Detection card and kit for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody

Abstract

The invention belongs to the technical field of fluorescent quantitative immunochromatography, and relates to a detection card and a kit for quantitative joint detection of a tyrosine phosphatase antibody and a zinc transporter 8 antibody. A sample pad, a combination pad, a reaction film, a water absorption pad, and a first detection line, a second detection line and a quality control line which are arranged on the reaction film are sequentially arranged on the bottom plate; the combination pad contains a tyrosine phosphatase recombinant protein antigen marked by fluorescent microspheres, a zinc transporter 8 recombinant protein antigen and a sheep-derived IgY antibody marked by fluorescent microspheres; the first detection line is coated with a tyrosine phosphatase recombinant protein antigen, the second detection line is coated with a zinc transporter 8 recombinant protein antigen, and the quality control line is coated with an IgY antibody. According to the present invention, the tyrosine phosphatase antibody and the zinc transporter 8 antibody can be accurately quantified by using the fluorescence quantitative immunochromatography technology, such that the detection sensitivity is high, the detection result is accurate, and the advantages of simple operation, rapid detection and strong portability are provided.

Description

Technical Field

[0001] The present application relates to the technical field of fluorescent quantitative immunochromatography, and in particular to a detection card and a kit for quantitative joint detection of tyrosine phosphatase antibodies and zinc transporter 8 antibodies. Background Art

[0002] Patients with type 1 diabetes mellitus (TIDM) already have a variety of autoantibodies in their bodies before the onset of the disease, such as tyrosine phosphatase (IA-2) antibodies, zinc transporters member 8 (ZnT8) antibodies, and other insulin autoantibodies. Pancreatic β-cell and immune damage often take several years, resulting in a long clinical prodromal period. The positive rate of Anti-IA-2 in patients with prediabetes and type 1 diabetes is 50%-75%, and the positive rate in young patients with the first onset is higher, and is related to the speed of initial onset. Tyrosine phosphatase is a major target antigen of type 1 diabetes with high specificity. Zinc transporter 8 is mainly located in pancreatic β-cells and can transport cytoplasmic zinc ions to insulin storage secretory vesicles. Reduced transport function will affect insulin synthesis, storage and secretion, and can increase the risk of type 1 diabetes. ZnT8 protein can also cause β-cell autoimmune damage and induce type 1 diabetes. Therefore, the detection of IA-2 and ZnT8 autoantibodies has important clinical significance for the diagnosis, differential diagnosis, treatment monitoring and prediction of type I diabetes. At present, the common methods for detecting IA-2 and ZnT8 antibodies are radioimmunoassay, enzyme-linked immunosorbent assay and immunoassay. Among them, radioimmunoassay is relatively complicated to operate, requires a centrifuge and a radiometric detection device, has high detection cost, high background and poor specificity; enzyme-linked immunosorbent assay is also extremely cumbersome to operate, has poor detection precision, and is random. At the same time, there is cross-contamination between various reagents, which affects the accuracy of the test results; immunoassay can only perform qualitative detection, the analysis results are not objective enough, and the detection cost is high. Summary of the invention

[0003] The purpose of the present application is to propose a detection card and a kit for quantitative joint detection of tyrosine phosphatase antibodies and zinc transporter 8 antibodies, so as to solve the technical problems in the prior art of cumbersome and complicated operation, low detection efficiency, and low detection accuracy and sensitivity in the process of detecting tyrosine phosphatase and zinc transporter 8.

[0004] In order to solve the above technical problems, the present application embodiment provides a test card for quantitative joint detection of tyrosine phosphatase antibodies and zinc transporter 8 antibodies, which adopts the following technical solution:

[0005] The test strip comprises a bottom plate, on which a sample pad, a binding pad, a reaction membrane, a water-absorbing pad, and a first detection line, a second detection line and a quality control line arranged on the reaction membrane are arranged in sequence;

[0006] The conjugate pad contains tyrosine phosphatase recombinant protein antigens and zinc transporter 8 recombinant protein antigens labeled with fluorescent microspheres, and sheep-derived IgY antibodies labeled with fluorescent microspheres;

[0007] The first detection line is coated with tyrosine phosphatase recombinant protein antigen, the second detection line is coated with zinc transporter 8 recombinant protein antigen, and the quality control line is coated with IgY antibody.

[0008] Furthermore, the sample pad is prepared by the following preparation method:

[0009] The phosphate buffer, bovine serum albumin, sucrose and human anti-erythrocyte antibody are mixed to prepare a treatment solution;

[0010] The treatment solution is evenly applied on the glass cellulose membrane and dried to obtain a sample pad.

[0011] Furthermore, the conjugate pad is prepared by the following preparation method:

[0012] Taking fluorescent microspheres for activation to obtain an activated microsphere solution;

[0013] The tyrosine phosphatase recombinant protein antigen and the zinc transporter 8 recombinant protein antigen are added to the activated microsphere solution to prepare fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution and fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution;

[0014] Adding the sheep-derived IgY antibody to the activated microsphere solution to prepare a fluorescent microsphere-labeled sheep-derived IgY antibody solution;

[0015] The fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution, the fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution and the fluorescent microsphere-labeled sheep-derived IgY antibody solution are mixed for reaction to obtain a fluorescently labeled antigen-antibody solution;

[0016] The fluorescent labeled antigen-antibody solution is sprayed onto the glass fiber and dried to obtain a conjugate pad.

[0017] Furthermore, the activated microsphere solution is prepared by the following preparation steps:

[0018] Take a fluorescent microsphere suspension with a solid content of 1%, add it to a centrifuge tube, add MES buffer and mix well to obtain a latex solution;

[0019] Measure the activation solution, add it into a centrifuge tube, mix it with the latex solution, and perform a rotation reaction to obtain a rotation reaction solution;

[0020] Ultrasonicate the rotating reaction solution to obtain a microsphere resuspension;

[0021] The microsphere resuspension is centrifuged, the supernatant is discarded, MES buffer is added, and ultrasonic dispersion is performed to obtain an activated microsphere solution.

[0022] Furthermore, the fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution is prepared by the following preparation steps:

[0023] adding tyrosine phosphate recombinant protein antigen to the activated microsphere solution, rotating the reaction to obtain a microsphere antigen complex;

[0024] After the reaction is completed, a blocking agent is added for blocking to obtain a blocked microsphere solution;

[0025] The blocked microsphere solution is centrifuged, and after the centrifugation is completed, the supernatant is discarded and the precipitate is retained;

[0026] Add Tris buffer to the precipitate, centrifuge after sonication, and repeat this step once;

[0027] After removing the supernatant, adding the microsphere reconstitution solution, ultrasonicating and centrifuging, removing the precipitate, and obtaining the fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution;

[0028] The fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution is prepared by the following preparation steps:

[0029] adding zinc transporter 8 recombinant protein antigen to the activated microsphere solution, rotating the reaction to obtain a microsphere antigen complex;

[0030] After the reaction is completed, a blocking agent is added for blocking to obtain a blocked microsphere solution;

[0031] The blocked microsphere solution is centrifuged, and after the centrifugation is completed, the supernatant is discarded and the precipitate is retained;

[0032] Add Tris buffer to the precipitate, centrifuge after sonication, and repeat this step once;

[0033] After removing the supernatant, the microsphere reconstitution solution was added, ultrasonicated and centrifuged, and the precipitate was removed to obtain the fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution.

[0034] Furthermore, the fluorescent microsphere-labeled sheep-derived IgY antibody solution is prepared by the following preparation steps:

[0035] Adding sheep IgY antibody to the activated microsphere solution and rotating the reaction;

[0036] After the reaction is completed, a blocking agent is added for blocking to obtain a blocked microsphere antibody solution;

[0037] The blocked microsphere antibody solution is centrifuged, and after the centrifugation is completed, the supernatant is discarded and the precipitate is retained;

[0038] Add Tris buffer to the precipitate, sonicate and centrifuge;

[0039] The supernatant was removed, and the microsphere reconstitution solution was added, and ultrasonic centrifugation was performed to obtain the fluorescent microsphere-labeled sheep IgY antibody solution.

[0040] Furthermore, the concentration of the tyrosine phosphatase recombinant protein antigen on the first test line is 1-1.5 mg / mL; the concentration of the zinc transporter 8 recombinant protein antigen on the second test line is 1-1.5 mg / mL; and the concentration of the IgY antibody on the quality control line is 1-1.5 mg / mL.

[0041] Furthermore, the reaction film is prepared by the following preparation steps:

[0042] Prepare coating diluent using phosphate buffer and trehalose;

[0043] Adding tyrosine phosphatase recombinant protein antigen, zinc transporter 8 recombinant protein antigen and IgY antibody to the coating diluent for dilution to obtain a first coating working solution, a second coating working solution and a quality control line coating working solution;

[0044] Using a gold-spraying film scribing instrument, the first coating working solution, the second coating working solution and the quality control line coating working solution are respectively scribed onto preset positions of the nitrocellulose membrane according to a preset scribing amount to form a first test line, a second test line and a quality control line;

[0045] The nitrocellulose membrane after the filming is dried to obtain a reaction membrane.

[0046] Furthermore, the detection card also includes a card shell, the card shell includes an upper cover and a bottom cover, the test strip is loaded into the bottom cover, and the upper cover is covered on the bottom cover;

[0047] The upper cover is provided with a sample adding port and an observation window; the sample adding port corresponds to the sample pad area, and the observation window corresponds to the reaction membrane area.

[0048] In order to solve the above technical problems, the present application also provides a kit for quantitative joint detection of tyrosine phosphatase antibodies and zinc transporter 8 antibodies, which adopts the following technical solution:

[0049] The kit comprises a sample diluent, a chip and a detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody as described above; wherein the chip is provided with standard curve data for determining tyrosine phosphatase antibody and zinc transporter 8 antibody.

[0050] Compared with the prior art, this application has the following beneficial effects:

[0051] The quantitative combined detection card for tyrosine phosphatase antibodies and zinc transporter 8 antibodies provided in the present application utilizes fluorescent quantitative immunochromatography technology to accurately quantify tyrosine phosphatase antibodies and zinc transporter 8 antibodies, has higher detection sensitivity, more accurate test results, and has the advantages of simple operation, rapid detection, and strong portability. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] 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.

[0053] Figure 1 It is a schematic diagram of the structure of a test strip for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody of the present application;

[0054] Figure 2 It is a schematic diagram of the structure of a test card for quantitative joint detection of tyrosine phosphatase antibodies and zinc transporter 8 antibodies of the present application. DETAILED DESCRIPTION

[0055] 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.

[0056] 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.

[0057] 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.

[0058] The present application provides a test card for quantitative joint detection of tyrosine phosphatase antibodies and zinc transporter 8 antibodies, including a test strip, see Figure 1 As shown, the test strip comprises a base plate 1 on which a sample pad 2, a binding pad 3, a reaction membrane 4, a water absorbent pad 5, and a first detection line 6, a second detection line 7 and a quality control line 8 arranged on the reaction membrane 4 are arranged in sequence.

[0059] The conjugate pad 3 contains tyrosine phosphatase recombinant protein antigen (IA-2 antigen), zinc transporter 8 recombinant protein antigen (ZnT8 antigen) and sheep IgY antibody labeled with fluorescent microspheres; the first detection line 6 is coated with tyrosine phosphatase recombinant protein antigen, the second detection line 7 is coated with zinc transporter 8 recombinant protein antigen, and the quality control line 8 is coated with IgY antibody.

[0060] The sheep-derived IgY antibody can be selected from sheep anti-chicken IgY antibody and sheep anti-mouse IgY antibody. Specifically, when the sheep-derived IgY antibody is a sheep anti-chicken IgY antibody, the IgY antibody is a chicken IgY antibody; when the sheep-derived IgY antibody is a sheep anti-mouse IgY antibody, the IgY antibody is a mouse IgY antibody.

[0061] The detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody provided in the present application adopts the double antigen sandwich method and utilizes the technical principle of fluorescent immunochromatography. During the test, the sample is dripped onto the sample pad 2, and flows from the sample pad 2 to the conjugate pad 3 under the capillary effect. The IA-2 antibody and ZnT8 antibody in the sample can specifically bind to the fluorescently labeled IA-2 antigen and ZnT8 antigen on the conjugate pad 3 to form a fluorescent microsphere-antigen-antibody reaction complex, which flows to the detection line position on the reaction membrane 4 through capillary action. At this time, the complex with IA-2 antibody and ZnT8 antibody will be captured by the IA-2 antigen coated on the first detection line and the ZnT8 antigen coated on the second detection line. The concentrations of IA-2 antibodies and ZnT8 antibodies in the sample are proportional to the fluorescence intensity of the complex on the test line. The more IA-2 antibodies and ZnT8 antibodies there are in the sample, the more complexes will be captured by the corresponding test line and the higher the fluorescence signal intensity will be. The content of IA-2 antibodies and ZnT8 antibodies in the sample can be quantitatively calculated by processing the fluorescence signal with an immunofluorescence detector.

[0062] It should be noted that regardless of whether the sample to be tested contains IA-2 antibodies and ZnT8 antibodies, the sheep-derived IgY antibodies labeled with fluorescent microspheres will specifically bind to the quality control line coated with IgY antibodies, that is, the sheep-derived IgY antibodies labeled with fluorescent microspheres will be captured by the quality control line and produce a fluorescent signal of a certain intensity as a quality control signal, thereby judging whether the test strip is valid, preventing detection errors caused by deterioration of the test strip, improving the reliability of the test, and ensuring the normal progress of the test.

[0063] In this embodiment, the fluorescence quantitative immunochromatography technology is based on the matching quantitative instrument to directly detect the excitation fluorescence signal, and the content of the analyte in the sample can be calculated through the built-in standard curve of the instrument. It has the following advantages: ① It has higher detection sensitivity; ② The detection results are accurate; ③ The instrument realizes quantitative calculation and eliminates the influence of human factors on the results; ④ Compared with traditional quantitative detection technology, this technology has the advantages of being fast and low-cost, and is more suitable for clinical quantitative rapid detection.

[0064] In some embodiments, the sample pad 2 is prepared by the following preparation method:

[0065] Phosphate buffer (PB buffer), bovine serum albumin (BSA), sucrose and human anti-red blood cell antibody (RBC) are mixed to prepare a treatment solution;

[0066] The treatment liquid is evenly applied on the glass cellulose membrane, and a sample pad 2 is obtained after drying.

[0067] Example, preparation of treatment solution:

[0068] The treatment solution was prepared by mixing 20 MM PB buffer, 1% BSA, 1% sucrose and 0.1 mg / mL RBC.

[0069] The above-mentioned treatment liquid is evenly applied on a glass cellulose membrane at an amount of 50 mL / sheet (width: 25 cm, length: 30 cm), and dried overnight (16 to 24) hours at a temperature of (37 to 50)°C and a relative humidity of ≤30% to obtain a sample pad 2.

[0070] At a temperature of (18-28)°C and a relative humidity of ≤30%, cut off 5 mm from the upper and lower long sides of the dried sample pad 2, and then cut it into a size of (23±1) mm×(300±10) mm for later use.

[0071] In some embodiments, the conjugate pad 3 is prepared by the following preparation method:

[0072] Step S11, taking fluorescent microspheres for activation to obtain an activated microsphere solution;

[0073] Step S12, adding the tyrosine phosphatase recombinant protein antigen and the zinc transporter 8 recombinant protein antigen to the activated microsphere solution respectively, to prepare fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution and fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution;

[0074] Step S13, adding the sheep-derived IgY antibody to the activated microsphere solution to prepare a fluorescent microsphere-labeled sheep-derived IgY antibody solution;

[0075] Step S14, mixing the fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution, the fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution and the fluorescent microsphere-labeled sheep-derived IgY antibody solution to obtain a fluorescently labeled antigen-antibody solution;

[0076] Step S15, spraying the fluorescent labeled antigen-antibody solution onto the glass fiber, and obtaining the conjugate pad 3 after drying.

[0077] Wherein, step S11 is the activation of fluorescent microspheres, and the specific steps are as follows:

[0078] A fluorescent microsphere suspension with a solid content of 1% is taken, added to a centrifuge tube, and MES buffer (2-(N-morpholino)ethanesulfonic acid buffer) is added and mixed to obtain a latex solution; an activation solution is measured and added to a centrifuge tube, mixed with the latex solution, and a rotating reaction is obtained to obtain a rotating reaction solution; the rotating reaction solution is ultrasonically subjected to a microsphere resuspension; the microsphere resuspension is centrifuged, the supernatant is discarded, MES buffer is added, and ultrasonic dispersion is uniformly performed to obtain an activated microsphere solution.

[0079] For example, 100 μL of a fluorescent microsphere suspension with a solid content of 1% is added to a centrifuge tube, 1 mL of MES buffer is added, and mixed to obtain a latex solution; 30 μL of EDC (1-ethyl-carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) (1:1) are measured as an activation solution, mixed with the latex solution in the centrifuge tube, and rotated on a rotary mixer for 20 minutes to obtain a rotating reaction solution; the rotating reaction solution after the reaction is sonicated with ultrasound to re-suspend the microspheres on the tube wall in the aqueous solution, and then the microsphere suspension is centrifuged at 15000 r / min for 30 minutes, the supernatant is discarded, 1 mL of MES buffer is added, and the mixture is evenly dispersed by ultrasound to obtain an activated microsphere solution.

[0080] Step S12 is to cross-link the recombinant protein antigen with fluorescent microspheres, which specifically includes the following steps:

[0081] Add tyrosine phosphatase recombinant protein antigen or zinc transporter 8 recombinant protein antigen) to the activated microsphere solution, rotate to react, and obtain a microsphere antigen complex; after the reaction is completed, add a blocking agent to block to obtain a blocked microsphere solution; centrifuge the blocked microsphere solution, and after the centrifugation is completed, discard the supernatant and retain the precipitate; add Tris buffer to the precipitate, sonicate and then centrifuge, and repeat this step once; after removing the supernatant, add the microsphere complex solution, sonicate and centrifuge, remove the precipitate, and obtain a fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution or a fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution.

[0082] Among them, the blocking agent contains a blocking group, which is a thiol blocking group. The thiol blocking group is connected to the thiol groups in the tyrosine phosphate recombinant protein antigen and the zinc transporter 8 recombinant protein antigen, and plays a blocking role to prevent the tyrosine phosphate recombinant protein antigen or the zinc transporter 8 recombinant protein antigen from polymerizing again through the thiol group, affecting the activity of the tyrosine phosphate recombinant protein antigen or the zinc transporter 8 recombinant protein antigen, and reducing the binding efficiency of the tyrosine phosphate recombinant protein antigen or the zinc transporter 8 recombinant protein antigen and the fluorescent microspheres.

[0083] In this embodiment, the blocking agent can be selected from bovine serum albumin. For example, 0.5 mg IA-2 recombinant protein antigen or ZnT8 recombinant protein antigen is added to the above-mentioned activated microsphere solution, and the reaction is rotated for 2 hours. After the reaction is completed, 10% bovine serum albumin (BSA) is added for blocking for 30 minutes, the microsphere solution is blocked, and the blocked microspheres in the blocked microsphere solution are centrifuged at 4°C, the centrifugal speed is 14000r / min, and the centrifugal time is 20min. After the centrifugation is completed, the supernatant is discarded, the precipitate is retained, 1 mL Tris buffer (tris hydroxymethylaminomethane buffer) is added, ultrasonicated for 3min, then 12000r / min, 4°C, centrifuged for 15min, and this step is repeated once. After removing the supernatant, 1 mL of microsphere re-solution is added, ultrasonicated for 3min, centrifuged at 2000r / min for 3min, and the precipitate is removed and stored at 4°C in the dark.

[0084] Step S13 is to cross-link the sheep-derived IgY antibody with fluorescent microspheres, and the specific steps are as follows:

[0085] Add sheep IgY antibody to the above-mentioned activated microsphere solution and rotate to react; after the reaction is completed, add a blocking agent for blocking to obtain a blocked microsphere antibody solution; centrifuge the blocked microsphere antibody solution, and after the centrifugation is completed, discard the supernatant and retain the precipitate; add Tris buffer to the precipitate, sonicate and centrifuge; remove the supernatant, add the microsphere re-solution, and sonicate and centrifuge to obtain a fluorescent microsphere-labeled sheep IgY antibody solution.

[0086] The blocking agent has the same blocking principle as the above-mentioned blocking IA-2 recombinant protein antigen or ZnT8 recombinant protein antigen.

[0087] For example, 0.5 mg of goat anti-chicken IgY antibody was added to the above activated microsphere solution, and the reaction was rotated for 2 hours. After the reaction was completed, 10% bovine serum albumin was added for blocking for 30 minutes. The blocked microspheres were centrifuged at 4°C, at a speed of 14000r / min, for 20 minutes. After the centrifugation, the supernatant was discarded, the precipitate was retained, 1 mL of Tris buffer was added, ultrasonicated for 3 minutes, and then centrifuged at 12000r / min, 4°C for 15 minutes, and this step was repeated once. After removing the supernatant, 1 mL of microsphere re-solution was added, ultrasonicated for 3 minutes, centrifuged at 2000r / min for 3 minutes, and the precipitate was removed and stored at 4°C in the dark.

[0088] The fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution, the fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution and the fluorescent microsphere-labeled sheep IgY antibody solution were mixed evenly in a volume ratio of 1:1:1, and ultrasonicated at 90W for 2s, rest for 5s, and repeated 3 times to obtain a fluorescently labeled antigen-antibody solution.

[0089] In step S15, the glass fiber 8964 is cut into (10±0.5) mm×(300±10) mm, and the fluorescently labeled antigen-antibody solution is loaded into the gold spray scribing machine and sprayed onto the cut glass fiber at a spray volume of 4.0 μL / cm; the sprayed glass fiber is placed in an environment at (37±2)°C and humidity ≤30% to dry overnight (16 to 24) hours.

[0090] In some embodiments, the concentration of the tyrosine phosphatase recombinant protein antigen on the first test line is 1-1.5 mg / mL; the concentration of the zinc transporter 8 recombinant protein antigen on the second test line is 1-1.5 mg / mL; and the concentration of the IgY antibody on the quality control line is 1-1.5 mg / mL.

[0091] In some embodiments, the reaction film 4 is prepared by the following preparation steps:

[0092] Prepare coating diluent using phosphate buffer and trehalose;

[0093] Adding tyrosine phosphatase recombinant protein antigen, zinc transporter 8 recombinant protein antigen and IgY antibody into coating diluent for dilution respectively to obtain a first coating working solution, a second coating working solution and a quality control line coating working solution;

[0094] Using a gold-spraying film scribing instrument, the first coating working solution, the second coating working solution and the quality control line coating working solution are respectively scribed onto preset positions of the nitrocellulose membrane according to a preset scribing amount to form a first test line, a second test line and a quality control line;

[0095] The nitrocellulose membrane after the filming is dried to obtain a reaction membrane.

[0096] The coating diluent is prepared by mixing 0.05 M PB buffer and 1% trehalose to obtain the coating diluent.

[0097] For example, a nitrocellulose membrane (NC membrane) is attached to a PVC base plate, and the IA-2 recombinant protein antigen T1 and the ZnT8 recombinant protein antigen on the test line are diluted to a concentration of 1 mg / mL with the above-mentioned coating diluent, and the chicken IgY antibody on the quality control line is diluted to a coating concentration of 1 mg / mL. Use a gold spray film scribing instrument to draw the first coating working solution, the second coating working solution and the quality control line coating working solution at a predetermined position on the NC membrane to obtain the first test line T1, the second test line T2 and the quality control line C.

[0098] Preferably, the scratching volume is 1 μL / cm, the scratching length is 300 mm, the distance between the T1 line and the T2 line is (5±0.5) mm, the distance between the C line and the T2 line is (5±0.5) mm, the distance between the C line and the lower edge of the nitrocellulose membrane is (7±0.5) mm, and the distance between the T1 line and the upper edge of the nitrocellulose membrane is (7±0.5) mm. After the scratching is completed, the NC membrane is placed in a drying oven at a temperature of (37±2)°C and a humidity of ≤30% and dried for (24 to 48) hours to obtain a reaction membrane 4.

[0099] In some embodiments, the absorbent pad 5 is prepared by the following steps:

[0100] Take H-1 absorbent paper and cut it into a size of (25±1) mm×(300±10) mm to obtain absorbent pad 5.

[0101] The function of the absorbent pad 5 is to increase the flow rate of the test sample, thereby improving the speed and reliability of the test. Meanwhile, the absorbent pad 5 can also prevent the backflow contamination of the sample solution left after the test is completed.

[0102] The prepared sample pad 2, conjugate pad 3, reaction membrane 4 and absorbent pad 5 are assembled. The assembly process is as follows:

[0103] Lay the base plate 1 flat on the work surface, peel off the protective film where the absorbent pad 5 is pasted on the base plate 1, and adhere the absorbent pad 5 thereon, ensuring that the top of the absorbent pad 5 is aligned with the top of the base plate 1, and the other side partially covers the reaction membrane 4 by 2 to 3 mm; peel off the protective film where the binding pad 3 and the sample pad 2 are pasted on the base plate 1, and adhere the binding pad 3 to its upper end, preferably with the front side of the binding pad 3 facing upward and one end of the binding pad 3 covering the reaction membrane 4 by 1 to 2 mm; adhere the sample pad 2 to the binding pad 3, aligning the lower end of the sample pad 2 with the place where the sample pad 2 is pasted on the base plate 1, and the upper end of the sample pad 2 covers the binding pad 3, and assemble to obtain a test strip.

[0104] In some embodiments, the test card further comprises a card housing, see Figure 2 As shown, the card case includes an upper cover 9 and a bottom cover 10, the test strip is loaded into the bottom cover 10, and the upper cover 9 covers the bottom cover 10; the upper cover 9 is provided with a sample addition port 11 and an observation window 12; the sample addition port 11 corresponds to the sample pad 2 area, and the observation window 12 corresponds to the reaction membrane 4 area.

[0105] Specifically, each test strip is loaded into the bottom cover 10 of the card case, and after loading, the upper cover 9 is covered, and the card is placed on the card press, and the height of the card press is adjusted to 3.5 mm, so that the upper and lower covers of the card case are tightly combined, and the test card is obtained. Each test card is loaded into an aluminum foil bag, and a pack of desiccant is added to each bag, and the bag is sealed to be a semi-finished product.

[0106] In this embodiment, the width of the test strip is (3.85±0.05) mm, and the length can be adapted to the card housing.

[0107] The test card of the present application utilizes fluorescent quantitative immunochromatography technology to simultaneously and accurately quantify tyrosine phosphatase antibodies and zinc transporter 8 antibodies, with higher detection sensitivity and more accurate test results. It is also easy to operate, fast to detect, and highly portable. In addition, the test cost is low, and it can be widely used in clinical testing.

[0108] The present application also provides a kit for quantitative joint detection of tyrosine phosphatase antibodies and zinc transporter 8 antibodies, which comprises a sample diluent, a chip, and a detection card for quantitative joint detection of tyrosine phosphatase antibodies and zinc transporter 8 antibodies as described above; wherein standard curve data for determining tyrosine phosphatase antibodies and zinc transporter 8 antibodies are provided on the chip.

[0109] In this example, the sample diluent is prepared as follows:

[0110] Measure 20 mM Tris buffer, 1% Tween20 and 3% Triton and mix them to obtain the sample dilution solution.

[0111] Sample test: Take 10μL of the serum sample to be tested and add it to 1000μL of the above sample diluent. After mixing evenly, add it to the test card for immunochromatographic reaction; then use the emission wavelength corresponding to the fluorescent microspheres for fluorescence detection under the fluorescence detector. When the fluorescence detector is used for fluorescence detection, a signal is detected on the quality control line, proving that the test result is valid.

[0112] Among them, the detection principle of the detection card is as follows:

[0113] During the test, the sample to be tested is mixed with the sample diluent and dripped into the sample well. Chromatography is performed under the capillary effect. The IA-2 antibody and ZnT8 antibody in the sample are combined with the fluorescently labeled IA-2 and ZnT8 recombinant protein antigens to form a reaction complex, which diffuses to the test area. The fluorescently labeled IA-2 antigen and ZnT8 antigen combined with the test object are captured by the IA-2 and ZnT8 recombinant antigens coated on the detection line of the reaction membrane 4; the concentration of IA-2 and ZnT8 antibodies in the sample is proportional to the fluorescence intensity of the complex on the detection line. According to the set standard curve, the immunofluorescence detector converts the fluorescence signal value into the concentration of IA-2 antibody and ZnT8 antibody in the sample. The concentration of IA-2 and ZnT8 antibodies in the sample can be detected by using a fluorescent immunoassay analyzer. The default measurement results of the instrument are in units of IU / mL and AU / mL.

[0114] The kit of the present application directly detects the excitation fluorescence signal based on the matching quantitative instrument, and the content of the analyte in the sample can be calculated through the built-in standard curve of the instrument. The advantages are as follows:

[0115] ①Compared with the colloidal gold method, fluorescent quantitative immunochromatography has higher detection sensitivity;

[0116] ②The results are accurate, the resistance to matrix interference is strong, and the reagents are stable;

[0117] ③The instrument realizes quantitative calculation and eliminates the influence of human factors on the results; ④Compared with traditional quantitative detection technology, this technology has the advantages of being fast and inexpensive, and is more suitable for bedside quantitative rapid detection.

[0118] Based on the above-mentioned tyrosine phosphatase antibody and zinc transporter 8 antibody quantitative joint detection kit, the present application also provides a detection method of the kit, comprising the following steps:

[0119] Sample collection;

[0120] Turn on the instrument and insert the chip with the same batch number as the reagent;

[0121] Use a pipette to draw 10 μL of sample, add it to the sample diluent, and mix by inverting;

[0122] Open the aluminum foil bag, take out the test card, and place it horizontally on the table;

[0123] Use a pipette to draw 100 μL of the diluted and mixed sample and add it to the sample well of the test card;

[0124] Select the sample type "plasma, serum or whole blood" on the supporting instrument;

[0125] Instant test: After 15 minutes of reaction at room temperature, put the test card into the instrument slot, select the "Instant Test" mode, and click "Test"; Standard test: Put the test card into the instrument slot, select the "Standard Test" mode, click "Test", the instrument will automatically start timing, and after the timing ends, it will automatically test and display the results;

[0126] Click "Print" to print the test result report.

[0127] The sample types in this application include serum and whole blood. Different samples are collected using different methods. It is not recommended to use samples with hemolysis, lipemia, or icterus.

[0128] In this embodiment, the sample collection method is plasma collection. Specifically, venous blood of the subject is collected using an anticoagulant tube containing heparin, EDTA or sodium citrate anticoagulant, and the collected blood sample is shaken for later use.

[0129] Preferably, 10 μL of serum or whole blood is used for testing. Fresh serum or whole blood is recommended for testing. Serum can be obtained by centrifugation after venous blood collection and coagulation. For immediate testing, blood can be collected from the finger and tested with whole blood. If the collected serum or whole blood is not tested on the same day, it should be stored at 4°C; if it exceeds seven days, it should be stored below -20°C.

[0130] The following is a more specific description of the content of the present application in conjunction with specific embodiments, and further elaboration of the present application, but these embodiments are by no means limiting the present application.

[0131] Example 1

[0132] This example evaluates the performance of a kit for quantitative joint detection of tyrosine phosphatase antibodies and zinc transporter 8 antibodies.

[0133] (1) Linear detection:

[0134] Tyrosine phosphatase (IA-2) detection: dilute the high-value samples close to the upper limit of the linear range to 3IU / mL, 50IU / mL, 100IU / mL, 150IU / mL, 250IU / mL, and 300IU / mL in a certain proportion, take 10μL of each and add it to the sample diluent and mix evenly, take 100μL of the mixed liquid and add it to the sample well of the test card, and repeat each reference product 3 times. After standing for 15 minutes, read the concentration value on the fluorescent immunoassay analyzer.

[0135] The linear correlation coefficient was calculated to be r=0.9963. In addition, the linear range of the kit for detecting anti-tyrosine phosphatase (IA-2) antibody samples was 3-300 IU / mL.

[0136] Zinc transporter 8 (ZnT8) detection: dilute the high-value samples close to the upper limit of the linear range to 5AU / mL, 50AU / mL, 100AU / mL, 200AU / mL, 800AU / mL, and 2000AU / mL in a certain proportion, take 10μL of each and add it to the sample diluent and mix evenly, take 100μL of the mixed liquid and add it to the sample well of the test card, and repeat each reference product 3 times. After standing for 15 minutes, read the concentration value on the fluorescent immunoassay analyzer.

[0137] The linear correlation coefficient was calculated to be r=0.9986. In addition, the linear range of the kit for detecting zinc transporter 8 antibody samples was 5-2000 AU / mL.

[0138] (2) Blank limit detection experiment

[0139] Blank limit: Mix 0IU / mL IA-2 and 0AU / mL ZnT8 reference products and repeat the measurement 20 times. The relative T / C average value is and standard deviation (SD), we get The corresponding numerical value is substituted into the linear equation to obtain the corresponding concentration value, which is the blank limit. The test results are shown in Table 1. The results show that the blank limit of IA-2 of the kit of this embodiment is not higher than 0.8 IU / mL, and the blank limit of ZnT8 is not higher than 1.6 IU / mL.

[0140] Table 1 Blank limit detection results

[0141]

[0142]

[0143] (3) Precision performance evaluation test

[0144] ① Take two IA-2 samples with concentrations of 50 IU / mL and 250 IU / mL, make 3 parallels for each sample and each concentration, and use three batches of kits for detection. Calculate the intra-batch and inter-batch differences of the kit. The results show that the intra-batch and inter-batch differences of the kit of this embodiment are less than 5%.

[0145] ② Take two ZnT8 samples with concentrations of 50AU / mL and 800AU / mL, make 3 parallels for each sample and each concentration, use three batches of test kits for testing, calculate the intra-batch and inter-batch differences of the test kits, and the results show that the intra-batch and inter-batch differences of the test kits are less than 5%.

[0146] In summary, the test kit of the present application has the characteristics of short detection time, high detection sensitivity, good stability and precision, and is also easy to operate and low cost.

[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 quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody, characterized in that: The test strip comprises a bottom plate, on which a sample pad, a binding pad, a reaction membrane, a water-absorbing pad, and a first detection line, a second detection line and a quality control line arranged on the reaction membrane are arranged in sequence; The conjugate pad contains tyrosine phosphatase recombinant protein antigens and zinc transporter 8 recombinant protein antigens labeled with fluorescent microspheres, and sheep-derived IgY antibodies labeled with fluorescent microspheres; The first detection line is coated with tyrosine phosphatase recombinant protein antigen, the second detection line is coated with zinc transporter 8 recombinant protein antigen, and the quality control line is coated with IgY antibody.

2. The detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody according to claim 1, characterized in that: The sample pad is prepared by the following preparation method: The phosphate buffer, bovine serum albumin, sucrose and human anti-erythrocyte antibody are mixed to prepare a treatment solution; The treatment solution is evenly applied on the glass cellulose membrane and dried to obtain a sample pad.

3. The detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody according to claim 1, characterized in that: The conjugate pad is prepared by the following preparation method: Taking fluorescent microspheres for activation to obtain an activated microsphere solution; The tyrosine phosphatase recombinant protein antigen and the zinc transporter 8 recombinant protein antigen are added to the activated microsphere solution to prepare fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution and fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution; Adding the sheep-derived IgY antibody to the activated microsphere solution to prepare a fluorescent microsphere-labeled sheep-derived IgY antibody solution; The fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution, the fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution and the fluorescent microsphere-labeled sheep-derived IgY antibody solution are mixed for reaction to obtain a fluorescently labeled antigen-antibody solution; The fluorescent labeled antigen-antibody solution is sprayed onto the glass fiber and dried to obtain a conjugate pad.

4. The detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody according to claim 3, characterized in that: The activated microsphere solution is prepared by the following preparation steps: Take a fluorescent microsphere suspension with a solid content of 1%, add it to a centrifuge tube, add MES buffer and mix well to obtain a latex solution; Measure the activation solution, add it into a centrifuge tube, mix it with the latex solution, and perform a rotation reaction to obtain a rotation reaction solution; Ultrasonicate the rotating reaction solution to obtain a microsphere resuspension; The microsphere resuspension is centrifuged, the supernatant is discarded, MES buffer is added, and ultrasonic dispersion is performed to obtain an activated microsphere solution.

5. The detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody according to claim 3, characterized in that: The fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution is prepared by the following preparation steps: adding tyrosine phosphate recombinant protein antigen to the activated microsphere solution, rotating the reaction to obtain a microsphere antigen complex; After the reaction is completed, a blocking agent is added for blocking to obtain a blocked microsphere solution; The blocked microsphere solution is centrifuged, and after the centrifugation is completed, the supernatant is discarded and the precipitate is retained; Add Tris buffer to the precipitate, centrifuge after sonication, and repeat this step once; After removing the supernatant, adding the microsphere reconstitution solution, ultrasonicating and centrifuging, removing the precipitate, and obtaining the fluorescent microsphere-labeled tyrosine phosphatase recombinant protein antigen solution; The fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution is prepared by the following preparation steps: adding zinc transporter 8 recombinant protein antigen to the activated microsphere solution, rotating the reaction to obtain a microsphere antigen complex; After the reaction is completed, a blocking agent is added for blocking to obtain a blocked microsphere solution; The blocked microsphere solution is centrifuged, and after the centrifugation is completed, the supernatant is discarded and the precipitate is retained; Add Tris buffer to the precipitate, centrifuge after sonication, and repeat this step once; After removing the supernatant, the microsphere reconstitution solution was added, ultrasonicated and centrifuged, and the precipitate was removed to obtain the fluorescent microsphere-labeled zinc transporter 8 recombinant protein antigen solution.

6. The detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody according to claim 3, characterized in that: The fluorescent microsphere-labeled sheep-derived IgY antibody solution is prepared by the following preparation steps: Adding sheep IgY antibody to the activated microsphere solution and rotating the reaction; After the reaction is completed, a blocking agent is added for blocking to obtain a blocked microsphere antibody solution; The blocked microsphere antibody solution is centrifuged, and after the centrifugation is completed, the supernatant is discarded and the precipitate is retained; Add Tris buffer to the precipitate, sonicate and centrifuge; The supernatant was removed, and the microsphere reconstitution solution was added, and ultrasonic centrifugation was performed to obtain the fluorescent microsphere-labeled sheep IgY antibody solution.

7. The detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody according to claim 1, characterized in that: The concentration of the tyrosine phosphatase recombinant protein antigen on the first test line is 1-1.5 mg / mL; the concentration of the zinc transporter 8 recombinant protein antigen on the second test line is 1-1.5 mg / mL; the concentration of the IgY antibody on the quality control line is 1-1.5 mg / mL.

8. The detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody according to claim 1, characterized in that: The reaction membrane is prepared by the following preparation steps: Prepare coating diluent using phosphate buffer and trehalose; Adding tyrosine phosphatase recombinant protein antigen, zinc transporter 8 recombinant protein antigen and IgY antibody to the coating diluent for dilution to obtain a first coating working solution, a second coating working solution and a quality control line coating working solution; Using a gold-spraying film scribing instrument, the first coating working solution, the second coating working solution and the quality control line coating working solution are respectively scribed onto preset positions of the nitrocellulose membrane according to a preset scribing amount to form a first test line, a second test line and a quality control line; The nitrocellulose membrane after the filming is dried to obtain a reaction membrane.

9. The detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody according to any one of claims 1 to 8, characterized in that: The test card also includes a card shell, the card shell includes an upper cover and a bottom cover, the test strip is loaded into the bottom cover, and the upper cover is covered on the bottom cover; The upper cover is provided with a sample adding port and an observation window; the sample adding port corresponds to the sample pad area, and the observation window corresponds to the reaction membrane area.

10. A kit for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody, characterized in that: The invention comprises a sample diluent, a chip and a detection card for quantitative joint detection of tyrosine phosphatase antibody and zinc transporter 8 antibody as claimed in any one of claims 1 to 9; wherein the chip is provided with standard curve data for determining tyrosine phosphatase antibody and zinc transporter 8 antibody.

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