CN109541234B - Preparation method of immunochromatographic test strip for accurate quantitative detection - Google Patents
Preparation method of immunochromatographic test strip for accurate quantitative detection Download PDFInfo
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
-
- 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
-
- 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
- G01N33/577—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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a preparation method of an immunochromatographic test strip for accurate quantitative detection, wherein the preparation of a bonding pad comprises the following steps: s1: cutting the combined pad: cutting the bonding pad into the size required for assembling into a finished product; s2: preparation of labeled complexes: after the antibody or the antigen is marked on the marker, placing the marker in a preservation solution for storage; s3: preparing a soak solution of the labeled compound: mixing and diluting a diluent and the preservation solution containing the labeled compound to obtain a soak solution of the labeled compound with a certain concentration; s4: infiltrating the bonding pad: adding a soaking solution of a standard marker compound into the combined pad for soaking; s5: combining a pad balancing process: spreading the soaked combined pad on a carrier, and standing in a constant temperature and humidity environment; s6: drying the combined pad: the equilibrated conjugate pad is dried. The method can effectively improve the precision of the test strip for accurate quantitative detection, and has the advantages of simple preparation method, high production efficiency, low production cost and convenience for industrial production.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a preparation method of an immunochromatographic test strip for accurate quantitative detection.
Background
The test strip technology is an immunological detection technology which is simple to operate, does not need professional training, is convenient and quick and has visual results. The method is widely applied to the fields of rapid disease diagnosis, environmental pollutant analysis, pathogenic biological factor detection and the like. The detection principle is that a specific antibody (or antigen) is fixed on a specific area of a nitrocellulose membrane as a capture reagent, after a sample to be detected is added, the sample to be detected moves forwards along the membrane under the pushing of capillary force, when the sample moves to the area where the capture reagent is fixed, the antigen (or antibody) in the sample is specifically combined with the capture reagent, and a labeling reagent displays a certain color, so that the immunoassay method for specific detection is realized. The traditional colloidal gold test strip displays a certain color through gold nanoparticles so as to realize immunoassay of specific detection, and although the detection result is visible by naked eyes, the method cannot finish accurate quantitative detection; the fluorescence immunochromatographic test strip detection technology is a new technology which is raised for realizing quantitative detection after the colloidal gold immunochromatographic test strip technology.
At present, the structure of the fluorescence immunochromatographic test strip consists of a sample pad, a combination pad, an NC membrane and a water absorption pad, which are sequentially overlapped and adhered on a PVC bottom plate, wherein a compound containing a labeled antibody/labeled antigen is sprayed or soaked and then dried and solidified on the combination pad, and the combination pad is usually made of glass fiber. During the test, the sample to be tested is directly added on the sample pad or the sample adding hole. The treatment method of the bonding pad mainly comprises a spraying method and a soaking method.
The method for processing the bonding pads by adopting the spraying mode comprises two modes (1) the whole uncut bonding pad is sprayed in a spraying instrument in a reciprocating mode, the quantity of the marker of each large bonding pad can be accurately quantified by adopting the mode, but the spraying parameters, particularly the spraying speed and the spraying flow rate, need to be accurately controlled; (2) the bonding pads are cut into proper sizes in advance, the bonding pads are sprayed once, the quantity of the markers on each bonding pad can be accurately quantified by adopting the method, the accurate alignment is needed in the spraying process, otherwise, the marking compound solution is easy to spray out of the bonding pads and stays on a spraying table top, and the spraying table top needs to be frequently wiped, so that the production efficiency is influenced, and mutual pollution among the bonding pads is easily caused. In addition, the complex is usually deposited on only one surface of the conjugate pad by spraying, and the label is unevenly distributed on the conjugate pad and even agglomerated on the conjugate pad, which causes difficulty in uniform release of the label during the process of testing the sample, and the label is easily remained on the conjugate pad, resulting in poor precision of product testing. The processing method has high requirements on the material of the sample pad, the bonding pad and the NC membrane, and the material used for different detection items is different, and in addition, the method generally needs to carry out pretreatment on the bonding pad in order to reduce CV.
The combination pad is processed by adopting a soaking mode, and the combination pad is generally applied to qualitative detection of a test strip, and is less applied to accurate quantitative detection, if the quantitative detection is needed, two modes are also adopted, (1) the whole combination pad is soaked in a solution containing a marker and then taken out for quick freeze-drying; (2) the label is quantitatively poured onto the whole bonding pad, the mixture is uniformly driven back and forth by a compression roller, and the drying oven is used for quickly drying. The soaking method has the advantages of simple method and higher production efficiency than the spraying method; however, the method has the disadvantages that the distribution of the labeling substance on the bonding pad is not uniform, especially the edge effect (that is, the distribution of the labeling compound on the bonding pad at the edge is different from that of the labeling compound on the bonding pad in the middle) is easily generated, the precision of the product test is poor, the CV is large, the peripheral part of the bonding pad needs to be cut off during the cutting process, and the rejection rate of the bonding pad is high.
Therefore, there is a need to develop a simple method for preparing an immunochromatographic test strip, so that the prepared immunochromatographic test strip has a small CV value and is suitable for accurate quantitative detection.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is that the immunochromatographic test paper in the prior art is poor in test precision and cannot be used for accurate quantitative detection, and the preparation method of the immunochromatographic test paper strip is further provided, can effectively improve the test paper strip precision for accurate quantitative detection, is simple in preparation method, high in production efficiency, low in production cost and convenient for industrial production.
In order to solve the technical problems, the invention provides a preparation method of an immunochromatographic test strip for accurate quantitative detection, which comprises the following steps: preparing a nitrocellulose membrane, preparing a sample pad, preparing a combined pad and assembling a test strip; wherein, in the preparation of the bonding pad, the following steps are included:
s1: cutting the combined pad: cutting the bonding pad into the size required for assembling into a finished product;
s2: preparation of labeled complexes: after the antibody or the antigen is marked on the marker, placing the marker in a preservation solution for storage;
s3: preparing a soak solution of the labeled compound: mixing and diluting a diluent and the preservation solution containing the labeled compound to obtain a soak solution of the labeled compound with a certain concentration;
s4: infiltrating the bonding pad: adding a soaking solution of a standard label compound into the bonding pad, and soaking the bonding pad;
s5: combining a pad balancing process: spreading the soaked combined pad on a carrier, and standing in a constant temperature and humidity environment;
s6: drying the combined pad: the equilibrated conjugate pad is dried.
Further, in step S2, the label is any one of a time-resolved fluorescent microsphere, a normal fluorescent microsphere, an upconversion luminescent microsphere, a magnetic microsphere, colloidal gold, a color latex microsphere, a quantum dot, a time-resolved fluorescent dye, a normal fluorescein, an enzyme, a biotin-avidin, and a microsphere amplification system.
Further, in step S3, the diluent contains buffer pair, protein, saccharide, high polymer, preservative and surfactant.
Further, the buffer pair of the diluent is one of Tris-HCL (Tris-hydroxymethyl-aminomethane-hydrochloric acid) buffer solution with the molar concentration of 0.015-0.025M, PB (phosphate) buffer solution with the molar concentration of 0.015-0.025M and HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) buffer solution with the molar concentration of 0.015-0.025M, the protein is BSA (bovine serum albumin) with the mass concentration of 0.5-2%, the saccharide is trehalose with the mass concentration of 2.0-15%, sucrose with the mass concentration of 2.0-15% and dextran 20000 with the mass concentration of 1.0-3%, the high polymer is PVP (polyvinylpyrrolidone) with the mass concentration of 0.25-3%, the preservative is Proclin300 with the mass concentration of 0.01-0.1% or NaN3 (sodium azide) with the mass concentration of 0.01-0.1%, the surfactant is TW-20 (Tween-20) with the mass concentration of 0.05-2% and PEG4000 (polyethylene glycol 4000) with the mass concentration of 0.05-2%.
Further, in step S3, the volume ratio of the preservation solution to the diluent of the labeled complex is 1:1000-20: 1000.
Further, in step S4, the quantitative determination means 10. mu.L to 50. mu.L.
Further, in step S4, the step of adding the soaking solution of the labeled complex to the bonding pad is to soak the bonding pad in the soaking solution of the labeled complex, or to drop the soaking solution of the labeled complex uniformly onto the bonding pad by using a solution taking device.
Further, in the step S5, the standing in the constant temperature and humidity environment means standing for 35min to 300min in an environment with a temperature of 20 ℃ to 50 ℃ and a humidity of 25% to 60%.
Furthermore, in step S5, the standing in the constant temperature and humidity environment includes two stages, wherein the first stage is standing for 5min to 60min at a temperature of 20 ℃ to 37 ℃ and a humidity of 40% to 60%; the second stage is to stand for 30-240 min under the environment of 25-50 ℃ and 25-40% of humidity. Therefore, the soaking solution can be well diffused in the combined pad, and the labeling compound is uniformly distributed in the combined pad.
Further, in step S5, the carrier is any one of a glass plate, a stainless steel plate and a plastic plate.
Further, in step S5, the carrier has a hydrophobic surface.
The invention has the beneficial effects that:
1. the preparation method of the invention also comprises a balancing process after the bonding pad is soaked, the bonding pad is placed on the carrier under the condition of constant temperature and constant humidity, the marking compound in the bonding pad placed on the flat carrier can be ensured to be fully soaked and diffused, the marking compound is uniformly distributed in the whole bonding pad three-dimensional structure after being dried, and the back-and-forth uniform driving and rapid drying by a press roller are not needed.
2. According to the preparation method, the bonding pad is cut into the size required by the bonding pad when the test strip finished product is assembled, and then a certain amount of soak solution is added, so that the amount of the labeled compound on each bonding pad with fixed size is the same.
3. The preparation method of the invention has the advantages that the carrier surface of the bonding pad is hydrophobic, the soak solution only flows on the bonding pad, the surface of the carrier has certain roughness, the complete bonding pad is convenient to take down after drying, the bonding pad is not easy to stick on the bonding pad carrier, and the hydrophobic surface carrier is easy to clean and is convenient to recycle.
4. According to the preparation method disclosed by the invention, for the mark which is invisible to naked eyes, the distribution condition of the marked compound after the bonding pad is dried can be directly observed by naked eyes by adding the non-toxic edible pigment with the color.
5. The immunochromatographic test strip prepared by the preparation method can be used for accurate quantitative detection, and has low inaccuracy degree and CV value less than 5%.
6. The preparation method is suitable for most of the bonding pads made of different materials, namely, the bonding pads have low requirements on the materials and are suitable for most of immunochromatography products, the bonding pads do not need pretreatment, the dried bonding pads can be stored and transported under the normal temperature condition, and the production cost is favorably reduced.
7. The preparation method of the invention has simple operation steps and equipment and can be produced in large batch.
Detailed Description
The invention provides a preparation method of an immunochromatographic test strip for accurate quantitative detection, which can effectively improve the compactness for accurate quantitative detection, and is simple in preparation method, high in production efficiency, low in production cost and convenient for industrial production. The method comprises the following steps: preparing a nitrocellulose membrane, preparing a sample pad, preparing a combined pad and assembling a test strip; wherein, in the preparation of the bonding pad, the following steps are included:
s1: cutting the combined pad: cutting the bonding pad into the size required for assembling into a finished product;
s2: preparation of labeled complexes: after the antibody or the antigen is marked on the marker, placing the marker in a preservation solution for storage;
s3: preparing a soak solution of the labeled compound: mixing and diluting a diluent and the preservation solution containing the labeled compound to obtain a soak solution of the labeled compound with a certain concentration;
s4: infiltrating the bonding pad: adding a soaking solution of a quantitative labeling compound into the bonding pad, and soaking the bonding pad;
s5: combining a pad balancing process: spreading the soaked combined pad on a carrier, and standing in a constant temperature and humidity environment;
s6: drying the combined pad: the equilibrated conjugate pad is dried.
In some embodiments, in step S1, the bonding pad is made of any one of glass fiber, polyester film, and non-woven fabric.
In some embodiments, the cutting of the bonding pad into the size required for assembling the test strip product in step S1 is to cut the bonding pad into the size required for assembling the test strip product, so that the bonding pad can be assembled directly without performing a secondary cutting process in the subsequent manufacturing process.
In some embodiments, step S2, the labeled complex is prepared by conventional labeling methods to couple the antibody or antigen to the label. The antibody or antibody refers to a substance capable of producing a specific reaction, such as an antibody conjugated with a label for detecting an antigen, an antigen conjugated with a label for detecting an antibody, or other substances capable of producing a specific reaction. A label refers to a substance that can produce a signal that can be perceived visually or using a sensor, either by the inherent properties of the label or by an external stimulus (e.g., fluorescence). The labeled complex is formed by labeling and covalently combining antigen/antibody/polypeptide/protein to form a labeled complex, the labeled complex not only retains the specificity of immunoreaction and can perform specific reaction with a substance to be detected to form a multi-element complex, but also does not affect the characteristics of the label, so that the substance to be detected can be identified and quantified through the characteristics of the label after immunoreaction.
In some embodiments, the label is any one of a time-resolved fluorescent microsphere, a normal fluorescent microsphere, an upconversion luminescent microsphere, a magnetic microsphere, colloidal gold, a colored latex microsphere, a quantum dot, a time-resolved fluorescent dye, a normal fluorescein, an enzyme, a biotin-avidin, and a microsphere amplification system. Microspheres or markers used in prior art immunochromatographic products can be used as the markers of the present invention.
In some embodiments, in step S3, the diluent comprises a buffer pair, a protein, a saccharide, a high polymer, a preservative, and a surfactant.
In some embodiments, the diluent buffer is selected from the group consisting of Tris-HCl buffer at a molarity of 0.015M to 0.025M, PB buffer at a molarity of 0.015M to 0.025M, HEPES buffer at a molarity of 0.015M to 0.025M, the protein is BSA with the mass concentration of 0.5-2%, the saccharide is trehalose with the mass concentration of 2.0-15%, the sucrose with the mass concentration of 2.0-15% and the glucan 20000 with the mass concentration of 1.0-3%, the high polymer is PVP with the mass concentration of 0.25-3%, the preservative is Proclin300 with the mass concentration of 0.01-0.1% or sodium azide with the mass concentration of 0.01-0.1%, the surfactant is TW-20 with the mass concentration of 0.05-2% and PEG4000 with the mass concentration of 0.05-2%.
Preferably, in some embodiments, the diluent formulation is 0.02M Tris-HCL, the BSA concentration is 0.5% -2% by mass, the trehalose concentration is 2.0% -5% by mass, the sucrose concentration is 2.0% -5% by mass, the dextran 20000 concentration is 1.0% -3% by mass, the PVP concentration is 0.5% -2% by mass, the Proclin300 concentration is 0.01% -0.1% by mass, the TW-20% concentration is 0.1% -1% by mass, and the PEG4000 concentration is 0.05% -1% by mass.
In some embodiments, the pH of the diluent is 7.4 to 8.5, and preferably, the pH of the diluent is 8.0 to 8.5.
In some embodiments, the labeled complex is mixed in a preservative to diluent volume ratio of 1:1000 to 20: 1000. When the dilution multiple is too low, the detection signal is low, the requirement on a detection instrument is high, and the detection sensitivity of the test strip is reduced; when the dilution factor is too high, the background of a detection signal is too high, the time for detection is too long, and meanwhile, the CV is larger due to the residue of the marker on the binding pad, and in addition, the number of labeled compounds is larger, so that the production cost of the test strip is increased.
In some embodiments, the diluent further comprises an edible pigment, and preferably, the edible pigment is 0.005% to 0.01% by mass. Generally, if the marker is invisible to naked eyes, edible pigment can be added into the diluent, and the distribution of the labeled compound after the conjugate pad is dried can be directly observed by naked eyes by adding the nontoxic colored edible pigment, so that the part of the conjugate pad, which is not uniformly distributed, of the labeled compound can be conveniently removed.
In some embodiments, in step S4, the quantification is 10 μ L-50 μ L. Therefore, each bonding pad can be fully infiltrated, and the quantity of the labeled compound on each bonding pad is the same, so that the accuracy of the immunochromatographic test strip in detection is improved, and the immunochromatographic test strip can be applied to accurate quantitative detection.
In some embodiments, the step S4, the step of adding the soaking solution of the labeled complex to the bonding pad is to soak the bonding pad in the soaking solution of the labeled complex, so that the bonding pad is sufficiently soaked.
In some embodiments, in step S4, the step of adding the soaking solution of the labeled complex to the bonding pad is to uniformly drop the soaking solution of the labeled complex onto the bonding pad by using a solution taking device, so as to fully wet the bonding pad.
In some embodiments, in step S5, the constant temperature and humidity environment includes two constant temperature and humidity environments, the temperature of the first constant temperature and humidity environment is 20 ℃ to 37 ℃, the humidity is 40% to 60%, and the standing time is 5min to 60 min; the temperature of the second constant-temperature constant-humidity environment is 25-50 ℃, the humidity is 25-40%, and the standing time is 30-240 min. Thus, in the first constant temperature and humidity environment, the labeled compound is further dispersed in the bonding pad until the labeled compound is uniformly distributed; and in the fully soaked combined pad, volatile substances in the soaking solution on the combined pad are uniformly volatilized, and the volatile substances in the soaking solution on the combined pad are not easily and uniformly solidified slowly.
In some embodiments, in step S5, the carrier has a flat surface, and the bonding pads are horizontally static on the surface of the carrier, so that it is better that the soaking solution is fully diffused in the bonding pads.
In some embodiments, in step S5, the carrier is any one of a glass plate, a stainless steel plate, and a plastic plate.
In some embodiments, in step S5, the support has a hydrophobic surface. Therefore, the soaking liquid only flows on the bonding pad and can not be absorbed by the carrier, and the labeling compound is further dispersed in the bonding pad in the balancing process until the labeling compound is uniformly distributed.
In some embodiments, in step S5, the carrier surface has a certain roughness, the carrier surface has regularly arranged micron-sized protrusions or depressions, which facilitates the removal of the complete bonding pad after drying, the bonding pad is not easily adhered to the bonding pad carrier, and the hydrophobic surface carrier is easily cleaned for recycling.
In some embodiments, in step S6, the drying is drying in an oven.
In some embodiments, in step S6, the drying is freeze drying.
The following detailed description of embodiments of the invention is intended to be illustrative, and not to be construed as limiting the invention.
Reagents and instrumentation:
TABLE 1
Example 1 alpha fetoprotein AFP fluorescence immunochromatographic test strip and preparation method thereof
A sample pad, a combination pad, an NC film and a water absorption pad are sequentially lapped on a polyvinyl chloride (PVC) bottom plate, then the PVC bottom plate and attached materials are cut into test strips with the width of 3.5mm, the test strips are loaded into a card shell, and a sample adding area and a test area are arranged on the card shell.
Wherein the sample pad is a glass fiber film CB 08; the combination pad contains a compound of europium ion fluorescent microspheres labeled AFP monoclonal antibodies, the PVC bottom plate is made of a polyvinyl chloride PVC plate, the NC film is made of UniSart 150 tape backing, and the film comprises: the T line (detection line) is coated with another AFP monoclonal antibody capable of being specifically combined with an AFP antigen to be detected, the C line (quality control line) is coated with a goat anti-mouse IgG antibody, the detection line is parallel to the quality control line, the distance is 3mm, and the europium ion fluorescent microspheres contain carboxyl with the particle size of 200 nm.
The test strip can be prepared by the following method:
(1) NC film preparation
Firstly, fixedly sticking an NC film to the middle part of a PVC bottom plate;
using a coating buffer solution (0.02M PB, pH7.4) to dilute the Roche AFP monoclonal antibody (clone number TU11) and the goat anti-mouse antibody to 1mg/ml, and obtaining a T-line AFP antibody and a C-line goat anti-mouse coating diluent;
coating the coated diluent obtained in the step two on an NC film by using a gold spraying and film scribing instrument, wherein the spraying parameter is 2 mu L/cm, and the interval between the T line and the C line is 3 mm;
placing the NC membrane in an oven at 37 ℃ for drying for 4 hours, adding a drying agent after drying, and sealing and storing;
(2) sample pad preparation
Sample pad buffer: 0.02M Tris-HCl, 0.5% Tween-20, 2% BSA, 2% sucrose, 1.5% PVP, pH 8.5.
② the sample pad material is CB08, soaking in the sample pad buffer solution for 1h at room temperature, and drying for 4h at 37 ℃.
(3) AFP antibody labelling
Cleaning microspheres: putting 50 mu L of europium ion fluorescent microspheres (solid content is 1%) in a 2mL centrifugal tube, performing 300W ultrasonic dispersion treatment for 1min by using 1mL MES buffer solution (0.05M pH5.5), centrifuging at 16000rpm for 20min, removing supernatant, and repeating the step for 3 times;
covalent activation: adding 0.5mL MES buffer (0.05M pH5.5) for ultrasonic resuspension, sequentially adding 40 μ g and 20 μ g of Sulfo-NHS and EDC, vortex mixing, incubating at 37 ℃ in a shaking table for 20min, centrifuging at 16000rpm for 20min, discarding the supernatant, and repeating the step for 2 times;
labeling the antibody: adding 0.5mL PB labeling buffer (0.02M pH7.4) and 300W ultrasonic dispersion for 1min, adding 70 μ g Roche AFP monoclonal antibody (clone number LJ738), and performing shaking table reaction at 37 deg.C for 2 h;
sealing the microspheres: adding 60uL BSA (10% by mass, diluted with pure water) microsphere blocking buffer, reacting for 1h at 37 ℃ by using a shaking table, centrifuging for 20min at 15000rpm, and removing the supernatant;
and (3) preserving the microspheres: 0.5mL of microsphere washing buffer (0.025M Tris-HCl, 0.9% NaCl (wt/wt), 0.2% Tween-20(wt/wt) and 0.05% Proclin-300(wt/wt), pH8.0) was added for ultrasonic resuspension, centrifuged at 12000rpm for 20min, the supernatant was discarded, the procedure was repeated 2 times, and finally 0.25mL of microsphere storage solution (0.025M Tris-HCl, 1% BSA (wt/wt)), 5% trehalose (wt/wt)), 20% sucrose (wt/wt)), 1% PVP (wt/wt)), 0.05% Tween-20(wt/wt)) and 0.05% Proclin-300(wt/wt)), pH8.5 was added for ultrasonic resuspension, and stored at 4 ℃ under sealed conditions. The solid content of the microspheres is 0.2%.
(4) Bond pad preparation
Cutting a combined pad: combining the glass fiber 8951 of the pad material, cutting the pad material into pieces of 10mm x 3.5 mm;
preparing a soaking solution for marking the antibody compound, wherein the formula of the diluting solution comprises the following steps: 0.015M Tris-HCl, BSA 0.5% (wt/wt), trehalose 2.0% (wt/wt), sucrose 15% (wt/wt), dextran 200001% (wt/wt), PVP 0.25% (wt/wt), proclin 3000.01% (wt/wt), TW-200.05% (wt/wt), PEG 40000.5% (wt/wt), pH8.5, food color (blue in color) 0.005% (wt/wt).
Dilution times are as follows: the volume ratio of the preservation solution of the labeled compound to the diluent is 1:1000, and the solid content of the microspheres is 0.002 per mill.
Then the infusion solution is subpackaged by moving a pump filling machine through BG600Y15, and 20ml of the infusion solution is added into each bottle.
And thirdly, soaking 750 pieces of 10 mm-3.5 mm bonding pads into 20ml of marked compound soaking solution for 20min, then taking out each bonding pad, laying the bonding pad on a flat-surface plastic plate in a tiling mode without overlapping, regularly arranging cylindrical bulges with the height of 2mm and the diameter of 2mm on the upper surface of the plastic plate, horizontally standing the bonding pad for 15min at the temperature of 20 ℃ and the humidity of 45%, horizontally placing the bonding pad and the plastic plate in an environment with the temperature of 30 ℃ and the humidity of 40% for natural drying for 2h, and then moving the bonding pad to a blast drying oven for drying for 4h at the temperature of 37 ℃.
Fourthly, the bonding pad stuck on the plastic plate is taken out by using stainless steel tweezers, and the bonding pad is placed in an aluminum foil bag at room temperature and sealed and stored by adding a drying agent.
(5) AFP test strip assembly
Tearing off a protective film at the pasting position of a water absorption pad on the upper edge of a PVC base plate, wherein an NC film is pasted in the middle, pasting the water absorption pad on the protective film, covering the water absorption pad on the NC film by 2mm, tearing off a protective film at the pasting position of a sample pad on the lower edge of the PVC base plate, and pasting the sample pad on the protective film;
cutting the assembled large plate obtained in the step one into test strips of 3.5mm by using a strip cutting device;
tearing off the protective film at the joint of the test strip NC film and the sample pad, and adhering the joint pad on the protective film, wherein the joint pad covers 2mm on the NC film, and the sample pad covers 2mm on the joint pad;
fourthly, the test paper strip with the width of 3.5mm obtained in the third step is put into a plastic card shell to form a detection card, and the detection card is put into an aluminum foil bag and is sealed and stored by adding a drying agent.
The detection principle of the chromatographic test strip is as follows:
after the sample to be detected is added to the sample adding region (sample pad), the antibody-fluorescent microsphere conjugate on the labeling membrane continuously flows after being combined with the antigen through capillary action, flows to the NC membrane and moves towards the water absorption end. If the sample to be detected contains AFP antigen, when the sample moves to a T line on an NC membrane, namely a coating line of AFP antibody marked by fluorescent microspheres, the conjugate of antigen-antibody-fluorescent microspheres is captured, and the conjugate of antibody-antigen-antibody-fluorescent microspheres is generated at the T line; when the sample continues to flow to the C line, namely the goat anti-mouse IgG antibody, the surplus antibody-fluorescent microsphere conjugate which is not bound by the AFP antigen is captured, and the effectiveness of the test strip is proved. If the AFP antigen does not exist in the detected actual sample and immune complex cannot be formed, no fluorescence detection signal exists on the T line, and only a fluorescence detection signal exists on the C line. If the C line has no fluorescence detection signal, the test strip is proved to be invalid, and the actual sample needs to be detected again.
Example 2 saccharide antigen CA199 fluorescent immunochromatographic test strip and preparation method thereof
A sample pad, a combination pad, an NC film and a water absorption pad are sequentially lapped on a polyvinyl chloride (PVC) bottom plate, then the PVC bottom plate and attached materials are cut into test strips with the width of 4mm, the test strips are loaded into a card shell, and a sample adding area and a test area are arranged on the card shell.
Wherein the sample pad is a glass fiber membrane RB 45; the combination pad is provided with a compound of a quantum dot nanosphere labeled CA199 monoclonal antibody, the PVC bottom plate is made of a polyvinyl chloride PVC plate, the NC membrane is made of UniSart 140 tape backing, and the membrane comprises: the T line (detection line) is coated with another CA199 monoclonal antibody capable of being specifically combined with the CA199 antigen to be detected, the C line (quality control line) is coated with a goat anti-mouse IgG antibody, the detection line is parallel to the quality control line, the distance is 4mm, and the carboxyl particle size of the quantum dot nanosphere is 130 nm.
The test strip can be prepared by the following method:
(1) NC film preparation
Firstly, fixedly sticking an NC film to the middle part of a PVC bottom plate;
using coating buffer solution (0.02M PB, pH7.4) to dilute meridian CA199 monoclonal antibody (product number M66106M) and goat anti-mouse antibody to 1mg/ml, so as to obtain T-line CA199 antibody and C-line goat anti-mouse coating diluent;
coating the coated diluent obtained in the step two on an NC film by using a gold spraying and film scribing instrument, wherein the spraying parameter is 2 mu L/cm, and the interval between the T line and the C line is 4 mm;
placing the NC membrane in an oven at 37 ℃ for drying for 4 hours, adding a drying agent after drying, and sealing and storing;
(2) sample pad preparation
Sample pad buffer: 0.02M Tris-HCl, 0.5% Tween-20, 2% BSA, 2% sucrose, 2.5% trehalose, 1.5% PVP, pH 8.5.
② the sample pad material is RB45, soaking in the sample pad buffer solution for 0.5h at room temperature, drying for 4h at 37 ℃.
(3) CA199 antibody labeling
Cleaning microspheres: putting 50 mu L of quantum dot nanospheres (solid content is 10mg/mL) into a 2mL centrifugal tube, performing 300W ultrasonic dispersion treatment for 1min by using 1mL MES buffer solution (0.05M pH5.5), centrifuging at 16000rpm for 20min, discarding the supernatant, and repeating the step for 3 times;
covalent activation: adding 0.5mL MES buffer (0.05M pH5.5) for ultrasonic resuspension, sequentially adding 40 μ g and 20 μ g of Sulfo-NHS and EDC, vortex mixing, incubating at 37 ℃ in a shaking table for 20min, centrifuging at 16000rpm for 20min, discarding the supernatant, and repeating the step for 2 times;
labeling the antibody: adding 0.5mL PB labeling buffer (0.02M pH7.4) and performing ultrasonic dispersion treatment for 1min at 300W, adding 60 μ g meridian CA199 monoclonal antibody (cat No. M66108M), and performing shake reaction at 37 ℃ for 3 h;
sealing the microspheres: adding 40uL BSA (10% by mass, diluted with pure water) microsphere blocking buffer, reacting for 1h at 37 ℃ by a shaking table, centrifuging at 15000rpm for 20min, and removing the supernatant;
and (3) preserving the microspheres: 0.5mL of microsphere washing buffer (0.025M Tris-HCl, 0.9% NaCl (wt/wt), 0.2% Tween-20(wt/wt) and 0.05% Proclin-300(wt/wt), pH8.0) was added for ultrasonic resuspension, centrifuged at 12000rpm for 20min, the supernatant was discarded, the procedure was repeated 2 times, and finally 0.25mL of microsphere preservation solution (0.025M Tris-HCl, 1% BSA (wt/wt), 5% trehalose (wt/wt), 20% sucrose (wt/wt), 1% PVP (wt/wt), 0.05% Tween-20(wt/wt) and 0.05% Proclin-300(wt/wt), pH8.5) was added for ultrasonic resuspension and stored at 4 ℃ in a sealed manner. The solid content of the microspheres is 0.2%.
(4) Bond pad preparation
Cutting a combined pad: bonding pad material glass fiber 6614, cutting the bonding pad into 6mm by 4mm in width;
preparing a soaking solution for marking the antibody compound, wherein the formula of the diluting solution comprises the following steps: 0.02M HEPES, BSA 2% (wt/wt), trehalose 10% (wt/wt), sucrose 2% (wt/wt), dextran 200001% (wt/wt), PVP 3% (wt/wt), Proclin 3000.1% (wt/wt), TW-202% (wt/wt), PEG 40002% (wt/wt), pH8.0, food color (blue in color) 0.01% (wt/wt).
Dilution times are as follows: the volume ratio of the labeled compound preserving fluid to the diluent is 5:1000, and the solid content of the microspheres is 0.01 per mill.
Then, 50ml of the soak solution is subpackaged by a BF600 subpackaging type peristaltic pump.
And thirdly, dipping 1500 pieces of bonding pads 6mm by 4mm into 50ml of marked compound soaking solution for 30min, taking out each bonding pad, laying each bonding pad on a flat-surface glass plate in a tiling way without overlapping, regularly arranging linear bulges with the height of 1mm and the width of 2mm on the upper surface of the glass plate, horizontally standing for 30min at the temperature of 25 ℃ and the humidity of 50%, horizontally placing the bonding pads together with a plastic plate in the environment with the temperature of 30 ℃ and the humidity of 30% for natural drying for 1h, and then moving the bonding pads to a blast drying oven for drying for 4h at the temperature of 37 ℃.
Fourthly, the bonding pad stuck on the plastic plate is taken out by using stainless steel tweezers, and the bonding pad is placed in an aluminum foil bag at room temperature and sealed and stored by adding a drying agent.
(5) CA199 test strip assembly
Tearing off a protective film at the pasting position of a water absorption pad on the upper edge of a PVC base plate, wherein an NC film is pasted in the middle, pasting the water absorption pad on the protective film, covering the water absorption pad on the NC film by 2mm, tearing off a protective film at the pasting position of a sample pad on the lower edge of the PVC base plate, and pasting the sample pad on the protective film;
cutting the assembled large plate obtained in the step one into test strips with the length of 4mm by using a strip cutting device;
tearing off the protective film at the joint of the test strip NC film and the sample pad, and adhering the joint pad on the protective film, wherein the joint pad covers 2mm on the NC film, and the sample pad covers 1.5mm on the joint pad;
fourthly, the test paper strip with the width of 4mm obtained in the third step is put into a plastic card shell to form a detection card, and the detection card is put into an aluminum foil bag and added with drying agent for sealing and storage.
Example 3 Procalcitonin PCT fluorescence immunochromatographic test strip and preparation method thereof
A sample pad, a combination pad, an NC film and a water absorption pad are sequentially lapped on a polyvinyl chloride (PVC) bottom plate, then the PVC bottom plate and attached materials are cut into test strips with the width of 4mm, the test strips are loaded into a card shell, and a sample adding area and a test area are arranged on the card shell.
Wherein the sample pad is Whatman brand FUSION 5; the combined pad contains a compound of a europium ion fluorescent microsphere labeled PCT monoclonal antibody, the PVC bottom plate is made of a polyvinyl chloride PVC plate, the NC film is made of UniSart 140 tape backing, and the film comprises: the detection line (quality control line) is parallel to the quality control line and is 4mm away, and the europium ion fluorescent microspheres contain carboxyl with the particle size of 300 nm.
The test strip can be prepared by the following method:
(1) NC film preparation
Firstly, fixedly sticking an NC film to the middle part of a PVC bottom plate;
using a coating buffer solution (0.02M PB, 0.5% trehalose, pH7.4) to dilute the BBI PCT monoclonal antibody (the cargo number BM448-M5C1) and the goat anti-mouse antibody to 1.5mg/ml and 1mg/ml to obtain a T-line PCT antibody and a C-line goat anti-mouse coating diluent;
coating the coated diluent obtained in the step two on an NC film by using a gold spraying and film scribing instrument, wherein the spraying parameter is 2 mu L/cm, and the interval between the T line and the C line is 4 mm;
placing the NC membrane in an oven at 37 ℃ for drying for 4 hours, adding a drying agent after drying, and sealing and storing;
(2) sample pad preparation
Sample pad buffer: 0.02M PB, 0.5% Tween-20, 2% BSA, 2% sucrose, 1.5% PVP, pH 7.4.
② the sample pad material is FUSION 5, soaking in the sample pad buffer solution for 1h at room temperature, drying for 4h at 37 ℃.
(3) PCT antibody labeling
Cleaning microspheres: taking 50 mu L of europium ion fluorescent microspheres (solid content is 10mg /) into a 2mL centrifugal tube, performing ultrasonic dispersion treatment for 1min by using 1mL MES buffer solution (0.05M pH5.5), centrifuging for 20min at 16000rpm, discarding the supernatant, and repeating the step for 3 times;
covalent activation: adding 0.5mL MES buffer (0.05M pH5.5) for ultrasonic resuspension, sequentially adding 40 μ g and 20 μ g of Sulfo-NHS and EDC, vortex mixing, incubating at 37 ℃ in a shaking table for 20min, centrifuging at 16000rpm for 20min, discarding the supernatant, and repeating the step for 2 times;
labeling the antibody: adding 0.5mL PB labeling buffer (0.02M pH7.4) and performing 300W ultrasonic dispersion treatment for 1min, adding 60 μ g of BBI PCT monoclonal antibody (with the product number BM448-H3D5), and performing shaking table reaction at 37 ℃ for 2H;
sealing the microspheres: adding 40uL BSA (10% by mass, diluted with pure water) microsphere blocking buffer, reacting for 2h at 37 ℃ by a shaking table, centrifuging at 15000rpm for 20min, and removing the supernatant;
and (3) preserving the microspheres: 0.5mL of microsphere washing buffer (0.02M Tris-HCl, 0.9% NaCl (wt/wt), 0.2% Tween-20(wt/wt) and 0.05% Proclin-300(wt/wt), pH8.0) was added for ultrasonic resuspension, centrifuged at 12000rpm for 20min, the supernatant was discarded, the step was repeated 2 times, and finally 0.25mL of microsphere preservation solution (0.02M Tris-HCl, 1% BSA (wt/wt), 5% trehalose (wt/wt), 15% sucrose (wt/wt), 1% PVP (wt/wt), 0.05% Tween-20(wt/wt) and 0.05% Proclin-300(wt/wt), pH8.5) was added for ultrasonic resuspension and stored at 4 ℃ in a sealed manner. The solid content of the microspheres is 0.2%.
(4) Bond pad preparation
Cutting a combined pad: combining the non-woven fabric FUSION 3 of the pad material, and cutting the combined pad into pieces with width of 7 mm/4 mm;
preparing a soaking solution for marking the antibody compound, wherein the formula of the diluting solution comprises the following steps: 0.025M PB, BSA 1.5% (wt/wt), trehalose 2.5% (wt/wt), sucrose 2.5% (wt/wt), dextran 200003% (wt/wt), PVP 2% (wt/wt), sodium azide 0.01% (wt/wt), TW-200.1% (wt/wt), PEG 40001% (wt/wt), pH8.5, food color (red in color) 0.01% (wt/wt).
Dilution times are as follows: the volume ratio of the preservation solution of the labeled compound to the diluent is 20:1000, and the solid content of the microspheres is 0.04 per mill.
Then the soak solution is subpackaged by a 10ml syringe pump, and each bottle contains 2 ml.
And thirdly, immersing 80 pieces of 7 mm-4 mm bonding pads into 2ml of marked compound soaking solution for 45min, taking out each bonding pad, laying each bonding pad on a flat-surface glass plate in a tiling and non-overlapping mode, enabling the bonding pads to be placed on the flat-surface glass plate in a 3mm inverted triangular pit with regular arrangement depth on the upper surface of the glass plate, horizontally standing for 30min at the temperature of 37 ℃ and the humidity of 60%, horizontally placing the bonding pads together with a plastic plate in an environment with the temperature of 50 ℃ and the humidity of 25% for natural drying for 2h, and then moving the bonding pads to a blast drying oven for drying for 4h at the temperature of 37 ℃.
Fourthly, the bonding pad stuck on the plastic plate is taken out by using stainless steel tweezers, and the bonding pad is placed in an aluminum foil bag at room temperature and sealed and stored by adding a drying agent.
(5) PCT test strip assembly
The test strips of PCT were assembled in the same manner as in example 2.
Example 4 neuron-specific enolase NSE fluorescence immunochromatographic test strip and preparation method thereof
A sample pad, a combination pad, an NC film and a water absorption pad are sequentially lapped on a polyvinyl chloride (PVC) bottom plate, then the PVC bottom plate and attached materials are cut into test strips with the width of 3.5mm, the test strips are loaded into a card shell, and a sample adding area and a test area are arranged on the card shell.
Wherein the sample pad is glass fiber BT 40; the combination pad contains a compound of a green microsphere labeled NSE monoclonal antibody, the PVC bottom plate is made of a PVC plate, the NC membrane is made of UniSart 150 tape backing, and the membrane comprises: the T line (detection line) is coated with another strain of NSE monoclonal antibody capable of being specifically combined with NSE antigen to be detected, the C line (quality control line) is coated with a goat anti-mouse IgG antibody, the detection line is parallel to the quality control line, the distance is 4mm, and the green microspheres contain carboxyl with the particle size of 150 nm.
The test strip can be prepared by the following method:
(1) NC film preparation
Firstly, fixedly sticking an NC film to the middle part of a PVC bottom plate;
secondly, diluting the Fitzgerald NSE monoclonal antibody (cargo number 10-7936) and the goat anti-mouse antibody to 1.5mg/ml by using a coating buffer solution (0.02M PB, pH7.4) to obtain a T-line PCT antibody and a C-line goat anti-mouse coating diluent;
coating the coated diluent obtained in the step two on an NC film by using a gold spraying and film scribing instrument, wherein the spraying parameter is 1 mu L/cm, and the interval between the T line and the C line is 3 mm;
placing the NC membrane in an oven at 37 ℃ for drying for 4 hours, adding a drying agent after drying, and sealing and storing;
(2) sample pad preparation
Sample pad buffer: 0.02M Tris-HCl, 0.5% Tween-20, 2% BSA, 2% sucrose, 1.5% PVP, pH 8.5.
② the sample pad is made of glass fiber BT40, and is soaked in the buffer solution of the sample pad for 1h at room temperature and dried for 4h at 37 ℃.
(3) NSE antibody labeling
Cleaning microspheres: taking 50 mu L of green fluorescent microspheres (solid content is 4%) in a 2mL centrifugal tube, performing 300W ultrasonic dispersion treatment for 1min by using 1mL MES buffer solution (0.05M pH5.5), centrifuging at 18000rpm for 20min, removing supernatant, and repeating the step for 3 times;
covalent activation: adding 0.5mL MES buffer (0.05M pH5.5) for ultrasonic resuspension, sequentially adding 40 μ g and 20 μ g of Sulfo-NHS and EDC, vortex mixing, incubating at 37 ℃ in a shaking table for 20min, centrifuging at 18000rpm for 20min, discarding the supernatant, and repeating the step for 2 times;
labeling the antibody: adding 0.5mL PB labeling buffer (0.02M pH7.4) and carrying out ultrasonic dispersion treatment for 1min under 300W, adding 30 mu g of Fitzgerald NSE monoclonal antibody (cat No. 10-7937), and carrying out shaking table reaction at 37 ℃ for 2 h;
sealing the microspheres: adding 30uL BSA (10% by mass, diluted with pure water) microsphere blocking buffer solution, reacting for 2h at 37 ℃ by a shaking table, centrifuging at 18000rpm for 20min, and removing the supernatant;
and (3) preserving the microspheres: 0.5mL of microsphere washing buffer (0.02M Tris-HCl, 0.9% NaCl (wt/wt), 0.5% Tween-20(wt/wt) and 0.05% Proclin-300(wt/wt), pH8.0) was added for ultrasonic resuspension, centrifuged at 16000rpm for 20min, the supernatant was discarded, the step was repeated 2 times, and finally 0.25mL of microsphere preservation solution (0.02M Tris-HCl, 2% BSA (wt/wt), 5% trehalose (wt/wt), 20% sucrose (wt/wt), 1.5% PVP (wt/wt), 0.05% Tween-20(wt/wt) and 0.05% Proclin-300(wt/wt), pH8.5) was added for ultrasonic resuspension and stored at 4 ℃ under sealed conditions. The solid content of the microspheres is 0.8%.
(4) Bond pad preparation
Cutting a combined pad: bonding pad material polyester film 6613, cutting the bonding pad into 10mm by 4mm in width;
preparing a soaking solution for marking the antibody compound, wherein the formula of the diluting solution comprises the following steps: 0.02M Tris-HCl, BSA 1% (wt/wt), trehalose 5% (wt/wt), sucrose 5% (wt/wt), dextran 200002% (wt/wt), PVP 1% (wt/wt), Proclin 3000.05% (wt/wt), TW-200.5% (wt/wt), PEG 40001.5% (wt/wt), pH 8.5.
Dilution times are as follows: the volume ratio of the preservation solution of the labeled compound to the diluent is 1:1000, and the solid content of the microspheres is 0.008 per mill.
Thirdly, laying each 10mm 4mm bonding pad on a flat-surface glass plate in a tiling and non-overlapping mode, regularly arranging regular triangular bulges with the height of 0.5mm on the upper surface of the glass plate, quantitatively and slowly dripping a marking compound on each bonding pad at a constant speed for 10 mu L by using a syringe pump, horizontally standing for 30min at the temperature of 25 ℃ and the humidity of 45 percent, horizontally placing the bonding pads together with a 304 stainless steel plate in an environment with the temperature of 50 ℃ and the humidity of 40 percent for natural drying for 1h, and then moving the bonding pads to a blast drying oven for drying for 4h at the temperature of 37 ℃.
Fourthly, the bonding pad stuck on the stainless steel plate is taken out by stainless steel tweezers, and the bonding pad is placed in an aluminum foil bag at room temperature and sealed and stored by adding a drying agent.
(5) NSE test strip assembly
The NSE test strip was assembled in the same manner as in example 1.
Example 5 cytokeratin 19CYFRA21-1 fluorescence immunochromatographic test strip and preparation method thereof
A sample pad, a combination pad, an NC film and a water absorption pad are sequentially lapped on a polyvinyl chloride (PVC) bottom plate, then the PVC bottom plate and attached materials are cut into test strips with the width of 4.5mm, the test strips are loaded into a card shell, and a sample adding area and a test area are arranged on the card shell.
Wherein the sample pad is glass fiber Oslon 6614; the combination pad contains a compound of europium ion fluorescent microsphere labeled CYFRA21-1 monoclonal antibody, the PVC bottom plate is made of PVC plate, the NC film is made of UniSart 140 tape backing, and the film comprises: the T line (detection line) is coated with another strain CYFRA21-1 monoclonal antibody capable of being specifically combined with CYFRA21-1 antigen to be detected, the C line (quality control line) is coated with a goat anti-mouse IgG antibody, the detection line is parallel to the quality control line, the distance is 3.5mm, and the europium ion fluorescent microsphere contains carboxyl with the particle size of 200 nm.
The test strip can be prepared by the following method:
(1) NC film preparation
Firstly, fixedly sticking an NC film to the middle part of a PVC bottom plate;
using coating buffer solution (0.02M PB, pH7.4) to dilute the biospacific CYFRA21-1 antibody monoclonal antibody (cargo number A82260) and the goat anti-mouse antibody to 1.5mg/ml, and obtaining T-line PCT antibody and C-line goat anti-mouse coating diluent;
coating the coated diluent obtained in the step two on an NC film by using a gold spraying and film scribing instrument, wherein the spraying parameter is 1 mu L/cm, and the interval between the T line and the C line is 4 mm;
placing the NC membrane in an oven at 37 ℃ for drying for 4 hours, adding a drying agent after drying, and sealing and storing;
(2) sample pad preparation
Sample pad buffer: 0.02M Tris-HCl, 0.5% Tween-20, 2% BSA, 2% sucrose, 1.5% PVP, pH 8.5.
② the sample pad is made of glass fiber 6614, and is soaked in the buffer solution of the sample pad for 1h at room temperature, and is dried for 4h at 37 ℃.
(3) CYFRA21-1 antibody marker
Cleaning microspheres: taking 50 mu L of europium ion fluorescent microspheres (solid content is 1%) in a 1.5mL centrifugal tube, performing 300W ultrasonic dispersion treatment for 1min by using 1mL MES buffer solution (0.05M pH5.5), centrifuging at 16000rpm for 20min, discarding the supernatant, and repeating the step for 3 times;
covalent activation: adding 0.5mL MES buffer (0.05M pH5.5) for ultrasonic resuspension, sequentially adding 40 μ g and 20 μ g of Sulfo-NHS and EDC, vortex mixing, incubating at 37 ℃ in a shaking table for 20min, centrifuging at 16000rpm for 20min, discarding the supernatant, and repeating the step for 2 times;
labeling the antibody: adding 0.5mL PB labeling buffer (0.02M pH7.4) for 300W ultrasonic dispersion treatment for 1min, adding 30 μ g of biospacific CYFRA21-1 antibody monoclonal antibody (cargo number A82261), and performing shake reaction at 37 ℃ for 4 h;
sealing the microspheres: adding 40uL BSA (10% by mass, diluted with pure water) microsphere blocking buffer, reacting for 2h at 37 ℃ by a shaking table, centrifuging at 15000rpm for 20min, and removing the supernatant;
and (3) preserving the microspheres: 0.5mL of microsphere washing buffer (0.025M Tris-HCl, 0.9% NaCl (wt/wt), 0.2% Tween-20(wt/wt) and 0.05% Proclin-300(wt/wt), pH8.5) was added for ultrasonic resuspension, centrifuged at 12000rpm for 20min, the supernatant was discarded, the procedure was repeated 2 times, and finally 0.25mL of microsphere preservation solution (0.025M Tris-HCl, 1% BSA (wt/wt), 5% trehalose (wt/wt), 20% sucrose (wt/wt), 1% PVP (wt/wt), 0.05% Tween-20(wt/wt) and 0.05% Proclin-300(wt/wt), pH8.5) was added for ultrasonic resuspension and stored at 4 ℃ in a sealed manner. The solid content of the microspheres is 0.2%.
(4) Bond pad preparation
Cutting a combined pad: cutting the bonding pad into pieces 8mm by 4.5mm in width by using the bonding pad glass fiber 8980;
preparing a soaking solution for marking the antibody compound, wherein the formula of the diluting solution comprises the following steps: 0.02M Tris-HCl, BSA 0.5% (wt/wt), trehalose 2.0% (wt/wt), sucrose 10% (wt/wt), dextran 200002.5% (wt/wt), PVP 0.5% (wt/wt), proclin 3000.05% (wt/wt), TW-201% (wt/wt), PEG 40001.5% (wt/wt), pH 8.5.
Dilution times are as follows: the volume ratio of the preservation solution of the labeled compound to the diluent is 2:1000, and the solid content of the microspheres is 0.004 per mill.
Thirdly, each 10 mm-4 mm bonding pad needs to be tiled and placed on a flat-surface glass plate without overlapping, linear scratches with the depth of 1mm are regularly arranged on the upper surface of the glass plate, a marking compound is slowly and uniformly dripped on each bonding pad by a JT-D3210 full-automatic dispensing machine for 30 microlitres, the bonding pads and the marking compound are horizontally placed for 30 minutes at the temperature of 30 ℃ and the humidity of 55 percent, then the bonding pads and the glass plate are horizontally placed in the environment with the temperature of 30 ℃ and the humidity of 30 percent for natural drying for 2 hours, and then the bonding pads and the glass plate are moved to a blast drying oven for drying for 4 hours at the temperature of 37 ℃.
Fourthly, the bonding pad stuck on the glass plate is taken out by using stainless steel tweezers, and the bonding pad is placed in an aluminum foil bag at room temperature and sealed and stored by adding a drying agent.
(5) CYFRA21-1 test strip assembly
Tearing off a protective film at the pasting position of a water absorption pad on the upper edge of a PVC base plate, wherein an NC film is pasted in the middle, pasting the water absorption pad on the protective film, covering the water absorption pad on the NC film by 1.5mm, tearing off a protective film at the pasting position of a sample pad on the lower edge of the PVC base plate, and pasting the sample pad on the protective film;
cutting the assembled large plate obtained in the step one into test strips of 4.5mm by using a strip cutting device;
tearing off the protective film at the joint of the test strip NC film and the sample pad, and adhering the joint pad on the protective film, wherein the joint pad covers 1.5mm of the NC film, and the sample pad covers 2mm of the joint pad;
fourthly, the test paper strip with the width of 4.5mm obtained in the third step is put into a plastic card shell to form a detection card, and the detection card is put into an aluminum foil bag and is sealed and stored by adding a drying agent.
Example 6 myoglobin MYO fluorescence immunochromatographic test strip and preparation method thereof
A sample pad, a combination pad, an NC film and a water absorption pad are sequentially lapped on a polyvinyl chloride (PVC) bottom plate, then the PVC bottom plate and attached materials are cut into test strips with the width of 4mm, the test strips are loaded into a card shell, and a sample adding area and a test area are arranged on the card shell.
Wherein the sample pad is glass fiber RB 65; the combination pad contains a compound of a magnetic bead marked MYO monoclonal antibody with carboxyl on the surface, the PVC bottom plate is made of a polyvinyl chloride PVC plate, the NC membrane is made of UniSart 95 tape backing, and the membrane comprises: the T line (detection line) is coated with another MYO monoclonal antibody capable of being specifically combined with the PCT antigen to be detected, the C line (quality control line) is coated with a goat anti-mouse IgG antibody, the detection line is parallel to the quality control line, the distance is 3.5mm, and the particle size of the magnetic beads is 300 nm.
The test strip can be prepared by the following method:
(1) NC film preparation
Firstly, fixedly sticking an NC film to the middle part of a PVC bottom plate;
using a coating buffer solution (0.02M PB, pH7.4) to dilute the Boyi biological MYO antibody monoclonal antibody (cargo number M020603) and the goat anti-mouse antibody to 1mg/ml, and obtaining a T-line PCT antibody and a C-line goat anti-mouse coating diluent;
coating the coated diluent obtained in the step two on an NC film by using a gold spraying and film scribing instrument, wherein the spraying parameter is 1 mu L/cm, and the interval between the T line and the C line is 4 mm;
placing the NC membrane in an oven at 37 ℃ for drying for 4 hours, adding a drying agent after drying, and sealing and storing;
(2) sample pad preparation
Sample pad buffer: 0.02M Tris-HCl, 0.5% Tween-20, 2% BSA, 2% sucrose, 1.5% PVP, pH 8.5.
② the sample pad material is glass fiber RB65, soaking in the sample pad buffer solution for 1h at room temperature, drying for 4h at 37 ℃.
(3) MYO antibody labeling
Cleaning microspheres: taking 20 mu L of magnetic beads (solid content is 10%) with carboxyl on the surface into a 2mL centrifugal tube, using 1mL MES buffer solution (0.05M pH5.5), carrying out heavy suspension on the magnetic suspension by a vortex oscillator, separating the magnetic suspension by a magnetic separator, discarding the supernatant, and washing the supernatant for 2 times by the MES buffer solution;
covalent activation: adding 0.25mL MES buffer (0.05M pH5.5) into a vortex oscillator for heavy suspension, sequentially adding 10 mu g and 8 mu g of Sulfo-NHS and EDC respectively, carrying out vortex mixing, incubating for 20min by the vortex oscillator at room temperature, separating by a magnetic separator, discarding supernatant, washing for 2 times by the MES buffer, and carrying out vortex mixing and washing for 2 times by 0.25mL BST labeling buffer (0.02M pH 7.4);
labeling the antibody: adding 0.25mL of BST marking buffer solution, mixing uniformly, adding 15 mu g of Boyi biological MYO antibody monoclonal antibody (product number M020603), reacting for 2h in a room-temperature vortex oscillator, separating and separating by a magnetic separator after marking is finished, and discarding supernatant;
sealing the microspheres: adding 0.25mL of BST buffer solution, mixing uniformly in a vortex mode, adding 50uL of BSA (the mass fraction is 10%, diluting with pure water), mixing uniformly in a vortex oscillator, rotating and sealing for 1h, separating by using a magnetic separator, and then removing supernatant;
and (3) preserving the microspheres: adding 0.25mL of magnetic bead BST preservation solution buffer (0.02M BST, 0.2% Tween-20(wt/wt) and 0.05% Proclin-300(wt/wt), 0.5% BSA (wt/wt)), 0.5% trehalose (wt/wt)), pH8.0) for vortex resuspension, separating by a magnetic separator, abandoning the supernatant, repeating the step for 2 times, and finally adding 0.20mL of BST preservation solution buffer (vortex resuspension and dispersion, sealing and preserving at 4 ℃). The solid content of the magnetic bead microspheres is 1%.
(4) Bond pad preparation
Cutting a combined pad: combining the glass fiber GL0194 made of the pad material, and cutting the combining pad into the glass fiber GL0194 with the width of 7mm 4 mm;
preparing a soaking solution for marking the antibody compound, wherein the formula of the diluting solution comprises the following steps: 0.02M Tris-HCl, BSA 1.5% (wt/wt), trehalose 4.5% (wt/wt), sucrose 5% (wt/wt), dextran 200001.4% (wt/wt), PVP 1.5% (wt/wt), proclin 3000.1% (wt/wt), TW-201.5% (wt/wt), PEG 40000.5% (wt/wt), pH 8.1.
Dilution times are as follows: the volume ratio of the preservation solution of the labeled compound to the diluent is 8:1000, and the solid content of the magnetic bead microspheres is 0.08 per mill.
Thirdly, laying each 10 mm-4 mm bonding pad on a stainless steel plate in a tiling and non-overlapping mode, regularly arranging S-shaped scratches with the depth of 1mm on the upper surface of the stainless steel plate, slowly dropwise adding a marking compound soaking solution to the bonding pad by using a liquid transfer machine, horizontally standing for 30min at the temperature of 27 ℃ and the humidity of 54%, horizontally placing the bonding pad and a plastic plate in an environment with the temperature of 30 ℃ and the humidity of 30% for natural drying for 2h, and then moving the bonding pad to a blast drying oven for drying for 4h at the temperature of 37 ℃.
Fourthly, the bonding pad stuck on the stainless steel plate is taken out by stainless steel tweezers, and the bonding pad is placed in an aluminum foil bag at room temperature and sealed and stored by adding a drying agent.
(5) MYO test paper strip assembly
Tearing off a protective film at the pasting position of a water absorption pad on the upper edge of a PVC base plate, wherein an NC film is pasted in the middle, pasting the water absorption pad on the protective film, covering the water absorption pad on the NC film by 2mm, tearing off a protective film at the pasting position of a sample pad on the lower edge of the PVC base plate, and pasting the sample pad on the protective film;
cutting the assembled large plate obtained in the step one into test strips with the length of 4mm by using a strip cutting device;
tearing off the protective film at the joint of the test strip NC film and the sample pad, and adhering the joint pad on the protective film, wherein the joint pad covers the NC film for 1mm, and the sample pad covers the joint pad for 1 mm;
fourthly, the test paper strip with the width of 4mm obtained in the third step is put into a plastic card shell to form a detection card, and the detection card is put into an aluminum foil bag and added with drying agent for sealing and storage.
Measurement of imprecision
Further, the human serum samples tested on the test strips of examples 1 to 6 were used as subjects to study the imprecision (CV value); the test strips of the control group 1 were sprayed with a bonding pad using a streaking apparatus, and the remaining steps were the same as in examples 1 to 6; the test strips of the control group 2 were soaked in the solution using the conjugate pad, taken out, left at room temperature for 15min, and then placed in an oven at 37 ℃ to be dried for 4h, with the remaining steps being the same as those in examples 1-6. The imprecision was compared and is shown in the following table:
TABLE 2
As can be seen from table 2, the test strip of the present invention can effectively reduce CV and improve the accuracy of the test result, and in examples 1 to 5, the CV value of the method of the present invention is lower than that of the spray method, which indicates that the method of the present invention has a better performance of accurate measurement.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (7)
1. A preparation method of an immunochromatographic test strip for accurate quantitative detection comprises the following steps: the method comprises the following steps of preparing a nitrocellulose membrane, preparing a sample pad, preparing a combined pad and assembling a test strip, and is characterized in that the preparation of the combined pad comprises the following steps:
s1: cutting the combined pad: cutting the bonding pad into the size required for assembling into a finished product;
s2: preparation of labeled complexes: after the antibody or the antigen is marked on the marker, placing the marker in a preservation solution for storage;
s3: preparing a soak solution of the labeled compound: mixing and diluting the diluent and a preservation solution containing the labeled compound into a soak solution of the labeled compound with a certain concentration;
s4: infiltrating the bonding pad: adding a soaking solution of a quantitative labeled compound into the bonding pad to soak the bonding pad, wherein the step of adding the soaking solution of the labeled compound into the bonding pad is to soak the bonding pad in the soaking solution of the labeled compound, or uniformly dripping the soaking solution of the labeled compound onto the bonding pad by adopting a solution taking device;
s5: combining a pad balancing process: spreading the infiltrated bonding pad on a carrier, and standing in a constant-temperature and constant-humidity environment, wherein the standing in the constant-temperature and constant-humidity environment refers to standing for 35-300 min in an environment with the temperature of 20-50 ℃ and the humidity of 25-60%, the standing in the constant-temperature and constant-humidity environment comprises two stages, and the first stage refers to standing for 5-60 min in an environment with the temperature of 20-37 ℃ and the humidity of 40-60%; the second stage is to stand for 30-240 min under the environment that the temperature is 25-50 ℃ and the humidity is 25-40%, and the carrier is provided with a hydrophobic surface;
s6: drying the combined pad: the equilibrated conjugate pad is dried.
2. The method for preparing an immunochromatographic test strip for precise quantitative detection according to claim 1, wherein in step S2, the label is any one of a time-resolved fluorescent microsphere, a normal fluorescent microsphere, an upconversion luminescent microsphere, a magnetic microsphere, colloidal gold, a colored latex microsphere, a quantum dot, a time-resolved fluorescent dye, a normal fluorescein, an enzyme, biotin-avidin and a microsphere amplification system.
3. The method for preparing an immunochromatographic test strip for precise quantitative detection according to claim 1, wherein in step S3, the diluent comprises a buffer pair, a protein, a saccharide, a high polymer, a preservative and a surfactant.
4. The method for preparing an immunochromatographic test strip for precise quantitative detection according to claim 3, characterized in that the buffer pair of the diluent is one of Tris-HCL buffer solution with the molar concentration of 0.015-0.025M, PB buffer solution with the molar concentration of 0.015-0.025M and HEPES buffer solution with the molar concentration of 0.015-0.025M, the protein is BSA with the mass concentration of 0.5-2%, the saccharide is trehalose with the mass concentration of 2.0-15%, the sucrose with the mass concentration of 2.0-15% and the glucan 20000 with the mass concentration of 1.0-3%, the high polymer is PVP with the mass concentration of 0.25-3%, the preservative is Proclin300 with the mass concentration of 0.01-0.1% or sodium azide with the mass concentration of 0.01-0.1%, the surfactant is TW-20 with the mass concentration of 0.05-2% and PEG4000 with the mass concentration of 0.05-2%.
5. The method for preparing an immunochromatographic test strip for precise quantitative detection according to claim 1, wherein in step S3, the volume ratio of the preservation solution to the diluent of the labeled complex is 1:1000 to 20: 1000.
6. The method for preparing an immunochromatographic test strip for precise quantitative detection according to claim 1, wherein in step S4, the quantitative determination means 10 μ L to 50 μ L.
7. The method for preparing an immunochromatographic test strip for precise quantitative detection according to claim 1, wherein in step S5, the carrier is any one of a glass plate, a stainless steel plate and a plastic plate.
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