microspheres were combined in the coupling buffer for 120 min. The coupling buffer was 0.02M phosphate buffer.
S2, preparing a microsphere diluent:
s21, preparing raw materials containing Tween, bovine serum albumin, PEG4000, sucrose, proclin300 and Tris-HCL buffer solution according to weight parts (see Table 2).
S22, sequentially adding Tween, bovine serum albumin, PEG4000, sucrose and proclin300 into a Tris-HCL buffer solution in a stirrer, and uniformly stirring to obtain the microsphere diluent.
S3, preparing a fluorescence labeling antibody compound working solution: uniformly mixing the fluorescent microspheres coupled with the antibody with the microsphere diluent to obtain a fluorescence labeling antibody compound working solution;
s4, coating antibody: diluting a C-reactive protein (CRP) detection antibody (CRP monoclonal antibody-2) to a fixed concentration (1.0mg/ml) by using a coating buffer solution, diluting a quality control antibody (goat anti-mouse IgG antibody) to a fixed concentration (0.2mg/ml), and spraying the solution on a nitrocellulose membrane 34 by adopting a membrane-scribing and gold-spraying integrated machine. The coating buffer is 0.01M phosphate buffer plus 3 wt% sucrose as a protective agent.
S5, coating a fluorescent labeled antibody complex: and spraying the working solution of the fluorescence labeling antibody compound on the nitrocellulose membrane 34 coated with the detection line antibody and the quality control line antibody through a membrane scratching and gold spraying integrated machine.
S6, placing the NC membrane coated with the antibody and the fluorescence labeled antibody compound in a drying oven at 45 ℃ for drying for 24 hours for later use.
S7, assembling a detection test strip: the coated nitrocellulose membrane 34, the sample pad and the absorption pad are adhered together and fixed on the substrate area 1, and cut into the detection test paper strips with the required width;
s8, and the use method of the test strip comprises the following steps: samples were taken from the tip of a common pipette and mixed with the sample diluent, 75. mu.L of the diluted sample was applied to the wells and the data was read by a fluorescence instrument after a reaction for a period of time. The sample diluent was 20mmol/L phosphate buffer pH7.4 containing 0.5 wt% Tween-20.
Embodiment 2-3, a method for preparing an immunochromatographic test strip, which is different from embodiment 1 in that: the amounts of the respective components added and the reaction conditions are shown in Table 2.
Example 4 is a human serum amyloid (SAA) assay, which differs from example 1 in that:
replacing the CRP-labeled antibody (CRP monoclonal antibody-1) in the S1 step with a human serum amyloid (SAA) labeled antibody (SAA monoclonal antibody-2); the C-reactive protein (CRP) detection antibody (CRP monoclonal antibody-2) in the S4 step was replaced with human serum amyloid (SAA) detection antibody (SAA monoclonal antibody-2), and the standing time in the S8 step was 180S replaced with 900S.
TABLE 2
Comparative example 1: an immunochromatographic test strip disclosed in publication No. CN108226466B was used as a reference.
The detection method 1: taking the test strips of example 1 and comparative example 1, taking 2 groups of human C-reactive protein (CRP) control reagents (data provided by Kjek Biotechnology Co., Ltd., Zhejiang), each group of control reagents comprising 34 parts of control reagents with different concentrations, the concentrations of which are shown in Table 3, dropping 2 groups of control reagents on the sample pads of the test strips of example 1 and comparative example 1, respectively, standing for 180s, and obtaining the results through a fluorescence instrument (shown in Table 3).
TABLE 3
The detection method 2 comprises the following steps: taking the test strips in the example 1 and the comparative example 1, taking 2 groups of human serum amyloid (SAA) control reagents (the concentration of the control reagents is detected by a Siemens immunoturbidimetry method), wherein each group of the control reagents comprises 34 parts of control reagents with different concentrations, the concentration of the control reagents is shown in the table 4, respectively dripping the 2 groups of the control reagents on the sample pads of the test strips in the example 4 and the comparative example 1, placing the test strips for 900s, and obtaining the result through a fluorescence instrument (shown in the table 4).
TABLE 4
As can be seen from table 3, in sample number 25, the concentration of the control reagent is 95.2, and the detection results of the test strips in example 1 and comparative example 1 are 96.464 and 52.959, respectively, it can be seen that the test strip in the present application can more accurately detect the concentration of the sample in sample number 25, while the detection result in comparative example 1 is greatly different from the actual result.
As shown in the detection results of example 1 and comparative example 1 in sample numbers 26 to 34 in Table 3, the detection test strip prepared by the method can obtain more accurate results along with the increase of the sample concentration, while the detection test strip in comparative example 1 cannot obtain accurate results.
As can be seen from table 4, in sample number 27, the concentration of the control reagent is 111.27, and the detection results of the test strips in example 4 and comparative example 1 are 126.09 and 83.21, respectively, it can be seen that the test strip in the present application can more accurately detect the concentration of the sample in sample number 27, while the detection result in comparative example 1 is greatly different from the actual result.
As shown by the detection results of example 4 and comparative example 1 in sample numbers 28 to 34 in Table 4, the detection test strip prepared by the method can obtain more accurate results along with the increase of the sample concentration, while the detection test strip in comparative example 1 cannot obtain accurate results.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. A preparation method of an immunochromatography detection test strip is characterized by comprising the following steps:
s1, preparing fluorescent microspheres coupled with the antibodies;
s2, preparing microsphere diluent;
s3, preparing a working solution of the fluorescent microsphere labeled antibody complex: mixing the fluorescent microspheres coupled with the antibody with a microsphere diluent according to the ratio of 1: (4-7) mixing the components in a volume ratio to obtain a working solution of the fluorescent microsphere labeled antibody compound;
s4, coating antibody: coating the detection antibody on the nitrocellulose membrane in a dot spraying manner to form a detection line, and coating the quality control antibody on the nitrocellulose membrane in a dot spraying manner to form a quality control line;
s5, coating a fluorescent labeled antibody complex: coating the fluorescent microsphere labeled antibody compound working solution on a nitrocellulose membrane in a dot spraying manner to form a fluorescent line;
s6, assembling a detection test strip: the coated nitrocellulose membrane is adhered to the sample pad and the absorbent pad and fixed on the substrate region (1), and cut to a desired width.
2. The method for preparing an immunochromatographic test strip according to claim 1, which is characterized in that: the specific implementation manner of the step S1 is as follows:
and (3) mixing the antibody and the fluorescent microspheres according to the following ratio of (0.3-1.5): 1 for 60-180min in a coupling buffer at pH 5-8.
3. The method for preparing an immunochromatographic test strip according to claim 2, characterized in that: the fluorescent microspheres are polystyrene-embedded lanthanide microspheres, polyethylene-embedded quantum dot microspheres or polystyrene-embedded organic fluorescent dye microspheres.
4. The method for preparing an immunochromatographic test strip according to claim 2, characterized in that: the coupling buffer was 0.02M phosphate buffer.
5. The method for preparing an immunochromatographic test strip according to claim 2, characterized in that: the particle size of the fluorescent microsphere is 200-300 nm.
6. The method for preparing an immunochromatographic test strip according to claim 1, wherein the microsphere diluent in the step S2 is prepared from the following raw materials in parts by weight:
10-100 parts of Tris-HCL buffer solution;
0.2-1 part of tween;
0.3-1 part of bovine serum albumin;
0.3-1 part of polyethylene glycol;
10-30 parts of cane sugar;
0.1-0.2 part of preservative.
7. The method for preparing an immunochromatographic test strip according to claim 6, wherein the step S2 is specifically realized by:
s1, preparing raw materials comprising Tween, bovine serum albumin, polyethylene glycol, sucrose, a preservative and a Tris-HCL buffer solution in parts by weight;
s2, sequentially adding Tween, bovine serum albumin, polyethylene glycol, sucrose and a preservative into the Tris-HCL buffer solution, and uniformly stirring to obtain the microsphere diluent.
8. The method for preparing an immunochromatographic test strip according to claim 1, which is characterized in that: the stirring speed in the step S3 is 40-60r/min, and the temperature is 25-30 ℃.
9. The method for preparing an immunochromatographic test strip according to claim 1, wherein the step S4 is specifically realized by:
the quality control line is obtained by spraying the quality control antibody on a nitrocellulose membrane at a spraying speed of 0.8-1.5 mu L/cm through a membrane scratching and gold spraying integrated machine and drying; the detection line is obtained by spraying the detection antibody on a nitrocellulose membrane at a spraying speed of 0.8-1.5 muL/cm through a membrane-scratching and gold-spraying integrated machine and drying.
10. The method for preparing an immunochromatographic test strip according to claim 1, wherein the step S5 is specifically realized by:
the fluorescent line is obtained by spraying the working solution of the fluorescence labeling antibody compound on a nitrocellulose membrane at a spraying speed of 0.8-1.5 mu L/cm through a membrane-scribing and gold-spraying integrated machine and drying.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113391062A (en) * | 2021-05-25 | 2021-09-14 | 黄淮学院 | Morphine immunofluorescence chromatography rapid detection test paper strip, preparation method and detection method thereof |
| CN114236118A (en) * | 2021-11-29 | 2022-03-25 | 桂林理工大学 | Method for immobilizing fluorescent dye on nitrocellulose membrane |
| CN115575632A (en) * | 2022-12-07 | 2023-01-06 | 广州国家实验室 | Detection test strip and application |
| CN113391062B (en) * | 2021-05-25 | 2024-06-07 | 黄淮学院 | Morphine immunofluorescence chromatography rapid detection test strip, preparation method and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103226143A (en) * | 2013-04-07 | 2013-07-31 | 南京基蛋生物科技有限公司 | Dry-type immunoassay test strip and preparation method and application thereof |
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| CN113391062A (en) * | 2021-05-25 | 2021-09-14 | 黄淮学院 | Morphine immunofluorescence chromatography rapid detection test paper strip, preparation method and detection method thereof |
| CN113391062B (en) * | 2021-05-25 | 2024-06-07 | 黄淮学院 | Morphine immunofluorescence chromatography rapid detection test strip, preparation method and detection method thereof |
| CN114236118A (en) * | 2021-11-29 | 2022-03-25 | 桂林理工大学 | Method for immobilizing fluorescent dye on nitrocellulose membrane |
| CN115575632A (en) * | 2022-12-07 | 2023-01-06 | 广州国家实验室 | Detection test strip and application |
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