Detection card, kit and method for determining type III procollagen N-terminal peptide

Abstract

The embodiment of the invention belongs to the technical field of detection, and relates to a detection card, kit and method for determining type III procollagen N-terminal peptide, the detection card comprises a card shell and a test strip, and the test strip is arranged in the card shell; the test strip comprises a PVC (polyvinyl chloride) bottom plate, and an absorption pad, a nitrocellulose membrane and a sample pad which are sequentially overlapped on the PVC bottom plate, wherein the nitrocellulose membrane is provided with a detection line and a quality control line, and the detection line is coated with a PIINP antibody; the detection card further comprises a fluorescence labeled antibody packaging area, a fluorescence labeled antibody solution is packaged in the fluorescence labeled antibody packaging area, and the fluorescence labeled antibody solution comprises a fluorescence labeled PIINP antibody. The method is short in detection time and high in sensitivity.

Description

Technical Field

[0001] The present application relates to the field of detection technology, and in particular to a detection card, a kit and a method for determining N-terminal peptide of type III procollagen. Background Art

[0002] Liver fibrosis is a common cause of various chronic liver diseases and is an inevitable stage in the development of liver cirrhosis. Procollagen Ⅲ N-terminal peptide (PⅢNP) is the amino-terminal tail peptide of type Ⅲ procollagen that is cut off by endonucleases before the formation of procollagen outside the cell and enters the blood circulation. The complete type Ⅲ collagen peptide is mainly cleared by the liver through endocytosis mediated by sinusoidal endothelial cell receptors, while small fragments are mainly excreted and cleared by the kidneys. Therefore, when collagen metabolism is active and liver function is damaged, blood PⅢNP increases. The increase in the level of PⅢNP in the blood of people with liver fibrosis is associated with the degree of fibrosis activity.

[0003] At present, the common methods for detecting type III procollagen N-terminal peptide mainly include enzyme-linked immunosorbent assay, radioimmunoassay, enzymatic chemiluminescence, latex immunoturbidimetry, etc.

[0004] Common methods for detecting type III procollagen N-terminal peptide:

[0005] (1) Enzyme-linked immunosorbent assay (ELISA) is the most widely used technique in enzyme immunoassay technology. Its basic method is to adsorb known antigens or antibodies on the surface of a solid phase carrier (polystyrene microplate), allow the enzyme-labeled antigen-antibody reaction to proceed on the solid phase surface, and use a washing method to remove free components in the liquid phase. Commonly used ELISA methods include double antibody sandwich method, double antigen sandwich method, indirect method, and competitive method. This method has some defects: ELISA generally uses 12×8, 6×8, 8×12 or whole plate 96-well dedicated microplates, and cannot perform independent single-person sample testing; during the test process, the test reagents are in an open space, which can easily cause cross-contamination between various reagents and affect the accuracy of the test results; the test process is mostly manual, which can easily cause errors.

[0006] (2) Radioimmunoassay is an in vitro microanalysis method using radioactive isotopes that competitively inhibit the reaction between isotope-labeled and unlabeled antigens and antibodies. This method also has certain shortcomings: high radioactive pollution; cumbersome sample addition steps, complex operation, long operation time, unstable measurement results, short reagent storage time, low degree of automation of kit operation, and expensive supporting instruments.

[0007] (3) Chemiluminescence is a type of molecular luminescence spectroscopic analysis. It is mainly based on the principle that the concentration of the analyte in the chemical detection system and the chemiluminescence intensity of the system are linearly quantitatively related under certain conditions. It uses instruments to detect the chemiluminescence intensity of the system to determine the content of the analyte. According to the luminescence principle, it can be divided into direct chemiluminescence and enzymatic chemiluminescence. The enzymatic reaction kinetics of enzymatic chemiluminescence are complex, there are many influencing factors, and the structure is not stable enough. The direct chemiluminescence of acridinium ester as a marker is easy to bind to proteins and affect the experimental results.

[0008] (4) The latex immunoturbidimetry method is to coat the antibody corresponding to the substance to be tested on latex particles with a diameter of 15 to 60 nm, and then combine with the substance to be tested to form an insoluble immune complex, which forms turbidity in a specific buffer environment. The turbidity is proportional to the content of the substance to be tested within the detection range. By comparing it with the calibration product operated under the same conditions, the content of the substance to be tested in the sample can be calculated. This method requires the use of various large-scale fully automatic biochemical analyzers for testing. Large-scale biochemical analyzers are expensive and complex to operate. They require relevant professional knowledge and operators who have received corresponding training. In addition, they have high requirements for supporting conditions and need to be equipped with a stabilized power supply, a pure water machine, etc. It occupies a large area and requires regular maintenance.

[0009] In summary, the current detection method takes a long time and the sensitivity is not high enough. Summary of the invention

[0010] The purpose of the embodiments of the present application is to provide a detection card, a kit and a method for determining the N-terminal peptide of type III procollagen, which has a short detection time and high sensitivity.

[0011] In order to solve the above technical problems, the present application embodiment provides a detection card for determining the N-terminal peptide of type III procollagen, which adopts the following technical solution:

[0012] A detection card for determining N-terminal peptide of type III procollagen, the detection card comprising a card shell and a test strip, wherein the test strip is arranged in the card shell;

[0013] The test strip comprises a PVC base plate and an absorption pad, a nitrocellulose membrane and a sample pad which are sequentially overlapped and arranged on the PVC base plate;

[0014] Wherein, a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is coated with PIIINP antibody, and the quality control line is coated with sheep anti-chicken IgY antibody;

[0015] The detection card also includes a fluorescently labeled antibody encapsulation area, wherein the fluorescently labeled antibody encapsulation area encapsulates a fluorescently labeled antibody solution, and the fluorescently labeled antibody solution includes a PIIINP antibody labeled with fluorescent microspheres and a chicken IgY antibody labeled with fluorescent microspheres.

[0016] Furthermore, the test card further comprises a diluent packaging area, wherein the diluent packaging area is packaged with a diluent;

[0017] Wherein, the diluent includes 0.05% Proclin 300 and 0.05M Tris buffer with pH 8.0 by mass fraction.

[0018] Further, the concentration of the PIIINP antibody coated on the test line is 1.0 mg / mL;

[0019] The concentration of the goat anti-chicken IgY antibody coated on the quality control line is 1.0 mg / mL.

[0020] Furthermore, the fluorescently labeled antibody solution is obtained by mixing a fluorescent microsphere-labeled PIIINP antibody solution and a fluorescent microsphere-labeled chicken IgY antibody solution in a volume ratio of 1:1.

[0021] Furthermore, the fluorescent microsphere-labeled PIIINP antibody solution is prepared by the following steps:

[0022] Add fluorescent microspheres with a solid content of 1% into the labeling buffer, mix well, and then add the labeling activation solution to obtain activated fluorescent microspheres;

[0023] Add PIIINP antibody, rotate to react, add labeling blocking solution, rotate to react after sonication;

[0024] After centrifugation, the supernatant is discarded, and a labeling preservation solution is added. After ultrasonication, the PIIINP antibody solution labeled with the fluorescent microspheres is obtained.

[0025] Further, the labeling buffer comprises, by mass fraction, 0.05 M MES at pH 6.2;

[0026] The marking activation solution includes 2% EDC;

[0027] The labeling blocking solution includes 10% BSA;

[0028] The labeling preservation solution includes 2% BSA, 5% trehalose, 2% sucrose, 0.5% casein sodium salt, 0.1% Proclin 300 and 0.2M pH 8.0 Tris buffer.

[0029] Furthermore, the nitrocellulose membrane is prepared by the following steps:

[0030] Diluting the PIIINP antibody and the goat anti-chicken IgY antibody respectively with an antibody coating diluent to obtain a test line coating working solution and a quality control line coating working solution respectively, wherein the antibody coating diluent includes 1% sucrose and 0.01M pH 7.4 PBS buffer by mass fraction;

[0031] The detection line is coated with the working solution to be smeared to the position of the detection line of the original nitrocellulose membrane, and the quality control line is coated with the working solution to be smeared to the position of the quality control line of the original nitrocellulose membrane to obtain the nitrocellulose membrane.

[0032] Furthermore, the sample pad is prepared by the following steps:

[0033] The sample pad is obtained by impregnating the glass fiber with a sample pad treatment solution and drying the glass fiber;

[0034] Wherein, the sample pad treatment solution includes 1% BSA, 0.5% Tween-20 and 0.2M pH 8.0 boric acid borax buffer solution by mass fraction.

[0035] In order to solve the above technical problems, the present application also provides a kit for determining the N-terminal peptide of type III procollagen, which adopts the following technical solution:

[0036] A kit for measuring type III procollagen N-terminal peptide comprises the above-mentioned detection card.

[0037] In order to solve the above technical problems, the present application also provides a method for determining the N-terminal peptide of type III procollagen, which adopts the following technical solution:

[0038] A method for determining N-terminal peptide of type III procollagen, using the above-mentioned kit, comprises the following steps:

[0039] Add the sample to be tested into the sample area of ​​the test card;

[0040] Placing the test card in a fluorescent immunoassay analyzer and starting it;

[0041] The fluorescence signal of the detection line of the detection card is read, and the PIIINP concentration is calculated based on the fluorescence signal.

[0042] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0043] The present application adopts a double antibody sandwich method and utilizes the technical principle of fluorescent immunochromatography to rapidly detect the content of type III procollagen N-terminal peptide (PIIINP) in human serum in vitro for quantitative determination. The operation process is simple, the detection time is short, and the detection cost is low. At the same time, the kit of the present application has good sensitivity, accuracy, precision and linearity, can fully meet the detection requirements, and is worthy of popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0045] Figure 1 It is a schematic structural diagram of an embodiment of a kit for determining N-terminal peptide of type III procollagen according to the present application;

[0046] Figure 2 The figure is a flow chart of an embodiment of the method for determining the N-terminal peptide of type III procollagen according to the present application.

[0047] Reference numerals: 101, sample placement area; 102, pipette tip placement area; 103, diluent packaging area; 104, fluorescent labeled antibody packaging area. DETAILED DESCRIPTION

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0049] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0050] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0051] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0052] The solutions used in this application are prepared as follows, wherein the percentages are calculated by mass fraction:

[0053] (1) 0.01M pH7.4 PBS buffer (1000mL): Dissolve 8.000g sodium chloride, 0.200g potassium chloride, 1.140g disodium hydrogen phosphate, and 0.270g potassium dihydrogen phosphate in 800mL purified water. After dissolution, adjust the pH to 7.40±0.05 with 1mol / L HCl or 1mol / L NaOH, and make up to 1000mL; store at (4-30)℃ for 3 months.

[0054] (2) Labeling buffer (100 mL) including 0.05 M MES, pH = 6.2: Dissolve 1.0666 g MES in 80 mL purified water. After dissolution, adjust the pH to 6.2 ± 0.05 with 1 mol / L HCl or 1 mol / L NaOH and make up to 100 mL. Store at (2-8)°C for 3 months.

[0055] (3) 0.05 M borax solution (1000 mL): Dissolve 19.07 g sodium tetraborate in 800 mL purified water and make up to 1000 mL. Store at (4-30)°C for 3 months.

[0056] (4) 0.2 M boric acid solution (1000 mL): Dissolve 12.37 g of boric acid in 800 mL of purified water and make up to 1000 mL. Store at (4-30)°C for 3 months.

[0057] (5) 0.2 M boric acid borax buffer (1000 mL): Mix 300 mL of 0.05 M borax solution and 700 mL of 0.2 M boric acid solution, and adjust the pH to (8.0 ± 0.05) with borax solution or boric acid solution; store at (4-30) °C for 3 months.

[0058] (6) Labeling blocking solution (10 mL) including 10% BSA: Dissolve 1.000 g of bovine serum albumin (BSA) in 10 mL of purified water. After fully dissolved, filter through a microporous filter membrane with a pore size of 0.22 μm and store at (2-8)°C for a period of 1 month.

[0059] (7) Labeling activation solution (1 mL) including 2% EDC: Dissolve 0.02 g EDC in 1 mL purified water and prepare it for use immediately.

[0060] (8) 0.05M pH 8.0 Tris buffer (1000 mL): Dissolve 6.057 g of tris(hydroxymethyl)aminomethane in 800 mL of purified water. After dissolution, adjust the pH to 8.0 ± 0.05 with 1 mol / L HCl and make up to 1000 mL. Store at (4-30)°C for 3 months.

[0061] (9) The labeling storage solution includes 2% BSA, 5% trehalose, 2% sucrose, 0.5% casein sodium salt, 0.1% Proclin 300 and 0.2M pH8.0 Tris buffer: 2.000g bovine serum albumin, 5.000g trehalose, 2.000g sucrose, and 0.5000g casein sodium salt are dissolved in 100mL 0.2M pH8.0 Tris buffer, and then 0.1mL Proclin 300 solution is added and mixed. Store at (2-8)℃, with a shelf life of 3 months.

[0062] (10) Antibody coating diluent (100 mL) includes 1% sucrose and the above-mentioned 0.01M pH 7.4 PBS buffer: Dissolve 1.00 g sucrose in 100 mL 0.01M pH 7.4 PBS buffer. Store at (2-8)°C, with a shelf life of 3 months.

[0063] (11) Sample pad treatment solution (1000 mL) includes 1% BSA, 0.5% Tween-20 and the above-mentioned 0.2 M pH 8.0 boric acid borax buffer: dissolve 10.00 g of bovine serum albumin in 1000 mL 0.2 M pH 8.0 boric acid borax buffer, add 5.000 mL of Tween-20, and continue stirring until all dissolved; store at (2-8)°C, with a shelf life of 3 months.

[0064] (12) Dilution solution (1000 mL) includes 0.05% Proclin 300 and the above-mentioned 0.05 M pH 8.0 Tris buffer: 0.5 mL of Proclin 300 is added to 1000 mL of 0.05 M pH 8.0 Tris buffer.

[0065] The present application provides a kit for determining N-terminal peptide of type III procollagen, including a detection card, such as Figure 1 As shown, Figure 1 It is a schematic diagram of the structure of an embodiment of a kit for determining N-terminal peptide of type III procollagen according to the present application, specifically a schematic diagram of the structure of a test card. The test card comprises a sample placement area 101 for placing a sample to be tested.

[0066] The test card comprises a card shell and a test strip, wherein the test strip is arranged in the card shell; the test strip comprises a PVC bottom plate and an absorption pad, a nitrocellulose membrane and a sample pad which are sequentially overlapped and arranged on the PVC bottom plate; wherein a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is coated with PIIINP antibody, and the quality control line is coated with sheep anti-chicken IgY antibody;

[0067] The detection card further comprises a fluorescently labeled antibody encapsulation area 104, wherein the fluorescently labeled antibody encapsulation area 104 encapsulates a fluorescently labeled antibody solution, wherein the fluorescently labeled antibody solution comprises a fluorescently labeled PIIINP antibody and a fluorescently labeled chicken IgY antibody.

[0068] As an option of the present application, the detection card further includes a diluent packaging area 103, wherein the diluent packaging area 103 is packaged with a diluent; wherein, by mass fraction, the diluent includes 0.05% Proclin 300 and 0.05M Tris buffer at pH 8.0.

[0069] As an option in the present application, the card housing of the above-mentioned detection card further includes a pipette tip placement area 102 for placing a pipette tip for absorbing the solution.

[0070] As an option in this application, the concentration of the PIIINP antibody coated on the test line is 1.0 mg / mL;

[0071] The concentration of the goat anti-chicken IgY antibody coated on the quality control line is 1.0 mg / mL.

[0072] As an option of the present application, the diluent packaging area is provided with a diluent cup, and the diluent is packaged in the diluent cup.

[0073] In this embodiment, the diluent cup is prepared by the following steps: (1) Material preparation: According to the production batch, the required amount is calculated according to the specifications of 0.3 mL / cup and 1 cup / person, and the diluent and the reaction cup are prepared according to the required amount. The diluent is prepared according to the above method; (2) Packaging and coating of the diluent: Take the diluent to be packaged, use an aluminum foil sealing filling machine to pack the diluent into the reaction cup according to the specification of 0.3 mL / cup and coat it to obtain the diluent cup. Store at 4-30°C, the validity period is 18 months.

[0074] As an option of the present application, the fluorescently labeled antibody solution is obtained by mixing a fluorescent microsphere-labeled PIIINP antibody solution and a fluorescent microsphere-labeled chicken IgY antibody solution in a volume ratio of 1:1.

[0075] In this embodiment, the fluorescent microsphere-labeled PIIINP antibody solution and the fluorescent microsphere-labeled chicken IgY antibody solution were mixed evenly at a ratio of 1:1 (V:V), ultrasonicated at 90W for 2s, rested for 5s, repeated 3 times, and mixed evenly to obtain the fluorescent microsphere-labeled antibody solution. The solution was stored at 2-8°C and was valid for 15 days.

[0076] As an option of the present application, the fluorescent microsphere-labeled PIIINP antibody solution is prepared by the following steps:

[0077] Add fluorescent microspheres with a solid content of 1% into the labeling buffer, mix well, and then add the labeling activation solution to obtain activated fluorescent microspheres;

[0078] Add PIIINP antibody, rotate to react, add labeling blocking solution, rotate to react after sonication;

[0079] After centrifugation, the supernatant is discarded, and a labeling preservation solution is added. After ultrasonication, the PIIINP antibody solution labeled with the fluorescent microspheres is obtained.

[0080] As an option in the present application, the labeling buffer comprises, by mass fraction, 0.05M MES, pH 6.2; the labeling activation solution comprises 2% EDC; the labeling blocking solution comprises 10% BSA; the labeling preservation solution comprises 2% BSA, 5% trehalose, 2% sucrose, 0.5% casein sodium salt, 0.1% Proclin 300 and 0.2M pH 8.0 Tris buffer.

[0081] In this example, the preparation of fluorescently labeled PIIINP antibody solution, fluorescent microspheres labeled type III procollagen N-terminal peptide (PIIINP) antibody, taking 100 μL microspheres as an example: (1) Take a 2 mL centrifuge tube and add the labeling buffer (containing 0.05 M (1) 1 mL of MES (pH = 6.2); after taking out the fluorescent microspheres and vortexing them, take 100 μL of fluorescent microspheres with a solid content of 1% and put them into the labeling buffer and vortex again to mix them; (2) add 100 μL of the labeling activation solution to the above centrifuge tube, cover it tightly, and slowly mix it upside down 10 to 20 times; (3) after mixing, place the centrifuge tube on a rotary mixer and rotate it for 30 minutes; (4) after the reaction is completed, centrifuge at 4°C, with a centrifugal speed of 10,000 rpm and a centrifugal time of 30 minutes; (5) after centrifugation, gently take out the centrifuge tube, use a pipette to slowly remove the supernatant, and keep the precipitate; use a pipette to take 1000 μL of the labeling buffer and add it to the centrifuge tube; use an ultrasonic cell crusher to completely break it up at 90W ultrasound, work for 2s, rest for 5s, and repeat the mode 3-5 times; (6) add 50 μg PIIINP antibody was mixed by inverting 20 times, and the tube was placed on a rotary mixer for rotation reaction for 30 minutes; (7) After the rotation reaction was completed, 100 μL of the labeling blocking solution was pipetted into the above-mentioned centrifuge tube using a pipette, and the tube was completely broken up by using an ultrasonic cell crusher at 90 W for 2 seconds, 5 seconds, and repeated 3-5 times; the tube was placed on a rotary mixer again for rotation reaction for 2 hours; (8) After the blocking was completed, the tube was centrifuged at 4°C with a centrifugal speed of 10,000 rpm and a centrifugal time of 30 minutes; (9) After the centrifugation was completed, the supernatant was discarded, and the precipitate was retained. 1 mL of the labeling preservation solution was pipetted into a centrifuge tube using a pipette, and the tube was completely broken up by using an ultrasonic cell crusher at 90 W for 2 seconds, 5 seconds, and repeated 10 times; (10) After the precipitate was evenly broken up, it was transferred into a 10 mL centrifuge tube, 4 mL of the labeling preservation solution was transferred to clean the 2 mL centrifuge tube used for labeling, and after cleaning, it was transferred into a 10 mL centrifuge tube and vortexed to mix. The solution is stored at 2-8°C and has a validity period of 15 days to obtain a test line labeled antibody solution.

[0082] As an option of the present application, the chicken IgY antibody solution labeled with fluorescent microspheres is prepared by the following steps:

[0083] Add fluorescent microspheres with a solid content of 1% into the labeling buffer, mix well, and then add the labeling activation solution to obtain activated fluorescent microspheres;

[0084] Add chicken IgY antibody, rotate to react, add labeling blocking solution, rotate to react after sonication;

[0085] After centrifugation, the supernatant is discarded, and a labeling preservation solution is added. After ultrasonication, the chicken IgY antibody solution labeled with the fluorescent microspheres is obtained.

[0086] In this embodiment, a chicken IgY antibody solution labeled with fluorescent microspheres was prepared: the chicken IgY antibody labeled with fluorescent microspheres was prepared, taking 100 μL microspheres as an example, and the specific steps were as follows: (1) Take a 2mL centrifuge tube and use a pipette to draw 1mL of the labeling buffer; vortex the fluorescent microspheres to mix, take 100 μL of fluorescent microspheres with a solid content of 1% in the labeling buffer, and vortex again to mix; (2) Add 100 μL of the labeling activation solution to the above centrifuge tube, cover the lid tightly, and slowly mix it upside down 10 to 20 times; (3) After the mixing is completed, place the centrifuge tube on a rotating mixer and rotate for 30 minutes; (4) After the reaction is completed, centrifuge at 4°C; the centrifugal speed is 10000 rpm and the centrifugal time is 30 minutes; (5) After the centrifugation is completed, gently take out the centrifuge tube and slowly draw the supernatant with a pipette; retain the precipitate and use a pipette to draw 1000 μL of the labeling buffer; use an ultrasonic cell crusher to ultrasonicate 3-5 times to completely break up the precipitate ; (6) Add 80 μg chicken IgY antibody, then mix it upside down 20 times, place it on a rotary mixer, and rotate it for 30 minutes; (7) After the rotation reaction is completed, use a pipette to draw 100 μL of the labeled blocking solution into the above-mentioned centrifuge tube, and use an ultrasonic cell crusher to sonicate 3-5 times to completely break up the precipitate; place it on a rotary mixer again and rotate it for 2 hours; (8) After the blocking is completed, centrifuge at 4°C; the centrifugal speed is 10000rpm and the centrifugal time is 30 minutes; (9) After the centrifugation is completed, discard the supernatant; retain the precipitate, use a pipette to draw 1 mL of the labeled preservation solution and add it to the centrifuge tube, and use an ultrasonic cell crusher to sonicate 3-5 times to completely break up the precipitate; (10) After the precipitate is evenly broken up, transfer it to a 10 mL centrifuge tube, draw 4 mL of the labeled preservation solution to clean the 2 mL centrifuge tube used for labeling, and after cleaning, transfer it to a 10 mL centrifuge tube, vortex and mix to obtain a chicken IgY antibody solution labeled with fluorescent microspheres.

[0087] As an option of the present application, the fluorescent-labeled antibody encapsulation area is provided with a fluorescent reaction cup, and the fluorescent-labeled antibody solution is encapsulated in the fluorescent reaction cup.

[0088] As an option in this application, the fluorescent reaction cup is prepared by the following steps:

[0089] Load the fluorescent labeled antibody solution into the streaking machine and spray it into the original reaction cup at a rate of 3.0 μL / drop;

[0090] After drying, the mixture is coated to obtain the fluorescent reaction cup.

[0091] In this embodiment, (1) Packaging: Load the fluorescently labeled antibody solution into the scribing machine and spray it into the original reaction cup at a rate of 3.0 μL / drop. (2) Drying: Place the original reaction cup containing the fluorescently labeled antibody solution in an environment of (37±2)°C and humidity ≤30% and dry for (3-5) hours. (3) Coating: Take out the dried reaction cup and coat it with an aluminum foil sealing and filling machine to obtain a fluorescent reaction cup. After the fluorescent reaction cup is prepared, place it in a sealed ziplock bag to which an appropriate amount of desiccant has been added. The validity period is 18 months and it must be assembled and used within 3 months.

[0092] As an option in this application, the nitrocellulose membrane is prepared by the following steps:

[0093] Diluting the PIIINP antibody and the goat anti-chicken IgY antibody respectively with an antibody coating diluent to obtain a test line coating working solution and a quality control line coating working solution respectively, wherein the antibody coating diluent includes 1% sucrose and 0.01M pH 7.4 PBS buffer by mass fraction;

[0094] The detection line is coated with the working solution to be smeared to the position of the detection line of the original nitrocellulose membrane, and the quality control line is coated with the working solution to be smeared to the position of the quality control line of the original nitrocellulose membrane to obtain the nitrocellulose membrane.

[0095] In this embodiment, the present application provides a specific implementation method as follows: (1) Preparation of materials: According to the production batch calculation requirements, prepare a PVC base plate and an original nitrocellulose membrane (NC membrane). (2) Cutting and pasting the original NC membrane: Cut the original NC membrane into 25mm×(300±5)mm, gently peel off the protective film at the NC membrane pasting point on the PVC base plate, and push the original NC membrane evenly from left to right to make it firm. (3) Preparation of the test line coating working solution (T line coating working solution) and the quality control line coating working solution (C line coating working solution): According to the production batch calculation requirements, use the antibody coating diluent to dilute the PIIINP antibody to 1.0mg / mL and the sheep anti-chicken IgY antibody to 1.0mg / mL, respectively, to obtain the test line coating working solution and the quality control line coating working solution; the prepared solution is stored at 2-8°C and has a shelf life of 72h. (4) Turn on the continuous film stripping instrument, set the film stripping volume to 1μL / cm, change the film stripping length to 300mm, adjust the film stripping needle position, the distance between the quality control line and the test line is (5±0.5)mm, the distance between the quality control line and the upper edge of the NC membrane is (10±0.5)mm, and the distance between the test line and the lower edge of the original NC membrane is (10±0.5)mm; the test line is coated with the working solution to the position of the test line of the original nitrocellulose membrane, and the quality control line is coated with the working solution to the position of the quality control line of the original nitrocellulose membrane to obtain a nitrocellulose membrane. (5) After the film stripping is completed, the PVC base plate with the NC membrane attached is used as a reaction plate, and is placed in a vertical forced air drying oven with a temperature of (37±2)℃ and a humidity of ≤30% to dry for (16-24)h; (6) Storage conditions: Take out the dried reaction plate, seal it in a ziplock bag, and put in an appropriate amount of desiccant. Store at 4-30℃ under relative humidity ≤30%. The shelf life is 18 months. Assemble and use within 3 months.

[0096] As an option of the present application, the sample pad is prepared by the following steps:

[0097] The sample pad is obtained by impregnating the glass fiber with a sample pad treatment solution and drying the glass fiber;

[0098] Wherein, the sample pad treatment solution includes 1% BSA, 0.5% Tween-20 and 0.2M pH 8.0 boric acid borax buffer solution by mass fraction.

[0099] In this embodiment, (1) Material preparation: prepare sample pad treatment liquid at a volume of 20 mL / sheet; calculate the required amount of glass fiber according to the production batch. (2) Sample pad treatment: first, spread an appropriate amount of glass fiber in a stainless steel container, then pour the calculated sample pad treatment liquid into the stainless steel container to allow the glass fiber to be fully soaked, and finally take out the treated glass fiber and place it on a clean, dry and flat drying rack. (3) Drying conditions: Place the above-mentioned drying rack at a temperature of (37±2)°C and a relative humidity of ≤30% and dry for (16 to 24) hours to obtain a sample pad. (4) Storage conditions: Seal the dried sample pad in a ziplock bag and add an appropriate amount of desiccant. The validity period is 18 months and it should be assembled and used within 3 months.

[0100] As an option in this application, the test strip is prepared by the following steps:

[0101] (1) Material preparation: Calculate the required quantity according to the production batch, the number of test strips = production batch (per serving) / 75, and prepare the corresponding number of sample pads, CH37 absorbent paper and reaction plates.

[0102] (2) Preparation of absorbent pad: Cut CH37 absorbent paper into a size of (18±1) mm×(300±10) mm to obtain an absorbent pad.

[0103] (3) Cutting the sample pad into strips: Cut off 5 mm from each of the upper and lower long sides of the sample pad, and then cut it into strips of (19 ± 1) mm × (300 ± 10) mm.

[0104] (4) Pasting of the absorbent pad: Lay the reaction plate flat on the work surface, gently peel off the protective film where the absorbent pad is pasted on the reaction plate, and adhere the absorbent pad on it. When pasting, operate gently and roll it evenly to avoid scratches or bubbles. Make sure that the top of the absorbent pad is aligned with the top of the base plate, and the other side is partially covered on the NC film by 1 to 2 mm.

[0105] (5) Pasting of sample pad: Lay the reaction plate flat on the work surface, and gently peel off the protective film where the sample pad is pasted on the reaction plate. Attach the sample pad after the strip is pasted on it. When pasting, operate it gently and roll it evenly to avoid scratches or bubbles. Make sure that the lower end of the sample pad is aligned with the lower end of the PVC bottom plate, and make sure that the upper end of the sample pad covers the NC film, and the type III procollagen N-terminal peptide large plate is obtained.

[0106] (6) Storage conditions: Place the pasted large board into an aluminum foil bag, add desiccant, and seal it with a sealing machine. The validity period is 18 months and it should be used within 3 months.

[0107] (7) Cut the prepared large plate into test strips with a width of (4.0±0.4) mm.

[0108] As an option in this application, the test card is prepared by the following steps:

[0109] (1) Material preparation: Calculate the required quantity according to the test requirements and prepare the corresponding number of large plates, card shells, fluorescent reaction cups, dilution cups, and test card bases.

[0110] (2) Each test strip is loaded into the cartridge. Specifically, the test strip is loaded into the first shell of the cartridge. After loading, the second shell of the cartridge is covered and placed on a cartridge press. The height of the cartridge press is adjusted to 3.5 mm so that the first shell and the second shell are tightly combined to obtain a reagent card.

[0111] (3) Assembling the test card: Place the assembled reagent card into the corresponding position of the base of the test card. The base of the test card also includes a first reagent area and a second reagent area. Place the diluent cup into the first reagent area, which is the diluent packaging area; place the fluorescent reaction cup into the second reagent area, which is the fluorescent labeled antibody packaging area; press tightly to obtain the test card.

[0112] The production environment of the test card: the temperature should be (18-30)℃ and the humidity should be ≤30%. The storage conditions of the test card: put the assembled test card into an aluminum foil bag, add desiccant, and seal it with a sealing machine. The validity period is 15 months and it should be used within 3 months.

[0113] The present application also provides a kit for determining the N-terminal peptide of type III procollagen, comprising the above-mentioned detection card for determining the N-terminal peptide of type III procollagen.

[0114] The kit also includes a standard curve data card and a quality control product, wherein the quality control product includes a recombinant antigen of the N-terminal peptide of type III procollagen.

[0115] The standard curve data card is prepared by the following steps:

[0116] (1) Prepare curve data: Take PIIINP enterprise calibrators (S1-S5); operate according to the reagent instructions on the dry fluorescent immunoassay analyzer (Auto-HFIAS1000) produced by Sichuan Xinjiancheng Biological Co., Ltd., select signal test-repeatability test in the experiment management tool, click New Reset Page, and then click Test to perform the test operation. Repeat the test for each concentration of sample 3 times, calculate the mean of the T / C value obtained by the test, and count the mean T / C values ​​of repeated measurements of each concentration. The prepared concentration and the corresponding T / C mean are used as linear data.

[0117] (2) Curve fitting: In the Lanbo AFS-1000 dry fluorescent immunoassay management tool, select project parameters, use Create or Edit to set relevant parameters, click Save after editing is completed and confirmed to be correct, and the project parameters are set. Select Curve Generation, fill in the curve data into the table, select the MMF Model four-parameter growth curve fitting as the fitting algorithm, and click Fit after confirmation; after fitting, the R value and the four-parameter fitting values ​​a, b, c, and d will be displayed on the right side of the fitting result. After confirming that the fitting result is accurate, the curve preparation is completed.

[0118] (3) Two-dimensional code encoding: Generate a two-dimensional code encoding for information such as product name, product batch number, peak position, and fitting value according to the two-dimensional code encoding rules, as shown in Table 1:

[0119] Table 1 QR code encoding

[0120]

[0121]

[0122] (4) QR code generation: Import the generated QR code into the software to generate a QR code.

[0123] (5) QR code verification: Read the QR code data to verify whether the data is correct. After the data and parameters are confirmed to be correct, they can be printed in batches to obtain the standard curve data card.

[0124] The present application also provides a method for determining the N-terminal peptide of type III procollagen, using the above-mentioned kit for determining the N-terminal peptide of type III procollagen

[0125] Continue to refer Figure 1 , Figure 1 The figure is a schematic flow chart of the method for determining the N-terminal peptide of type III procollagen of the present application; the method comprises the following steps:

[0126] S1: Add the sample to be tested into the sample area of ​​the test card;

[0127] S2: placing the test card in a fluorescent immunoassay analyzer and starting it;

[0128] S3: reading the fluorescence signal of the detection line of the detection card, and calculating the PIIINP concentration according to the fluorescence signal.

[0129] In this embodiment, during the test, after the sample to be tested is added to the sample area, the fluorescent immunoassay analyzer automatically draws the sample to be tested into the diluent encapsulation area and mixes it with the sample diluent in the diluent cup in the diluent encapsulation area; after mixing, it is transferred to the fluorescent labeled antibody encapsulation area, and the PIIINP in the sample to be tested is first mixed and combined with the PIIINP antibody labeled with the fluorescent microspheres in the fluorescent labeled antibody encapsulation area to form a reaction complex; the chicken IgY antibody labeled with the fluorescent microspheres in the fluorescent labeled antibody encapsulation area and the reaction complex are transferred by the instrument to the reaction area of ​​the detection card, and under the action of chromatography, when the reaction complex is chromatographed to the nitrocellulose membrane (NC membrane), it is captured by the PIIINP antibody coated on the detection line (T line) on the NC membrane to form a "fluorescent labeled antibody-antigen-coated antibody" complex; the chicken IgY antibody labeled with the fluorescent microspheres is captured by the sheep anti-chicken IgY antibody coated on the quality control line (C line). The fluorescence intensity of the detection line (T line) is positively correlated with the concentration of the analyte in the sample to be tested. After reading the fluorescence signal using the matching analyzer, the fluorescence signal is converted into PIIINP concentration according to the standard curve information.

[0130] This kit adopts the double antibody sandwich method and the principle of fluorescence immunochromatography to quantitatively determine the content of type III procollagen N-terminal peptide (PIIINP) in human serum in vitro.

[0131] Compared with the prior art, the beneficial effects of this application are:

[0132] Type III procollagen N-terminal peptide is the amino-terminal tail peptide of type III procollagen that is cut off by endonucleases before forming procollagen outside the cell and enters the blood circulation. The complete type III collagen peptide is mainly cleared by the liver through endocytosis mediated by sinusoidal endothelial cell receptors, while small fragments are mainly excreted and cleared by the kidneys. When collagen metabolism is active and liver function is damaged, blood PIIINP increases. The double antibody sandwich method used in this application uses the technical principle of fluorescent immunochromatography to quickly detect the content of type III procollagen N-terminal peptide (PIIINP) in human serum in vitro and quantitatively determine it. It has the advantages of simple operation process, short detection time and low detection cost. At the same time, the kit of this application has good sensitivity, accuracy, precision and linearity, which can fully meet the detection requirements, and this kit is worthy of promotion and application. This application belongs to point-of-care testing (POCT). This detection method can quickly obtain results, and it is simple to operate, easy to use, miniaturized, cost-saving, and can widely cover various detection institutions.

[0133] The present application also provides experimental data: the liquid migration velocity results of the type III procollagen N-terminal peptide determination kit are shown in Table 2; the detection limit results of the type III procollagen N-terminal peptide determination kit are shown in Table 3; the linearity results of the type III procollagen N-terminal peptide determination kit are shown in Table 4; the repeatability results of the type III procollagen N-terminal peptide determination kit are shown in Table 5.

[0134] Table 2 Results of liquid migration velocity of type III procollagen N-terminal peptide assay kit

[0135]

[0136]

[0137] Table 3 Detection limit results of type III procollagen N-terminal peptide assay kit

[0138]

[0139]

[0140] Table 4 Linearity results of type III procollagen N-terminal peptide assay kit

[0141]

[0142]

[0143] Table 5 Repeatability results of type III procollagen N-terminal peptide assay kit

[0144]

[0145]

[0146] It should be understood that, although the steps in the flowchart of the accompanying drawings are displayed in sequence as indicated by the arrows, these steps are not necessarily executed in sequence in the order indicated by the arrows. Unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least a part of the steps in the flowchart of the accompanying drawings may include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn or alternately with other steps or at least a part of the sub-steps or stages of other steps.

[0147] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A test card for measuring N-terminal peptide of type III procollagen, characterized in that: The test card comprises a card housing and a test strip, wherein the test strip is arranged in the card housing; The test strip comprises a PVC base plate and an absorption pad, a nitrocellulose membrane and a sample pad which are sequentially overlapped and arranged on the PVC base plate; Wherein, a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is coated with PIIINP antibody, and the quality control line is coated with sheep anti-chicken IgY antibody; The detection card also includes a fluorescently labeled antibody encapsulation area, wherein the fluorescently labeled antibody encapsulation area encapsulates a fluorescently labeled antibody solution, and the fluorescently labeled antibody solution includes a PIIINP antibody labeled with fluorescent microspheres and a chicken IgY antibody labeled with fluorescent microspheres.

2. The detection card for measuring N-terminal peptide of type III procollagen according to claim 1, characterized in that: The test card further comprises a diluent packaging area, wherein the diluent packaging area is packaged with a diluent; Wherein, the diluent includes 0.05% Proclin 300 and 0.05M Tris buffer with pH 8.0 by mass fraction.

3. The detection card for measuring N-terminal peptide of type III procollagen according to claim 1, characterized in that: The concentration of the PIIINP antibody coated on the test line is 1.0 mg / mL; The concentration of the goat anti-chicken IgY antibody coated on the quality control line is 1.0 mg / mL.

4. The detection card for measuring N-terminal peptide of type III procollagen according to claim 1, characterized in that: The fluorescently labeled antibody solution is obtained by mixing a fluorescent microsphere-labeled PIIINP antibody solution and a fluorescent microsphere-labeled chicken IgY antibody solution in a volume ratio of 1:

1.

5. The detection card for measuring N-terminal peptide of type III procollagen according to claim 4, characterized in that: The fluorescent microsphere-labeled PIIINP antibody solution is prepared by the following steps: Add fluorescent microspheres with a solid content of 1% into the labeling buffer, mix well, and then add the labeling activation solution to obtain activated fluorescent microspheres; Add PIIINP antibody, rotate to react, add labeling blocking solution, rotate to react after sonication; After centrifugation, the supernatant is discarded, and a labeling preservation solution is added. After ultrasonication, the PIIINP antibody solution labeled with the fluorescent microspheres is obtained.

6. The detection card for measuring N-terminal peptide of type III procollagen according to claim 5, characterized in that: By mass fraction, the labeling buffer includes 0.05 M MES at pH 6.2; The marking activation solution includes 2% EDC; The labeling blocking solution includes 10% BSA; The labeling preservation solution includes 2% BSA, 5% trehalose, 2% sucrose, 0.5% casein sodium salt, 0.1% Proclin 300 and 0.2M pH 8.0 Tris buffer.

7. The detection card for measuring N-terminal peptide of type III procollagen according to claim 1, characterized in that: The nitrocellulose membrane is prepared by the following steps: Dilute the PIIINP antibody and the goat anti-chicken IgY antibody respectively with the antibody coating diluent to obtain the test line coating working solution and the quality control line coating working solution respectively, wherein the antibody coating diluent includes 1% sucrose and 0.01M pH 7.4 PBS buffer by mass fraction; The detection line is coated with the working solution to be smeared to the position of the detection line of the original nitrocellulose membrane, and the quality control line is coated with the working solution to be smeared to the position of the quality control line of the original nitrocellulose membrane to obtain the nitrocellulose membrane.

8. The detection card for measuring type III procollagen N-terminal peptide according to claim 1, characterized in that: The sample pad is prepared by the following steps: The sample pad is obtained by impregnating the glass fiber with a sample pad treatment solution and drying the glass fiber; Wherein, the sample pad treatment solution includes 1% BSA, 0.5% Tween-20 and 0.2M pH 8.0 boric acid borax buffer solution by mass fraction.

9. A kit for determining N-terminal peptide of type III procollagen, characterized in that: Comprising a test card as described in any one of claims 1 to 8.

10. A method for determining N-terminal peptide of type III procollagen, using the kit according to claim 9, characterized in that: The steps include: Add the sample to be tested into the sample area of ​​the test card; Placing the test card in a fluorescent immunoassay analyzer and starting it; The fluorescence signal of the detection line of the detection card is read, and the PIIINP concentration is calculated based on the fluorescence signal.

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