Test strip and kit for joint detection of thyroid function markers and application of test strip and kit

Abstract

The embodiment of the invention belongs to the technical field of detection, and relates to a test strip for joint inspection of thyroid function markers, a kit and application, and the test strip comprises first detection test paper, second detection test paper and third detection test paper; a fluorescently-labeled TSH antibody, a fluorescently-labeled T3 antibody and a fluorescently-labeled T4 antibody are fixed on the first detection test paper, and the first detection test paper is coated with a TSH monoclonal antibody, a T3 monoclonal antibody and a T4 monoclonal antibody; a fluorescently-labeled FT3 antibody and a fluorescently-labeled FT4 antibody are fixed on the second detection test paper, and the second detection test paper is coated with an FT3 monoclonal antibody and an FT4 monoclonal antibody; a fluorescence-labeled TGA-BSA antigen and a fluorescence-labeled TPOA-BSA antigen are fixed on the third detection test paper, and the third detection test paper is coated with a TGA monoclonal antibody and a TPOA monoclonal antibody. According to the application, the contents of TSH, T3, T4, FT3, FT4, TGA and TPOA can be rapidly detected at the same time.

Description

Technical Field

[0001] The present application relates to the field of detection technology, and in particular to test strips, kits and applications for joint detection of thyroid function markers. Background Art

[0002] There have been many studies on the markers used in thyroid function testing, including thyroid stimulating hormone (TSH), triiodothyronine (T3), tetraiodothyronine (T4), free triiodothyronine (FT3), free thyroxine (FT4), anti-thyroglobulin antibodies (TGA) and anti-thyroid peroxidase antibodies (TPOA). The seven items of thyroid function are indicators of the physiological function status of the thyroid gland.

[0003] TSH test is a preliminary screening test to find out the thyroid function. It is a very sensitive and specific parameter for testing thyroid function. It is particularly suitable for early detection or exclusion of functional disorders of the hypothalamus-pituitary-thyroid central regulatory loop. People with pituitary tumors that secrete TSH have elevated serum TSH.

[0004] T3 serum total triiodothyronine, which is a commonly used indicator of hyperthyroidism or hypothyroidism, and a specific indicator of hyperthyroidism.

[0005] T4 thyroxine is a serum total thyroxine secreted by thyroid cells and can be converted into T3. Its blood content is higher than T3, but its activity is not as good as T3. It is a common indicator of hyperthyroidism (commonly known as "hyperthyroidism") or hypothyroidism (commonly known as "hypothyroidism").

[0006] FT3 and FT4 are physiologically active forms of T3 and T4, and are a true reflection of the metabolic state of the thyroid gland. FT3 and FT4 are more sensitive and meaningful than T3 and T4. The advantage of FT3 and FT4 determination is that they are not affected by changes in the concentration and binding characteristics of the binding protein, so there is no need to determine the binding parameters separately. The FT3 content is of great significance in differential diagnosis of whether the thyroid function is normal, hyperthyroid or hypothyroid. It is very sensitive to the diagnosis of hyperthyroidism and is a specific indicator for diagnosing T3-type hyperthyroidism. FT4 can be used as a monitoring method for thyroid suppression experiments.

[0007] TGA is the first autoantibody found in thyroid disease. It is highly species-specific and is a common indicator for detecting autoimmune thyroid disease (AITD). TGA concentrations can be found to be elevated in people with autoimmune thyroiditis, with a frequency of about 70-80%. The positive rate of TGA in Graves' disease is about 60%. Thyroid cancer is correlated to a certain extent with TGA, with a positive rate of 13%-65%. The increase in TGA values ​​is a sign of tumor progression.

[0008] TPOA (transthyretinase antibody) is the main autoantibody of thyroid tissue and the key enzyme in the synthesis of thyroid hormones. It is closely related to the immune damage of thyroid tissue. TPOA is closely related to the occurrence and development of autoimmune thyroid disease (AITD). It can cause autoimmune-related hypothyroidism by causing insufficient secretion of thyroid hormones through cell-mediated and antibody-dependent cytotoxicity. As a monitoring indicator of autoimmune thyroid disease, TPOA has better sensitivity, specificity, reliability and significance than TMA.

[0009] In the prior art, when the above-mentioned markers are rapidly detected, each item is usually detected independently, and it is impossible to test them simultaneously. The operation steps are complicated, the operation cycle is long, the detection efficiency is low, and it will cause higher detection costs and manpower expenses, which is not conducive to promotion and application. Therefore, it is urgent to find a test paper that can realize the combined test of thyroid function at the same time. Summary of the invention

[0010] The purpose of the embodiments of the present application is to propose a test strip, a kit and an application for joint detection of thyroid function markers. The present application realizes the simultaneous rapid detection of TSH, T3, T4, FT3, FT4, TGA and TPOA contents.

[0011] In order to solve the above technical problems, the present application embodiment provides a test strip for joint detection of thyroid function markers, which adopts the following technical solution:

[0012] A test strip for joint testing of thyroid function markers, the test strip comprising a first test strip, a second test strip and a third test strip;

[0013] Wherein, the first test paper, the second test paper and the third test paper all include a sample pad, a binding pad, an NC membrane and a water-absorbing paper on a PVC bottom plate and sequentially overlapped on the PVC bottom plate, and a quality control line is arranged on the NC membrane;

[0014] The binding pad of the first test strip is fixed with fluorescently labeled TSH antibody, fluorescently labeled T3 antibody and fluorescently labeled T4 antibody, the NC membrane of the first test strip is provided with a first detection line, a second detection line and a third detection line, the first detection line is coated with TSH monoclonal antibody, the second detection line is coated with T3 monoclonal antibody, and the third detection line is coated with T4 monoclonal antibody;

[0015] The conjugate pad of the second test strip is fixed with a fluorescently labeled FT3 antibody and a fluorescently labeled FT4 antibody, the NC membrane of the second test strip is provided with a fourth test line and a fifth test line, the fourth test line is coated with a FT3 monoclonal antibody, and the fifth test line is coated with a FT4 monoclonal antibody;

[0016] The conjugate pad of the third test strip is fixed with fluorescently labeled TGA-BSA antigen and fluorescently labeled TPOA-BSA antigen, the NC membrane of the third test strip is provided with a sixth test line and a seventh test line, the sixth test line is coated with TGA monoclonal antibody, and the seventh test line is coated with TPOA monoclonal antibody.

[0017] Furthermore, the conjugate pad is also fixed with sheep anti-chicken IgY antibody, and the quality control line is coated with chicken IgY antibody.

[0018] Furthermore, the fluorescent label is 3+ Lanthanide fluorescent microspheres are used to label the Eu 3+ The diameter of lanthanide fluorescent microspheres is 290nm-350nm.

[0019] Furthermore, the coating concentrations of the TSH monoclonal antibody, T3 monoclonal antibody, T4 monoclonal antibody, FT3 monoclonal antibody, FT4 monoclonal antibody, TGA monoclonal antibody and TPOA monoclonal antibody were all 1.0 mg / mL, and the streaking volumes were all 1 μL / cm.

[0020] Furthermore, the sample pad is prepared by the following steps:

[0021] Immersing the glass fiber in a sample pad treatment solution, wherein the sample pad treatment solution comprises, by mass fraction, 1% bovine serum albumin, 0.2M boric acid borax buffer, and 0.5% Tween-20;

[0022] The sample pad is dried for 16 to 24 hours at a temperature of 18 to 26° C. and a relative humidity of ≤30% to obtain the sample pad.

[0023] Furthermore, the conjugate pad is prepared by the following steps:

[0024] activating the fluorescent microspheres to obtain activated fluorescent microspheres;

[0025] The activated fluorescent microspheres are used to label the target substance and the goat anti-chicken IgY antibody, respectively, to obtain the fluorescently labeled target substance and the fluorescently labeled goat anti-chicken IgY antibody, respectively;

[0026] Mixing the fluorescently labeled target substance and the fluorescently labeled goat anti-chicken IgY antibody in equal volume ratios, and spraying the mixture onto the glass fiber to obtain the conjugate pad;

[0027] Wherein, the target substances include TSH antibodies, T3 antibodies and T4 antibodies; or

[0028] The target substances include FT3 antibodies and FT4 antibodies; or

[0029] The target substances include TGA-BSA antigen and TPOA-BSA antigen.

[0030] Furthermore, the step of activating the fluorescent microspheres to obtain the activated fluorescent microspheres comprises:

[0031] Adding fluorescent microspheres to a labeling activation solution, rotating the solution for reaction, centrifuging, and retaining the precipitate, wherein the labeling activation solution includes 50% by mass of EDC;

[0032] A labeling buffer was added, and ultrasonic operation was performed using an ultrasonic cell disruptor to obtain activated fluorescent microspheres, wherein the labeling buffer included MES with a mass fraction of 1.07%.

[0033] In order to solve the above technical problems, the present application also provides a kit for joint detection of thyroid function markers, which adopts the following technical solution:

[0034] A kit for joint testing of thyroid function markers, the kit comprising a test card, and the test card comprising the above-mentioned test strip for joint testing of thyroid function markers.

[0035] Furthermore, the kit also includes a sample diluent, wherein the sample diluent includes 0.5 mL of Proclin 300 and 0.01 M PBS buffer at pH 7.4.

[0036] In order to solve the above technical problems, the present application also provides the use of the above kit in detecting thyroid function markers for non-diagnostic purposes. The following technical solutions are adopted:

[0037] Use of the above kit in detecting thyroid function markers for non-diagnostic purposes

[0038] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0039] This application adopts the point-of-care testing (POCT) method, and uses time-resolved fluorescence immunoassay technology to simultaneously perform rapid, specific, simple and sensitive joint detection of TSH, T3, T4, FT3, FT4, TGA and TPOA. Fluorescence immunochromatography technology is used to quantitatively detect thyroid stimulating hormone (TSH), triiodothyronine (T3), tetraiodothyronine (T4), free triiodothyronine (FT3), free thyroxine (FT4), anti-thyroglobulin antibodies (TGA) and anti-thyroid peroxidase antibodies (TPOA) in specimens (whole blood / serum / plasma), which can achieve sensitive, specific and rapid detection of thyroid function markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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.

[0041] Figure 1 It is a schematic structural diagram of an embodiment of a test strip for joint detection of thyroid function markers according to the present application;

[0042] Figure 2 is a schematic structural diagram of another embodiment of a test strip for joint detection of thyroid function markers according to the present application;

[0043] Figure 3 is a schematic structural diagram of another embodiment of a test strip for joint detection of thyroid function markers according to the present application;

[0044] Figure 4 is a schematic structural diagram of another embodiment of a test strip for joint detection of thyroid function markers according to the present application;

[0045] Figure 5 This is a graph showing the experimental results of an embodiment of a kit for joint detection of thyroid function markers according to the present application;

[0046] Figure 6 is another experimental result diagram of one embodiment of the kit for joint detection of thyroid function markers according to the present application;

[0047] Figure 7 is another experimental result diagram of one embodiment of the kit for joint detection of thyroid function markers according to the present application;

[0048] Figure 8 is another experimental result diagram of one embodiment of the kit for joint detection of thyroid function markers according to the present application;

[0049] Fig. 9 is another experimental result diagram of one embodiment of the kit for joint detection of thyroid function markers according to the present application;

[0050] Fig.10 is another experimental result diagram of one embodiment of the kit for joint detection of thyroid function markers according to the present application;

[0051] Fig.11 This is another experimental result diagram of an embodiment of the kit for joint detection of thyroid function markers according to the present application.

[0052] Figure numerals: 101, first PCV plate; 102, first sample pad; 103, first conjugation pad; 104, first nitrocellulose membrane; 105, first absorbent paper; 201, second PCV plate; 202, second sample pad; 203, second conjugation pad; 204, second nitrocellulose membrane; 205, second absorbent paper; 301, third PCV plate; 302, third sample pad; 303, third conjugation pad; 304, third nitrocellulose membrane; 305, third absorbent paper; 401, first detection reagent; 402, second detection test paper; 403, third detection test paper. DETAILED DESCRIPTION

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] The present application provides a test strip for joint detection of thyroid function markers

[0058] The test strips include a first test strip, a second test strip and a third test strip;

[0059] Wherein, the first test paper, the second test paper and the third test paper all include a sample pad, a binding pad, an NC membrane and a water-absorbing paper on a PVC bottom plate and sequentially overlapped on the PVC bottom plate, and a quality control line is arranged on the NC membrane;

[0060] The binding pad of the first test strip is fixed with fluorescently labeled TSH antibody, fluorescently labeled FT3 antibody and fluorescently labeled FT4 antibody, the NC membrane of the first test strip is provided with a first detection line, a second detection line and a third detection line, the first detection line is coated with TSH monoclonal antibody, the second detection line is coated with T3 monoclonal antibody, and the third detection line is coated with T4 monoclonal antibody;

[0061] The conjugate pad of the second test strip is fixed with a fluorescently labeled FT3 antibody and a fluorescently labeled FT4 antibody, the NC membrane of the second test strip is provided with a fourth test line and a fifth test line, the fourth test line is coated with a FT3 monoclonal antibody, and the fifth test line is coated with a FT4 monoclonal antibody;

[0062] The conjugate pad of the third test strip is fixed with fluorescently labeled TGA-BSA antigen and fluorescently labeled TPOA-BSA antigen, the NC membrane of the third test strip is provided with a sixth test line and a seventh test line, the sixth test line is coated with TGA monoclonal antibody, and the seventh test line is coated with TPOA monoclonal antibody.

[0063] In this embodiment, the present application adopts time-resolved fluorescence labeling technology to achieve simultaneous joint detection of TSH, T3, T4, FT3, FT4, TGA, and TPOA content in serum. The above test strips have high sensitivity, a wide linear range, and accurate results.

[0064] As an option of the present application, sheep anti-chicken IgY antibody is also fixed on the conjugate pad, and the quality control line is coated with chicken IgY antibody.

[0065] In this embodiment, the quality control of the test results of the test strip is achieved by coating the chicken IgY antibody and fixing the goat anti-chicken IgY antibody.

[0066] As an option in this application, the fluorescent label is Eu 3+ Lanthanide fluorescent microspheres are used to label the Eu 3+ The diameter of lanthanide fluorescent microspheres is 290nm-350nm.

[0067] In this embodiment, non-isotope immunoassay technology is used, lanthanide elements are used to label antigens or antibodies, and fluorescence is measured using time-resolved technology based on the luminescence characteristics of lanthanide chelates. The wavelength and time parameters are detected simultaneously for signal resolution, which can effectively eliminate the interference of non-specific fluorescence and greatly improve the analysis sensitivity. Compared with ordinary fluorophores, the decay time of lanthanide chelate fluorescence is more than 100 times that of traditional fluorescence, which greatly reduces background fluorescence and achieves a high signal-to-noise ratio.

[0068] As an option in the present application, the coating concentrations of the TSH monoclonal antibody, T3 monoclonal antibody, T4 monoclonal antibody, FT3 monoclonal antibody, FT4 monoclonal antibody, TGA monoclonal antibody and TPOA monoclonal antibody are all 1.0 mg / mL, and the streaking volumes are all 1 μL / cm.

[0069] In this embodiment, the coating concentration of the above antibodies is 1.0 mg / mL, which ensures the accuracy of the test results and ensures that there are enough antibodies to bind to the target test substance in the test sample.

[0070] As an option of the present application, the sample pad is prepared by the following steps:

[0071] Immersing the glass fiber in a sample pad treatment solution, wherein the sample pad treatment solution comprises, by mass fraction, 1% bovine serum albumin, 0.2M boric acid borax buffer, and 0.5% Tween-20;

[0072] The sample pad is dried for 16 to 24 hours at a temperature of 18 to 26° C. and a relative humidity of ≤30% to obtain the sample pad.

[0073] In this embodiment, the sample pad is fully dried by drying for 16 to 24 hours under the condition of relative humidity ≤ 30%.

[0074] As an option in this application, the conjugate pad is prepared by the following steps:

[0075] activating the fluorescent microspheres to obtain activated fluorescent microspheres;

[0076] The activated fluorescent microspheres are used to label the target substance and the goat anti-chicken IgY antibody, respectively, to obtain the fluorescently labeled target substance and the fluorescently labeled goat anti-chicken IgY antibody, respectively;

[0077] Mixing the fluorescently labeled target substance and the fluorescently labeled goat anti-chicken IgY antibody in equal volume ratios, and spraying the mixture onto the glass fiber to obtain the conjugate pad;

[0078] Wherein, the target substances include TSH antibodies, T3 antibodies and T4 antibodies; or

[0079] The target substances include FT3 antibodies and FT4 antibodies; or

[0080] The target substances include TGA-BSA antigen and TPOA-BSA antigen.

[0081] As an option of the present application, the step of activating the fluorescent microspheres to obtain the activated fluorescent microspheres includes:

[0082] Adding fluorescent microspheres to a labeling activation solution, rotating the solution for reaction, centrifuging, and retaining the precipitate, wherein the labeling activation solution includes 50% by mass of EDC;

[0083] A labeling buffer was added, and ultrasonic operation was performed using an ultrasonic cell disruptor to obtain activated fluorescent microspheres, wherein the labeling buffer included MES with a mass fraction of 1.07%.

[0084] The present application also provides a kit for joint detection of thyroid function markers

[0085] The above-mentioned test kit includes a test card and a card shell, the test card includes the above-mentioned test strips for joint detection of thyroid function markers, the test strips are arranged in the card shell, and specifically, the first test strip, the second test strip, and the third test strip are sequentially arranged and fixed in the groove of the card shell. Alternatively, the card shell includes a first card shell, a second card shell, and a third card shell that are independent of each other, the first test strip is placed in the first card shell, the second test strip is placed in the second card shell, and the third test strip is placed in the third card shell.

[0086] As an option of the present application, the kit further includes a sample diluent, wherein the sample diluent includes 0.5 mL of Proclin 300 and 0.01 M PBS buffer at pH 7.4.

[0087] The present application also provides the use of the above-mentioned kit in detecting thyroid function markers for non-diagnostic purposes. Specifically, the following steps are included:

[0088] The test sample is dripped into the sample adding hole of the reagent card, and the reagent card is placed in a fully automatic fluorescence immunoassay analyzer for fluorescence detection to obtain the output test result.

[0089] The test samples of the present application include whole blood, serum or plasma.

[0090] In this embodiment, the present application adopts the point-of-care testing (POCT) method, and through time-resolved fluorescence immunoassay technology, TSH, T3, T4, FT3, FT4, TGA, TPOA are simultaneously detected rapidly, specifically, simply, and sensitively. Fluorescence immunochromatography is used to quantitatively detect thyroid stimulating hormone (TSH), triiodothyronine (T3), tetraiodothyronine (T4), free triiodothyronine (FT3), free thyroxine (FT4), anti-thyroglobulin antibodies (TGA), and anti-thyroid peroxidase antibodies (TPOA) in specimens (whole blood / serum / plasma), which can realize sensitive, specific, and rapid detection of thyroid function markers.

[0091] This application has the following advantages:

[0092] 1) The technology used in this application is a non-isotope immunoassay technology, which uses lanthanide elements to label antigens or antibodies, and measures fluorescence using time-resolved technology based on the luminescence characteristics of lanthanide chelates. The two parameters of wavelength and time are detected simultaneously for signal resolution, which can effectively eliminate the interference of non-specific fluorescence and greatly improve the analysis sensitivity.

[0093] 2) Time-resolved fluorescence immunoassay: Compared with common fluorophores, the decay time of lanthanide chelate fluorescence is more than 100 times that of traditional fluorescence, which greatly reduces the background fluorescence and achieves a high signal-to-noise ratio.

[0094] 3) This application detects 7 different thyroid markers at the same time, which can conduct comprehensive monitoring of the thyroid gland, making early detection of the thyroid gland faster and more accurate. Currently, there is no such multi-test strip for thyroid gland detection on the market;

[0095] 4) This application is simple to operate and has a short detection time: only three times of sample addition are required to obtain seven test results. After the sample addition is completed, the results can be obtained in 10-15 minutes, which is more suitable for grassroots applications;

[0096] 5) This application adopts time-resolved microsphere labeling technology. The detection card of this application has high sensitivity, wide linear range, and accurate results, which better meets the application.

[0097] The fluorescent microsphere technology is combined with lanthanide elements and applied to immunochromatography technology to achieve rapid, specific, simple and sensitive combined detection of TSH, T3, T4, FT3, FT4, TGA and TPOA.

[0098] The above scheme is further described below in conjunction with specific implementation examples, but these embodiments are by no means limiting the present application. The preferred embodiments of the present application are described in detail as follows:

[0099] Example 1

[0100] This example introduces the structure of the test strip for joint detection of thyroid function markers

[0101] like Figure 1 As shown, Figure 1 It is a structural schematic diagram of an embodiment of a test strip for joint detection of thyroid function markers according to the present application, specifically a structural schematic diagram of a first test strip; the first test strip comprises a first sample pad 102, a first conjugation pad 103, a first nitrocellulose membrane 104, and a first absorbent paper 105, which are sequentially pasted on a first PVC board 101, and each layer of the structure covers another layer of the structure by 1-2 cm, wherein T1 is a first detection line coated with TSH monoclonal antibody, T2 is a second detection line coated with T3 monoclonal antibody, T3 is a third detection line coated with T4 monoclonal antibody, and C is a quality control line coated with chicken IgY antibody.

[0102] like Figure 2 As shown, Figure 2 It is a structural schematic diagram of another embodiment of the test strip for joint detection of thyroid function markers according to the present application, specifically a structural schematic diagram of a second test strip; the second test strip includes a second sample pad 202, a second conjugation pad 203, a second nitrocellulose membrane 204, and a second absorbent paper 205, which are sequentially pasted on a second PVC board 201, and each layer of the structure covers another layer of the structure by 1-2 cm, wherein T4 is a fourth detection line coated with FT3 monoclonal antibody, T5 is a fifth detection line coated with FT4 monoclonal antibody, and C is a quality control line coated with chicken IgY antibody.

[0103] like Figure 3 As shown, Figure 3 It is a structural schematic diagram of another embodiment of the test strip for joint detection of thyroid function markers according to the present application, specifically a structural schematic diagram of a third test strip; the third test strip includes a third sample pad 302, a third binding pad 303, a third nitrocellulose membrane 304, and a third absorbent paper 305, which are sequentially pasted on a third PVC board 301, and each layer of the structure covers another layer of the structure by 1-2 cm, wherein T6 is the sixth detection line coated with TGA monoclonal antibody, T7 is the seventh detection line coated with TPOA monoclonal antibody, and C is a quality control line coated with chicken IgY antibody.

[0104] like Figure 4 As shown, Figure 4 It is a structural schematic diagram of another embodiment of a test strip for joint testing of thyroid function markers according to the present application. The test strip for joint testing of thyroid function markers includes a first test strip 401, a second test strip 402 and a third test strip 403.

[0105] The structure of the test strip of this application is specifically shown in Table 1:

[0106] Table 1 Test strips for joint detection of thyroid function markers

[0107]

[0108]

[0109] Example 2

[0110] The first test paper, the second test paper and the third test paper are prepared by the following steps:

[0111] 1. Take the preparation of the first test paper as an example

[0112] Step 1: Preparation method of solution required for this application

[0113] 0.01M pH7.4 PBS buffer: Dissolve 8g sodium chloride, 0.2g potassium chloride, 1.14g disodium hydrogen phosphate, and 0.27g 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.

[0114] Preparation of labeling buffer: Dissolve 1.07 g MES (2-morpholineethanesulfonic acid) 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.

[0115] Preparation of 0.05M borax solution: Dissolve 19.07g sodium tetraborate in 800mL purified water and adjust the volume to 1000mL after dissolution.

[0116] Preparation of 0.2M borax solution: Dissolve 12.37g sodium tetraborate in 800mL purified water and adjust the volume to 1000mL after dissolution.

[0117] Preparation of labeling blocking solution: Dissolve 1g of bovine serum albumin in 10mL of purified water, and filter through a microporous filter membrane with a pore size of 0.22μm after it is fully dissolved.

[0118] Preparation of labeling activation solution: Dissolve 0.5g EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) in 1mL purified water and use within 5 minutes.

[0119] Preparation of labeling preservation solution: add 2g bovine serum albumin, 2g sucrose, 0.5g casein sodium salt, and 5g trehalose into 100mL 0.05mL 0.02M pH8.0 Tris to dissolve, then add 0.05mL Proclin 300 solution and mix well.

[0120] Preparation of antibody coating diluent: Add 1g sucrose to 100mL 0.01M pH7.4 PBS buffer and dissolve and mix.

[0121] Preparation of sample pad treatment solution: Dissolve 10 g of bovine serum albumin in 1000 mL of 0.2 M boric acid borax buffer (pH 8.0), add 5 mL of Tween-20 and stir until completely dissolved.

[0122] Sample diluent preparation: Dissolve 0.5 mL Proclin 300 in 1000 mL 0.01 M pH 7.4 PBS buffer and mix well.

[0123] Step 2: Sample pad preparation

[0124] Set the wheel axle height of the paper impregnation pad press, the height of the left micrometer is 9mm, and the height of the right micrometer is 9.5mm; pour the sample pad treatment liquid into the stainless steel liquid storage container (use 50mL sample pad treatment liquid for each sample pad), and the solution cannot be too full to avoid overflow; take out the treated sample pad, place it on a clean, dry and flat drying rack, and dry it overnight (16-24)h at a temperature of (18-26)℃ and a relative humidity of ≤30%. After drying, cut it into a width of 22-24mm and a length of 290-310mm for use.

[0125] Step 3: Preparation of fluorescently labeled antibodies

[0126] (1) Take four clean 2 mL centrifuge tubes and add 1 mL of labeling buffer to each tube using a pipette; vortex the fluorescent microspheres to mix well, take 50 μL of fluorescent microspheres with a solid content of 1% into the labeling buffer and vortex again to mix well;

[0127] (2) Add 10 μL of the labeling activation solution to each of the above centrifuge tubes, cover the tubes tightly, and slowly invert upside down 10 to 20 times to mix;

[0128] (3) After mixing, place the centrifuge tube on a rotary mixer and rotate for 30 minutes;

[0129] (4) After the reaction is completed, centrifuge at 4°C at a speed of 12,000 rpm and a centrifugation time of 20 min;

[0130] (5) After centrifugation, gently remove the centrifuge tube and slowly aspirate the supernatant using a pipette; retain the precipitate and use a pipette to aspirate 900 μL-1000 μL of the labeling buffer; use an ultrasonic cell disruptor to sonicate 3-5 times to completely break up the precipitate;

[0131] (6) Add 50 μg of TSH antibody, T3 antibody, T4 antibody and 80 μg of goat anti-chicken IgY antibody to four tubes respectively, then mix them by inverting 20 times, place them on a rotating mixer, and rotate them for 1 h;

[0132] (7) After the rotation reaction is completed, use a pipette to draw 100 μL of the labeled blocking solution into the above centrifuge tubes, and use an ultrasonic cell crusher to sonicate 3-5 times to completely break up the precipitate; place it on a rotating mixer again and rotate for 1 hour;

[0133] (8) After blocking, centrifuge at 4°C at a speed of 12,000 rpm for 20 min.

[0134] (9) After centrifugation, discard the supernatant; retain the precipitate, use a pipette to draw 1 mL of labeling preservation solution and add it to a centrifuge tube. Use an ultrasonic cell disruptor to sonicate 3-5 times to completely break up the precipitate to obtain a fluorescent labeling solution. The solution is stored at 2-8°C for later use.

[0135] Step 4: Preparation of conjugate pad

[0136] (1) Mix the four tubes of fluorescent labeling solution in step 3 (9) at a ratio of 1:1:1:1, and sonicate at 90W for 2 seconds, rest for 5 seconds, and repeat 3 times.

[0137] (2) Cut glass fiber 8964 into (10±0.5) mm×(300±10) mm.

[0138] (3) Load the mixed fluorescent microsphere-labeled antibody solution into a gold spray scribing machine and spray it onto the cut conjugate pads at a spray rate of 4.0 μL / cm; place the sprayed conjugate pads in an environment of (37±2)°C and humidity ≤30% to dry overnight (16-24) hours, and after drying, cut into pieces with a width of 9.5-10.5 mm and a length of 290-310 mm.

[0139] Step 6: Preparation of reaction plate

[0140] Preparation of T-line and C-line coating working solution:

[0141] (1) Preparation of T line coating working solution: Dilute TSH monoclonal antibody, T3 monoclonal antibody, and T4 monoclonal antibody to 1 mg / mL with antibody coating diluent, namely, the first detection line coating working solution, the second detection line coating working solution, and the third detection line coating working solution, respectively; the prepared solutions are stored at 2 to 8°C for future use.

[0142] (2) Preparation of C-line coating working solution: Dilute chicken IgY antibody to 1.0 mg / mL with antibody coating diluent to obtain C-line coating working solution; store the prepared solution at 2-8°C for future use.

[0143] (3) Film cutting:

[0144] Cut the NC film into 25mm×(300±5)mm, peel off the protective film where the NC film is pasted on the PVC bottom plate, and push the NC film evenly from left to right to make it firm.

[0145] Turn on the gold spray film scribing machine, set the scribing volume to 1 μL / cm, change the scribing length to 300 mm, adjust the scribing needle position, the distance between the quality control line and the third test line is (5±0.5) mm, the distance between the quality control line and the lower edge of the nitrocellulose membrane is (10±0.5) mm, and the distance between the first test line and the upper edge of the nitrocellulose membrane is (10±0.5) mm;

[0146] After the film is cut, the NC film is placed in an electric blast drying oven with a temperature of (37±2)°C and a humidity of ≤30% to dry for (16-24) hours, and stored at 4-30°C with a relative humidity of ≤30% for later use.

[0147] Step 7: Assemble the test strips

[0148] (1) Lay the reaction plate flat on the work surface at a temperature of (18-26)°C and a humidity of ≤30%. Remove the protective film at the place where the absorbent pad is attached to the reaction plate, adhere the absorbent pad thereon, and partially cover the other side of the absorbent pad on the NC membrane by 2-3 mm. Remove the protective film at the place where the binding pad and the sample pad are attached to the reaction plate, adhere the binding pad to its upper end, making sure that the binding pad faces upward and one end of the binding pad covers the NC membrane by 1-2 mm. Adhere the sample pad thereon, making sure that the lower end of the sample pad is aligned with the place where the sample pad is attached to the PVC bottom plate, and making sure that the upper end of the sample pad covers the binding pad. Cut the attached reaction plate into reagent strips with a width of 3.80-3.90 mm and install them in the corresponding plastic card shell.

[0149] 2. Prepare the second test paper

[0150] The second test strip is prepared according to the preparation method of the first test strip, wherein the preparation of the second test strip is different from the preparation of the first test strip in that:

[0151] Step 3 (6) is: add 50 μg of FT3 antibody, FT4 antibody and 80 μg of goat anti-chicken IgY antibody to three tubes respectively, then mix them upside down 20 times, place them on a rotating mixer, and rotate them for 1 hour;

[0152] Step 4 (1) is: mix the three tubes of fluorescent labeling solution in step 3 (9) at a ratio of 1:1:1, sonicate at 90W, work for 2s, rest for 5s, and repeat 3 times.

[0153] Step six (1) is to prepare T-line coating working solution: dilute FT3 monoclonal antibody and FT4 monoclonal antibody to 1 mg / mL with antibody coating diluent, i.e., the fourth detection line coating working solution and the fifth detection line coating working solution respectively; the prepared solutions are stored at 2-8°C for future use.

[0154] The remaining steps are the same as the preparation process of the first test strip in Example 2 and will not be repeated here.

[0155] 3. Prepare the third test paper

[0156] The third test paper is prepared according to the preparation method of the first test paper, wherein the preparation of the third test paper is different from that of the first test paper in that:

[0157] Step 3 (6) is: add 50 μg of TGA-BSA antigen, TPOA-BSA antigen and 80 μg of goat anti-chicken IgY antibody to three tubes respectively, then mix them upside down 20 times, place them on a rotary mixer, and rotate them for 1 hour;

[0158] Step 4 (1) is: mix the three tubes of fluorescent labeling solution in step 3 (9) at a ratio of 1:1:1, sonicate at 90W, work for 2s, rest for 5s, and repeat 3 times.

[0159] Step six (1) is to prepare T-line coating working solution: dilute TGA monoclonal antibody and TPOA monoclonal antibody to 1 mg / mL with antibody coating diluent, i.e., the sixth detection line coating working solution and the seventh detection line coating working solution respectively; the prepared solutions are stored at 2-8°C for future use.

[0160] The remaining steps are the same as the preparation process of the first test strip in Example 2 and will not be repeated here.

[0161] 4. It should be noted that the test card composed of the first test paper, the second test paper, and the third test paper can be a complete card shell, wherein the first test paper, the second test paper, and the third test paper are sequentially arranged and fixed in the groove of the card shell. It can also be three card shells, namely the first card shell, the second card shell, and the third card shell, wherein the first test paper is placed in the first card shell, the second test paper is placed in the second card shell, and the third test paper is placed in the third card shell.

[0162] Example 3

[0163] This embodiment provides a thyroid function combined detection kit, which includes the combined detection test strip provided in Example 1. Using this kit, TSH, T3, T4, FT3, FT4, TGA and TPOA can be tested simultaneously.

[0164] First, TSH, T3, T4, FT3, FT4, TGA, and TPOA were dissolved into multiple gradient standard solutions, and blank solutions were prepared at the same time. Standards and samples to be tested were respectively dripped into the sample slot of the test strip, and the color-developed test strip was placed in the fluorescent immunochromatography reader, and the signal value of the T / C line was read and recorded, and the monitoring standard curves of TSH, T3, T4, FT3, FT4, TGA, and TPOA were drawn; the T / C value of the sample to be tested was brought into the standard curve to obtain the corresponding values ​​of thyroid-stimulating hormone TSH, triiodothyronine T3, thyroxine T4, free triiodothyronine T3, free thyroxine T4, thyroglobulin antibody TGA, and anti-thyroid peroxidase anti-TPOA.

[0165] Example 4

[0166] Clinically relevant tests for this application:

[0167] In order to illustrate the test results, the present application tested 50 clinically relevant samples with the kit obtained in Example 1, and the obtained data are shown in Table 2 below:

[0168] Table 2 Clinical relevance test results

[0169]

[0170]

[0171]

[0172] Further, in order to illustrate the test results, the present application simultaneously tested the above 50 relevant samples with the kit of Hongkui Biotechnology (China) Co., Ltd., and the obtained data are shown in Table 3 below:

[0173] Table 3 Test results of the above 50 related samples tested simultaneously by the kit of Hongkui Biotechnology (China) Co., Ltd.

[0174]

[0175]

[0176]

[0177] Furthermore, in order to illustrate the relevance of the test results, the present application conducted a consistency analysis on the 50 clinical samples tested simultaneously above. The results are as follows: Figures 5 to 11 As shown. Figures 5 to 11It can be seen that the kit of the present application obtained in Example 1 and the kit of Hongkui Biotechnology (China) Co., Ltd. for testing TSH, T3, T4, FT3, FT4, TGA, and TPOA have good clinical correlation, and r is greater than 0.975.

[0178] 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.

[0179] 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 strip for joint detection of thyroid function markers, characterized in that: The test strips include a first test strip, a second test strip and a third test strip; Wherein, the first test paper, the second test paper and the third test paper all include a sample pad, a binding pad, an NC membrane and a water-absorbing paper on a PVC bottom plate and sequentially overlapped on the PVC bottom plate, and a quality control line is arranged on the NC membrane; The binding pad of the first test strip is fixed with fluorescently labeled TSH antibody, fluorescently labeled T3 antibody and fluorescently labeled T4 antibody, the NC membrane of the first test strip is provided with a first detection line, a second detection line and a third detection line, the first detection line is coated with TSH monoclonal antibody, the second detection line is coated with T3 monoclonal antibody, and the third detection line is coated with T4 monoclonal antibody; The conjugate pad of the second test strip is fixed with a fluorescently labeled FT3 antibody and a fluorescently labeled FT4 antibody, the NC membrane of the second test strip is provided with a fourth test line and a fifth test line, the fourth test line is coated with a FT3 monoclonal antibody, and the fifth test line is coated with a FT4 monoclonal antibody; The conjugate pad of the third test strip is fixed with fluorescently labeled TGA-BSA antigen and fluorescently labeled TPOA-BSA antigen, the NC membrane of the third test strip is provided with a sixth test line and a seventh test line, the sixth test line is coated with TGA monoclonal antibody, and the seventh test line is coated with TPOA monoclonal antibody.

2. The test strip for joint detection of thyroid function markers according to claim 1, characterized in that: The conjugate pad is also fixed with sheep anti-chicken IgY antibody, and the quality control line is coated with chicken IgY antibody.

3. The test strip for joint detection of thyroid function markers according to claim 1, characterized in that: The fluorescent labeling is carried out by Eu 3+ Lanthanide fluorescent microspheres are used to label the Eu 3+ The diameter of lanthanide fluorescent microspheres is 290nm-350nm.

4. The test strip for joint detection of thyroid function markers according to claim 1, characterized in that: The coating concentrations of the TSH monoclonal antibody, T3 monoclonal antibody, T4 monoclonal antibody, FT3 monoclonal antibody, FT4 monoclonal antibody, TGA monoclonal antibody and TPOA monoclonal antibody were all 1.0 mg / mL, and the streaking volumes were all 1 μL / cm.

5. The test strip for joint detection of thyroid function markers according to claim 1, characterized in that: The sample pad is prepared by the following steps: Immersing the glass fiber in a sample pad treatment solution, wherein the sample pad treatment solution comprises, by mass fraction, 1% bovine serum albumin, 0.2M boric acid borax buffer, and 0.5% Tween-20; The sample pad is dried for 16 to 24 hours at a temperature of 18 to 26° C. and a relative humidity of ≤30% to obtain the sample pad.

6. The test strip for joint detection of thyroid function markers according to claim 1, characterized in that: The conjugate pad is prepared by the following steps: activating the fluorescent microspheres to obtain activated fluorescent microspheres; The activated fluorescent microspheres are used to label the target substance and the goat anti-chicken IgY antibody, respectively, to obtain the fluorescently labeled target substance and the fluorescently labeled goat anti-chicken IgY antibody, respectively; Mixing the fluorescently labeled target substance and the fluorescently labeled goat anti-chicken IgY antibody in equal volume ratios, and spraying the mixture onto the glass fiber to obtain the conjugate pad; Wherein, the target substances include TSH antibodies, T3 antibodies and T4 antibodies; or The target substances include FT3 antibody and FT4 antibody; or The target substances include TGA-BSA antigen and TPOA-BSA antigen.

7. The test strip for joint detection of thyroid function markers according to claim 6, characterized in that: The step of activating the fluorescent microspheres to obtain the activated fluorescent microspheres comprises: Adding fluorescent microspheres to a labeling activation solution, rotating the solution for reaction, centrifuging, and retaining the precipitate, wherein the labeling activation solution includes 50% by mass of EDC; A labeling buffer was added, and ultrasonic operation was performed using an ultrasonic cell disruptor to obtain activated fluorescent microspheres, wherein the labeling buffer included MES with a mass fraction of 1.07%.

8. A kit for joint detection of thyroid function markers, characterized in that: The kit comprises a test card, and the test card comprises the test strip for the joint detection of thyroid function markers as described in any one of claims 1 to 7.

9. The kit for joint detection of thyroid function markers according to claim 8, characterized in that: The kit further comprises a sample diluent, wherein the sample diluent comprises 0.5 mL of Proclin 300 and 0.01 M PBS buffer solution with a pH of 7.

4.

10. Use of the kit according to claim 8 or 9 in detecting thyroid function markers for non-diagnostic purposes.

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