Free triiodothyronine fluorescence immunochromatography detection kit and preparation method thereof

Abstract

The invention discloses a free triiodothyronine fluorescence immunochromatography detection kit and a preparation method thereof. The detection kit comprises a reagent card and a diluent, the reagent card comprises a bottom plate as well as a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially erected on the bottom plate, the combination pad is coated with a fluorescent microsphere labeled FT3 monoclonal antibody and a fluorescent microsphere labeled goat anti-chicken IgY antibody, and the mass ratio of the fluorescent microsphere labeled FT3 monoclonal antibody to the fluorescent microsphere labeled goat anti-chicken IgY antibody is 10: (15-35); the nitrocellulose membrane is provided with a detection line coated with an FT3 capture antibody and a quality control line coated with a chicken IgY (Immunoglobulin Y). The detection kit has the advantages of excellent sensitivity and specificity, good stability of detection results, and good application prospects.

Description

Technical Field

[0001] This invention relates to the field of immunochromatographic detection technology, and in particular to a free triiodothyronine fluorescence immunochromatographic detection kit and its preparation method. Background Technology

[0002] Free triiodothyronine (FT3) is a thyroid hormone that regulates metabolism. Measuring its levels is crucial for differentiating between normal, hyperthyroid, and hypothyroid function. Studies have shown that the vast majority of total triiodothyronine is bound to transport proteins, while free triiodothyronine is the physiologically active form of thyroid triiodothyronine. Methods using labeled antigens are often susceptible to changes in serum protein binding affinity, while methods using a series of labeled antibodies can reduce this influence. FT3-based detection is theoretically unaffected by the concentration and affinity of binding proteins, thus eliminating the need for additional tests reflecting binding parameters.

[0003] Currently, the main methods used clinically to measure FT3 include radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, and fluorescence immunoassay. Among these, the free triiodothyronine assay kit based on radioimmunoassay suffers from methodological limitations, resulting in insufficient sensitivity and interference resistance, and has largely been phased out of the market. ELISA uses enzymes as markers, catalyzing the substrate for luminescence detection. Qualitative or quantitative analysis can be performed based on the colorimetric reaction, but it suffers from high detection costs, numerous influencing factors affecting luminescence, and the presence of carcinogenic substances in the substrate solution. Chemiluminescence uses luminescent molecules as markers for direct luminescence or uses a luminescent agent as a substrate, catalyzing luminescence through enzymes. It offers advantages such as independence from external light source excitation and relatively stable signals, but the reaction time is relatively long, and the signal can be interfered with by other components in the sample, easily leading to errors. Fluorescence immunoassay uses fluorescein (such as isothiocyanate) as a marker to detect analytes in tissues or serum, offering advantages such as fast reaction speed and low cost, but its specificity and sensitivity need further improvement.

[0004] Therefore, there is still a need to find a free triiodothyronine kit with high specificity and sensitivity, and low cost. Summary of the Invention

[0005] The first objective of this invention is to provide a reagent card for the fluorescence immunochromatographic assay of free triiodothyronine.

[0006] The second objective of this invention is to provide a method for preparing a reagent card for the fluorescence immunochromatographic assay of free triiodothyronine.

[0007] The third objective of this invention is to provide a kit for the fluorescence immunochromatographic assay of free triiodothyronine.

[0008] The third objective of this invention is to provide a reagent card or kit for the fluorescence immunochromatographic assay of free triiodothyronine in the detection of free triiodothyronine.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a reagent card for the fluorescence immunochromatographic assay of free triiodothyronine, the reagent card comprising a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper sequentially disposed on the base plate, wherein:

[0011] The binding pad is coated with fluorescent microsphere-labeled FT3 monoclonal antibody and fluorescent microsphere-labeled goat anti-chicken IgY antibody, and the mass ratio of the fluorescent microsphere-labeled FT3 monoclonal antibody to the fluorescent microsphere-labeled goat anti-chicken IgY antibody is 10:15-35.

[0012] The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with FT3 capture antibody, and the control line is coated with chicken IgY.

[0013] In some embodiments of the present invention, the sample pad is prepared by soaking it in a sample pad treatment solution, wherein the sample pad treatment solution contains an anti-human RBC antibody, an inhibitor, bovine serum albumin, Tween-20, and borate-borax buffer.

[0014] In some embodiments of the present invention, per 100 mL, the sample pad treatment solution comprises 0.5–2.5 mg of anti-human RBC antibody, 1–2.5 mg of blocking agent, 0.5–1.5 g of bovine serum albumin, 0.2–0.8 mL Tween-20, and the balance being 0.15–0.25 M borate-borax buffer. For example, the content of the anti-human RBC antibody in the sample pad treatment solution may be 0.5, 1, 1.5, 2, or 2.5 mg; the content of the blocking agent may be 1, 1.5, 2, or 2.5 mg, and so on.

[0015] In some embodiments of the present invention, the binding pad is obtained by spraying a fluorescent microsphere-labeled antibody solution; wherein the fluorescent microsphere-labeled antibody solution contains the fluorescent microsphere-labeled FT3 monoclonal antibody, the fluorescent microsphere-labeled goat anti-chicken IgY antibody, and a labeling preservation solution.

[0016] In some embodiments of the present invention, the labeling preservation solution comprises bovine serum albumin, sucrose, sodium caseinate, alginate, Proclin 300, and Tris buffer.

[0017] In some embodiments of the present invention, the labeled preservation solution comprises, per 100 mL, 1–3 g bovine serum albumin, 1–3 g sucrose, 0.2–0.8 g sodium caseinate, 6–8 g alginate, 0.05–0.2 mL Proclin 300, and the balance being 0.015–0.03 M Tris buffer (pH 8.0).

[0018] In some embodiments of the present invention, the labeling preservation solution, per 100 mL, comprises 2 g bovine serum albumin, 2 g sucrose, 0.5 g sodium caseinate, 5 g alginate, 0.1 mL Proclin 300, and the balance being 0.02 M Tris buffer (pH 8.0).

[0019] In some embodiments of the present invention, the raw materials for preparing the fluorescent microsphere-labeled FT3 monoclonal antibody include fluorescent microspheres and FT3 monoclonal antibody.

[0020] In some embodiments of the present invention, the raw materials for preparing the fluorescent microsphere-labeled goat anti-chicken IgY antibody include fluorescent microspheres and goat anti-chicken IgY antibody.

[0021] In some embodiments of the present invention, the ratio of the FT3 monoclonal antibody to the goat anti-chicken IgY antibody is 5-20:8-32.

[0022] In some embodiments of the present invention, the particle size of the fluorescent microspheres is 150–350 nm.

[0023] In some embodiments of the present invention, the fluorescent microspheres include a first fluorescent microsphere and a second fluorescent microsphere; wherein the particle size of the first fluorescent microsphere is 180-220 nm; and the particle size of the second fluorescent microsphere is 280-320 nm.

[0024] In some embodiments of the present invention, the mass ratio of the first fluorescent microsphere to the second fluorescent microsphere is 1.5 to 2.5:1.

[0025] In some embodiments of the present invention, the coating concentration of the FT3 capture antibody on the detection line is 0.8 to 1.0 mg / mL.

[0026] In some embodiments of the present invention, the coating concentration of chicken IgY on the quality control line is 1.0 to 1.5 mg / mL.

[0027] A second aspect of the present invention provides a method for preparing a reagent card as described in the first aspect, comprising the following steps:

[0028] S1. Spray the antibody solution labeled with the fluorescent microspheres onto a glass cellulose membrane, and dry it to obtain a conjugate pad;

[0029] S2. Spray the first coating working solution containing the FT3 capture antibody and the second coating working solution containing the chicken IgY onto the detection line and the quality control line respectively, and dry them to obtain a nitrocellulose membrane.

[0030] S3. Assemble the nitrocellulose membrane, the sample pad, the conjugation pad, and the absorbent paper sequentially on the base plate to obtain the final product.

[0031] In some embodiments of the present invention, the amount of the fluorescent microsphere-labeled antibody solution sprayed is 3-5 μL / cm. 2 .

[0032] In some embodiments of the present invention, the spraying amounts of the first coating working fluid and the second coating working fluid are independently 1-1.5 μL / cm. 2 .

[0033] In some embodiments of the present invention, the first coating working solution and the second coating working solution further include a coating solution, wherein the coating solution is a 0.005-0.015M PBS buffer containing 0.5-2% sucrose.

[0034] In some embodiments of the present invention, the pH value of the PBS buffer is 7.2 to 7.5; preferably 7.4.

[0035] A third aspect of the present invention provides a free triiodothyronine fluorescence immunochromatographic assay kit, comprising the reagent card and diluent described in the first aspect.

[0036] In some embodiments of the present invention, the raw materials for preparing the diluent include Proclin 300 and PBS buffer;

[0037] Preferably, the pH value of the PBS buffer is 7.3 to 7.5.

[0038] In some embodiments of the present invention, the raw materials for preparing the diluent include 0.02% to 0.08% Proclin 300 and 0.08 to 0.12M PBS buffer.

[0039] A fourth aspect of the present invention provides the use of the reagent card as described in the first aspect or the free triiodothyronine fluorescence immunochromatographic assay kit as described in the third aspect in the detection of free triiodothyronine.

[0040] The reagent card or kit for the fluorescence immunochromatographic assay of free triiodothyronine in this invention has at least the following beneficial effects:

[0041] The free triiodothyronine fluorescence immunochromatographic assay kit of this invention, compared with the commonly used methods for detecting free triiodothyronine, such as enzyme-linked immunosorbent assay / immunoturbidimetric assay, not only greatly shortens the detection time and reduces the detection cost, but also improves the detection sensitivity. Compared with similar methods, it has the advantages of simple labeling process, high sensitivity, accurate and stable results.

[0042] Furthermore, this invention optimizes the preparation method of the free triiodothyronine fluorescence immunochromatographic assay kit. By selecting appropriate raw materials, rationally designing the dosage of each material, and selecting the optimal reaction conditions, the preparation process is simple, the product labeling process is simple, the sensitivity is high, the results are accurate and stable, the detection cost is low, the product is easy to use, and the cost is low, making it suitable for widespread application.

[0043] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0045] Figure 1 This is a schematic diagram of the structure of the reagent card for the fluorescence immunochromatographic assay of free triiodothyronine (FT3) of the present invention.

[0046] Figure 2 The T / C test results of the reagent cards prepared in Examples 1 to 9 of this invention under different concentrations of reference materials are shown.

[0047] Figure 3 The results show the gradient ratio detection of reagent cards prepared in Examples 1-9 of this invention under different concentrations of reference materials.

[0048] Figure 4 The T / C test results of reagent cards in Examples 39-41 of this invention at different reaction times are shown. Detailed Implementation

[0049] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0050] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0051] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0052] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0053] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0054] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0055] Inventive concept:

[0056] This invention provides a reagent kit for the determination of free triiodothyronine by fluorescence immunochromatography, comprising a reagent card and a diluent. A schematic diagram of the reagent card is shown below. Figure 1 As shown, it includes a PVC board and, sequentially disposed on the PVC board, a sample pad, a conjugation pad, a nitrocellulose membrane (NC membrane), and absorbent paper, wherein:

[0057] The NC membrane has a detection line and a control line. The detection line is coated with a free triiodothyronine capture antibody.

[0058] The mat is coated with an antibody solution labeled with fluorescent microspheres. The antibody solution contains free triiodothyronine labeled with fluorescent microspheres and goat anti-chicken IgY antibody labeled with fluorescent microspheres, wherein the particle size of the fluorescent microspheres is 200nm to 300nm.

[0059] I. The impact of control line-related antibodies on detection performance

[0060] This section examines the impact of the type of free triiodothyronine-labeled antibody coated on the binding pad and the type of capture antibody coated on the T line on the detection performance of the test card. The following is an explanation with reference to specific examples.

[0061] Example 1:

[0062] This embodiment provides a reagent card for the fluorescence immunochromatographic assay of free triiodothyronine (FT3) and its preparation method.

[0063] 1. FT3 Fluorescence Immunochromatographic Assay Reagent Card

[0064] The test strip card in this embodiment consists of a PVC board and a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper sequentially placed on the PVC board, wherein:

[0065] The combination pad was coated with FT3 monoclonal antibody 01 (purchased from Guangzhou Nuclear Source Biotechnology Co., Ltd.) labeled with fluorescent microspheres and goat anti-chicken IgY antibody labeled with fluorescent microspheres;

[0066] The nitrocellulose membrane has T and C lines. The T line is coated with free triiodothyronine capture antibody (i.e., T3-BSA01), and the C line is coated with chicken IgY.

[0067] 2. Preparation method of FT3 fluorescence immunochromatographic assay reagent card

[0068] The reagent card preparation method in this embodiment includes the following:

[0069] (1) Preparation of sample pad

[0070] Weigh 1 mg of anti-human RBC antibody, 2 mg of blocking agent 002 (purchased from Guangzhou Nuclear Source Biotechnology Co., Ltd., catalog number HY102), 1 g of bovine serum albumin and 0.5 mL of Tween-20, and then make up to 100 mL with 0.2 M borate-borax buffer (pH 8.0) to obtain the sample pad treatment solution.

[0071] Then, using a glass fiber membrane as the sample pad base material, and setting the wheel axle height of the paper impregnation and pressing machine, the sample pad treatment solution prepared above is poured in for treatment. The treated sample pad is placed on a drying rack and dried for 16–24 hours at a humidity ≤30% and a temperature of 37±2℃.

[0072] (2) Preparation of binding pad

[0073] The fluorescent microsphere-labeled antibody solution was sprayed onto the cut conjugate pad at a rate of 5 μL / cm using a gold spraying machine. The pad was then dried at 37±2℃ for 16–24 hours under conditions of ≤30% humidity.

[0074] The fluorescent microsphere-labeled antibody solution includes 1.0 mg / mL fluorescent microsphere-labeled FT3 monoclonal antibody 01, 1.6 mg / mL fluorescent microsphere-labeled goat anti-chicken IgY antibody, and labeling preservation solution.

[0075] The method for obtaining fluorescent microsphere-labeled FT3 monoclonal antibody 01 includes the following steps:

[0076] Add 1 mL of labeling buffer (MES) to a 2 mL centrifuge tube; add 20 μL of Eu-fluorescent microspheres (purchased from Nanjing Microtest Biotechnology, catalog number MD016) with a solid content of 1% to the labeling buffer, wherein the Eu-fluorescent microspheres consist of 200 nm and 300 nm microspheres in a mass ratio of 2:1, and vortex to mix again; add 100 μL of labeling activation solution and mix well, vortex to mix and react for 30 min; centrifuge for 30 min at a speed of 10000 rpm / min; Discard the supernatant, add 1000 μL of labeling buffer, and sonicate; add 10 μg of free triiodothyronine-labeled antibody, mix well, and vortex to mix for 30 min; add 100 μL of labeling blocking buffer, sonicate, and vortex to mix for 2 h; centrifuge for 30 min; discard the supernatant, add 1 mL of labeling preservation solution, and sonicate; transfer the label to a new 10 mL centrifuge tube, aspirate 4 mL of labeling preservation solution to wash the 2 mL centrifuge tube used for labeling, and then transfer the washed product to a 10 mL centrifuge tube and vortex to mix.

[0077] The method for obtaining fluorescent microsphere-labeled goat anti-chicken IgY antibody includes the following steps:

[0078] Add 1 mL of MES labeling buffer to a 2 mL centrifuge tube; add 20 μL of a 1% solids content fluorescent microsphere mixture (containing 200 nm and 300 nm microspheres in a 2:1 mass ratio) to the labeling buffer, and vortex again to mix; add 100 μL of labeling activation solution and mix, then vortex for 30 min; centrifuge for 30 min at 10000 rpm; discard the supernatant, add 1000 μL of labeling buffer, and sonicate. Add 16 μg of goat anti-chicken IgY antibody (purchased from Guangzhou Nuclear Source Biotechnology Co., Ltd., catalog number 20220230), mix well, and vortex to mix for 30 min; add 100 μL of labeling blocking solution, sonicate, and vortex to mix for 2 h; centrifuge for 30 min; discard the supernatant, add 1 mL of labeling preservation solution, and sonicate; transfer the label to a new 10 mL centrifuge tube, aspirate 4 mL of labeling preservation solution to wash the 2 mL centrifuge tube used for labeling, and then transfer it to a 10 mL centrifuge tube and vortex to mix.

[0079] Methods for preparing labeled preservation solutions include:

[0080] Weigh out 2g of bovine serum albumin, 2g of sucrose, 0.5g of sodium caseinate, 5g of alginate, and 0.1mL of Proclin 3000. Then, adjust the volume to 100mL with 0.02M Tris buffer (pH 8.0).

[0081] (3) Preparation of nitrocellulose membrane

[0082] Two coating solutions were prepared: one containing 1.0 mg / mL of free triiodothyronine capture antibody (FT3-coated antigen T3-BSA 01) and the other containing 1.0 mg / mL of chicken IgY (purchased from Guangzhou Nuclear Source Biotechnology Co., Ltd., catalog number 20220506). Then, the solutions were prepared at a concentration of 1.0 μL / cm³. 2 The coating amount is determined by marking the coating onto the T-line and C-line areas of the NC film using a gold marking machine. The NC film is then placed in an electric heating drying oven at 37±2℃ and humidity ≤30% for 16–24 hours to dry.

[0083] The base solution (i.e., coating solution) of the above-mentioned coating working solution is 0.01M PBS buffer (pH 7.4) containing 1% sucrose.

[0084] (4) Assembly

[0085] On a PVC adhesive base, the sample pad, conjugate pad, NC membrane, and absorbent paper (pure cotton pulp filter paper) obtained above are overlapped in sequence, with the NC membrane at the bottom. Each overlap is about 2mm. After assembly, the reagent card is obtained and ready for use.

[0086] Examples 2-9:

[0087] Examples 2-9 of this invention provide a fluorescent immunochromatographic assay reagent card for free triiodothyronine (FT3) and its preparation method, which differ from Example 1 in that the fluorescent microsphere-labeled FT3 monoclonal antibody and the free triiodothyronine capture antibody (i.e., FT3-coated antigen T3-BSA01) are different, while the other conditions are the same. Details are as follows:

[0088] Table 1:

[0089]

[0090] Among them, FT3 monoclonal antibody 01, FT3 monoclonal antibody 02 and FT3 monoclonal antibody 03 were purchased from Guangzhou Nuclear Source Biotechnology Co., Ltd., with product numbers T3-01, T3-02 and T3-03 respectively (T3 has different iodinated tyrosine residue recognition epitopes);

[0091] T3-BSA 01, T3-BSA 02 and T3-BSA 03 were purchased from Guangzhou Nuclear Source Biotechnology Co., Ltd., with product numbers T3-BSA01, T3-BSA02 and T3-BSA03 respectively (T3 has different iodinated tyrosine residue recognition epitopes).

[0092] Detection Example 1:

[0093] This test example detects the T / C, T / C coefficient of variation (CV), and linear gradient value of the fluorescence immunochromatographic assay reagent cards prepared in Examples 1-9 above. The specific method is as follows:

[0094] 1) Turn on the instrument (dry fluorescence immunoassay analyzer) and insert the chip with the same batch number as the reagent;

[0095] 2) Open the aluminum foil bag, take out the test card, and place it horizontally on the table;

[0096] 3) Use a pipette to draw 100 μL of the premixed system 1-30 and add it to the sample well of the test card;

[0097] 4) Select the sample type "plasma or whole blood" on the instrument.

[0098] 5) Testing:

[0099] Instant Test: After reacting for 10 minutes at an ambient temperature of 18-30℃, place the test card into the instrument slot, select the "Instant Test" mode, click "Test", and the instrument will automatically test and display the results;

[0100] Standard test: Insert the test card into the instrument's card slot, select the "Standard Test" mode, click "Test", the instrument will automatically start timing, and after the timing is over, the instrument will automatically test and display the results.

[0101] 6) Read / print the results, i.e., read / print the results from the display screen of the dry fluorescence immunoassay analyzer.

[0102] The reference standard for this test was free triiodothyronine, purchased from Guangzhou Nuclear Source Biotechnology Co., Ltd.

[0103] The relevant performance test results of Examples 1 to 9 above are shown in Table 2. Figure 2 and Figure 3 As shown:

[0104] Table 2:

[0105]

[0106] The test results show that the test reagent card of Example 4 has a stronger linear interval signal value, higher sensitivity, and the best effect. Specifically, the capture antibody coated on the detection line (i.e., the T line) is preferably the FT3-labeled monoclonal antibody 01 from Guangzhou Nuclear Source Biotechnology Co., Ltd.; the FT3-labeled antibody coated on the binding pad is preferably the FT3-coated antigen T3-BSA02. The combination of these antibodies has the best linear gradient and better T / C precision.

[0107] II. The impact of control line-related antibodies on detection performance

[0108] This section examines the impact of the type of goat anti-chicken IgY labeled antibody coated on the binding pad and the type of chicken IgY coated on the C line on the detection performance of the reagent card. The following is an explanation with reference to specific examples.

[0109] Example 10:

[0110] This embodiment provides a reagent card for the fluorescence immunochromatographic assay of free triiodothyronine (FT3) and its preparation method, which differs from Example 4 in that:

[0111] The goat anti-chicken IgY antibody on the binding pad was replaced with goat anti-mouse IgG antibody (purchased from Shanghai Lingchao Biotechnology Co., Ltd., catalog number B2448), and the chicken IgY on the quality control line was replaced with mouse IgG (purchased from Shanghai Lingchao Biotechnology Co., Ltd., catalog number B2721). All other preparation conditions remained the same.

[0112] Detection Example 2:

[0113] Referring to the method described in Example 1 above, fluorescent immunochromatographic assay reagent cards prepared in Examples 4 and 10 were randomly selected, and their T / C ratio, T / C coefficient of variation (CV), and linear gradient value were measured. The results are as follows:

[0114] Table 3:

[0115]

[0116] The results showed that, compared with the combination of mouse IgG antibody (secondary antibody) and goat anti-mouse IgG, the chicken IgY and goat anti-chicken IgY antibody of Example 10 had better detection performance.

[0117] III. The Influence of Fluorescent Nanospheres on Detection Performance

[0118] This section investigates the impact of fluorescent microspheres on the detection performance of reagent cards, which will be explained below with reference to specific examples.

[0119] Examples 11-19:

[0120] Examples 11-19 of this invention provide a free triiodothyronine (FT3) fluorescence immunochromatographic assay reagent card and its preparation method, which differ from Example 4 in that: the particle size ratio of the fluorescent microspheres is different, the source of purchase is different (the core luminescent material and process are different), and the proportion of different particle size mixing systems is different; all other aspects are the same. Details are as follows:

[0121] Table 4:

[0122]

[0123]

[0124] Detection Example 3:

[0125] The T / C, T / C coefficient of variation (CV), and linear gradient value of the fluorescence immunochromatographic assay reagent cards prepared in Examples 12-21 were detected using the method described in Example 1 above. The results are as follows:

[0126] Table 5:

[0127]

[0128]

[0129] The test results showed that the reagent card prepared by the method of Example 4 had the best linear gradient and the T / C precision was <5%. Therefore, Nanjing Microtest Biotechnology's Eu-fluorescent nanospheres of 200nm and 300nm were selected as fluorescent nanospheres for labeling.

[0130] IV. The Influence of Label Preservative Solution on Detection Performance

[0131] This section examines the impact of the labeling and preservation solution on the detection performance of the reagent card, which is illustrated below with specific examples.

[0132] Examples 20-22:

[0133] Examples 20-22 of this invention provide a reagent card for the fluorescence immunochromatographic assay of free triiodothyronine (FT3) and its preparation method. The difference between this reagent card and Example 4 is that the labeling and preservation solution is different; otherwise, they are the same. Details are as follows:

[0134] Table 6:

[0135]

[0136] Detection Example 4:

[0137] The T / C coefficient of variation (CV) of the fluorescent immunochromatographic assay reagent cards prepared in Examples 20-22 was detected using the method described in Example 1 above. The results are as follows:

[0138] Table 7:

[0139] Reference concentration (pmol / L) 50 25 15 5 1 Example 20 (T / CCV) 5.31％ 6.44％ 6.33％ 4.09％ 7.32％ Example 21 (T / CCV) 5.67％ 5.29％ 6.82％ 9.38％ 4.96％ Example 22 (T / CCV) 7.13％ 4.01％ 8.17％ 6.99％ 8.16％ Example 4 (T / CCV) 4.58％ 5.78％ 4.52％ 4.44％ 3.46％

[0140] The test results showed that in the labeling and preservation solution formulation system of the present invention, Tris buffer was more stable than PBS buffer, pH 8.0 was more conducive to the release of fluorescent microspheres, and the T / C precision was <6%. 2g bovine serum albumin, 2g sucrose, 0.5g sodium caseinate, 5g trehalose, 0.1mL Proclin 3000, and the remainder of 0.02M pH 8.0 Tris buffer (total volume 100mL) were selected as the labeling and preservation solution formulation for the FT3 assay reagent card of the present invention.

[0141] V. The Influence of Coating Solution on Detection Performance

[0142] This section examines the effect of the coating solution used in the preparation of nitrocellulose membranes on the detection performance of reagent cards. The following is an explanation with reference to specific examples.

[0143] Examples 23-25:

[0144] Examples 23-25 ​​of this invention provide a reagent card for the fluorescence immunochromatographic assay of free triiodothyronine (FT3) and its preparation method. The difference between this method and Example 4 is that the coating solution is different, while the other conditions are the same, as detailed below:

[0145] Table 8:

[0146] Sucrose (g) buffer solution Example 4 Wrapped 1 1.00 Dissolve in 100 mL of 0.01 M pH 7.4 PBS buffer Example 23 Wrapped 2 1.00 Dissolve in 100 mL of 0.01 M pH 8.0 PBS buffer Example 24 Wrapped 3 1.00 Dissolve in 100 mL of 0.02 M pH 7.4 PBS buffer Example 25 Wrapped 4 1.00 Dissolve in 100 mL of 0.02 M pH 8.0 PBS buffer

[0147] Detection Example 5:

[0148] The T / C, T / C coefficient of variation (CV), and linear gradient value of the fluorescence immunochromatographic assay reagent cards prepared in Examples 23-25 ​​were detected using the method described in Example 1 above. The results are as follows:

[0149] Table 9:

[0150]

[0151] The test results showed that Example 4 exhibited the highest linear gradient and lowest T / C precision, resulting in the best performance. The ionic strength of the coating solution had virtually no impact on the test results. Taking all factors into consideration, 0.01M PBS containing 1% sucrose at pH 7.4 was selected as the final formulation of the coating solution.

[0152] VI. The effect of sample pad treatment solution on detection performance

[0153] This section examines the impact of sample processing solution on the detection performance of reagent cards, which will be described below with reference to specific examples.

[0154] Examples 26-28:

[0155] Examples 26-28 of this invention provide a free triiodothyronine (FT3) fluorescence immunochromatographic assay reagent card and its preparation method, which differ from Example 4 in that the sample pad treatment solution formulation is different in the sample pad preparation process; all other aspects are the same. Details are as follows:

[0156] Table 10:

[0157]

[0158] Detection Example 6:

[0159] The T / C, T / C coefficient of variation (CV), and linear gradient value of the fluorescent immunochromatographic assay kits prepared in Examples 26-28 were detected using the method described in Example 1 above. Specifically, the fluorescent immunochromatographic assay kits prepared in Examples 26-28 were tested simultaneously with kits without added anti-human RBC antibodies and blocking agent 002. Evaluation was performed using healthy human samples with added interfering agents to ensure no false positives and no membrane climbing of whole blood samples. The detection results of the reference samples with added interfering agents using different sample pad treatment solutions are as follows:

[0160] Table 11:

[0161]

[0162] The test results showed that both 1 mg and 2 mg of the blocking agent 002 effectively blocked red blood cells. The T / C of the 2 mg blocking agent 002 test showed no significant difference between the reference standard with and without the interfering agent, indicating strong anti-interference ability. The sample pad treatment solutions of Examples 27 and 28 met the requirements. Considering the cost factors, the sample pad treatment solution of Example 4 was finally selected as the sample pad treatment solution formula (1 mg anti-human RBC antibody, 2 mg blocking agent 002, 1 g bovine serum albumin, 0.5 mL Tween-20, 0.2 M pH 8.0 borate and borax buffer, adjusted to 100 mL).

[0163] VII. The effect of the proportion of labeled antibodies on detection performance

[0164] This section examines the impact of the labeled antibody ratio on the detection performance of the reagent card, which will be explained below with reference to specific examples.

[0165] Examples 29-36:

[0166] Examples 29-36 of this invention provide a free triiodothyronine (FT3) fluorescence immunochromatographic assay reagent card and its preparation method, respectively. The difference between these and Example 4 lies in the amount of FT3-labeled antibody and goat anti-chicken IgY-labeled antibody added during the preparation of the binding pad; all other aspects are the same. Details are as follows:

[0167] Table 12:

[0168] FT3-labeled antibody (μg) Goat anti-chicken IgY labeled antibody (μg) Example 29 5 8 Example 30 5 16 Example 31 5 32 Example 32 10 8 Example 4 10 16 Example 33 10 32 Example 34 20 8 Example 35 20 16 Example 36 20 32

[0169] Detection Example 7:

[0170] The T / C coefficient of variation (CV) of the fluorescent immunochromatographic assay reagent cards prepared in Examples 29-36 was detected using the method described in Example 1 above. The T / C precision results of the reagent cards prepared with different T-line and C-line labeled antibody feed ratios are as follows:

[0171] Table 13:

[0172] T / CCV 50 pmol / L 25 pmol / L 15 pmol / L 5 pmol / L 1 pmol / L Example 29 9.06％ 8.36％ 5.87％ 6.89％ 6.80％ Example 30 4.04％ 4.97％ 5.64％ 6.18％ 7.47％ Example 31 6.01％ 6.26％ 8.98％ 8.64％ 7.88％ Example 32 8.29％ 7.25％ 9.42％ 9.32％ 6.72％ Example 4 5.37％ 3.90％ 4.70％ 5.07％ 3.26％ Example 33 6.76％ 6.00％ 5.46％ 6.14％ 7.59％ Example 34 5.97％ 9.12％ 5.60％ 5.69％ 5.03％ Example 35 5.53％ 8.90％ 8.96％ 6.50％ 7.61％ Example 36 7.36％ 5.44％ 5.83％ 7.51％ 5.70％

[0173] The test results showed that the concentration gradient ratio of each internal reference in Example 4 was the best, with high sensitivity, good linearity, and precision of <6%. Therefore, a combination of 10 μg of FT3-labeled antibody and 16 μg of goat anti-chicken IgY-labeled antibody was selected for feeding.

[0174] 8. Effect of the amount of antibody solution sprayed onto the conjugate pad on detection performance

[0175] This section examines the effect of the amount of antibody solution sprayed onto the binding pad on the detection performance of the reagent card, which is illustrated below with specific examples.

[0176] Examples 37-38:

[0177] Examples 37 and 38 of this invention provide a fluorescent immunochromatographic assay reagent card for free triiodothyronine (FT3) and its preparation method, which differ from Example 4 in that the amount of antibody solution sprayed onto the binding pad is different, while the rest are the same. Details are as follows:

[0178] Table 16:

[0179]

[0180]

[0181] Example 9:

[0182] The T / C coefficient of variation (CV) of the fluorescent immunochromatographic assay reagent cards prepared in Examples 37-38 was detected using the method described in Example 1 above. The T / C precision test results of the reagent cards under different amounts of labeled antibody solution sprayed are as follows:

[0183] Table 17:

[0184]

[0185] The test results showed that the T / C precision of the reagent card at a spray volume of 4 uL / cm was <6%, which was relatively optimal. There was no significant difference in the linear gradient of each spray volume. Considering both cost and performance, 4 uL / cm was selected as the spray volume of the labeled antibody solution.

[0186] IX. The Influence of Reaction Time on Detection Performance

[0187] This section examines the effect of reaction time on the detection performance of the reagent card, which is illustrated below with specific examples.

[0188] Examples 39-41:

[0189] Examples 39-41 of this invention provide a fluorescent immunochromatographic assay reagent card for free triiodothyronine (FT3) and its preparation method, which differ from Example 4 in that the reaction time is different, but all other aspects are the same. Details are as follows:

[0190] Table 18:

[0191] time Example 39 5min Example 4 10min Example 40 15min Example 41 20min

[0192] Example 10:

[0193] The coefficient of variation (CV) of the T / C values ​​of the fluorescent immunochromatographic assay reagent cards prepared in Examples 39-41 was detected using the method described in Example 1 above. The results of the T / C precision detection at different reaction times are as follows: Figure 4 As shown in Table 19:

[0194] Table 19:

[0195]

[0196] The test results showed that the reaction was completed in 10 minutes, the T / C ratio stabilized earlier, and the precision was all <6%. Therefore, 10 minutes was selected as the reaction time.

[0197] The following test examples, using Example 4 as an example, further tested its relevant performance, as detailed below:

[0198] Detection Example 11: Linear Establishment

[0199] Three batches of reagent cards prepared using the method in Example 4 (11 samples per batch) were randomly selected for testing. Each sample was tested four times. The relative deviation (B) and linear correlation coefficient (r) between the average value (M) of the four test results for each sample and the theoretical value were calculated according to the following formulas (1) and (2).

[0200] B = (MT) / T × 100% (1);

[0201] In the formula: B is the relative deviation; M is the average value of the test results; T is the theoretical value.

[0202]

[0203] In the formula: n is the number of samples measured; X i —Determine the concentration of the sample; Y i is the mean of the measured values ​​corresponding to the concentration of the measured sample from four repeated measurements; r is the linear correlation coefficient; i is 1, 2, 3, ..., n.

[0204] The test results are shown in Table 20:

[0205] Table 20: Linearity Validation

[0206]

[0207] As can be seen from the table, the establishment and verification of linearity meet the above design requirements. Therefore, the correlation coefficient r ≥ 0.9900 of the linear regression in the range of 1 to 50 pmol / L is taken as the linear performance index of this product.

[0208] Test Example 12: Accuracy Performance Evaluation

[0209] Three batches of free triiodothyronine (FT3) assay kits (fluorescence immunoassay chromatography) stably produced under the quality management system were selected for accuracy performance evaluation. Statistical analysis was performed on the test results. If the results met the acceptable criteria of the designed experiment, they were used as the accuracy performance indicators of the FT3 kits.

[0210] Determine the enterprise reference products of free triiodothyronine at 5 pmol / L, 15 pmol / L, and 50 pmol / L, and perform the detection according to the steps in the instruction manual. After each measurement is repeated 3 times, the test result of each time is recorded as (Xi). If the relative deviation of all 3 results does not exceed ±15%, it is judged as qualified. If the results of 2 or more times do not meet the requirements, it is judged as unqualified. If the result of 1 time does not meet the requirements, 20 consecutive tests should be performed again, and the relative deviation should be calculated according to formula (3). If the relative deviation of the results of 19 or more tests does not exceed ±15%, it is judged as qualified.

[0211] Bi = (Xi - T) / T × 100% (3);

[0212] In the formula: Bi is the relative deviation; Xi is the concentration measured each time; T is the labeled concentration.

[0213] The test results are shown in Table 21: <000​​​​​​​​​​​​​​​​​​Three batches of this kit that passed quality inspection were taken and tested by the same operator on the same instrument at concentrations of 5 pmol / L, 15 pmol / L, and 50 pmol / L using the FT3 enterprise reference standard. Each sample was measured twice, for a total of 20 days. At the end of the evaluation, there were 60 pairs, or 120 test results. Intra-analytical precision was calculated from 40 data points in each batch. Inter-batch precision was calculated from all 120 data points. The inter-batch coefficient of variation was calculated from all 120 data points. The test results are shown below.

[0221] Table 22: Record of raw data from precision experiments within low-value samples

[0222]

[0223]

[0224] Table 23: Record of raw data from the precision experiment within the median sample

[0225]

[0226]

[0227] Table 24: Record of raw data from precision experiments within high-value samples

[0228]

[0229]

[0230] Table 25: Calculation of raw data for intra-batch precision experiments on low-value samples

[0231]

[0232] Table 26: Calculation of raw data for intra-batch precision experiments of high-value samples

[0233]

[0234]

[0235] Table 27: Calculation of raw data for intra-batch precision experiments of high-value samples

[0236]

[0237]

[0238] The above experimental data shows that the intra-batch coefficient of variation (CV) for the three batches of FT3 reagent should be ≤15%. The inter-batch coefficient of variation (CV) should be ≤15%.

[0239] Referring to the series of guidelines for performance evaluation of in vitro diagnostic reagents, the formula for inter-batch precision is:

[0240] In the formula, n = the total number of tests.

[0241] According to the formula: Find the mean.

[0242] From Tables 20 and 21 above, we can conclude that:

[0243] Determine the inter-batch precision:

[0244]

[0245] From Tables 20 and 21 above, we can obtain...

[0246]

[0247] In the formula, n = the total number of tests.

[0248] Note:

[0249] (1) represents the sum of the results of the first batch of reagents measured over 20 days;

[0250] (2) represents the sum of the results of the first batch of reagents measured over 20 days;

[0251] (3) indicates the sum of the results of the second batch of reagents measured over 20 days;

[0252] (4) indicates the sum of the results of the second batch of reagents measured over 20 days;

[0253] (5) indicates the sum of the results of the third batch of reagents measured over 20 days;

[0254] (6) indicates the sum of the results of the third batch of reagents measured over 20 days;

[0255] (7) represents the sum of the squares of (mean - result 1) measured in the first batch of reagents over 20 days;

[0256] (8) represents the sum of the squares of (mean - result 2) measured in the first batch of reagents over 20 days;

[0257] (9) represents the sum of the squares of (mean - result 1) measured over 20 days in the second batch of reagents;

[0258] (10) represents the sum of the squares of (mean - result 2) measured over 20 days in the second batch of reagents;

[0259] (11) represents the sum of the squares of (mean - result 1) measured over 20 days in the third batch of reagents;

[0260] (12) means: the sum of the squares of (mean - result 2) measured in the third batch of reagents over 20 days.

[0261] The results of the intra-batch and inter-batch indicators, as summarized in Table 28, are obtained from the above data analysis.

[0262] Table 28: Results of Intra-batch and Inter-batch Indicators

[0263]

[0264] Under repeatability conditions, this kit was used to test two samples at different concentration levels: a low concentration level of 5 pmol / L, a medium concentration level of 15 pmol / L, and a high concentration level of 50 pmol / L. Each test was repeated 10 times (n=10). As shown in Table 29, the intra-batch coefficient of variation (CV) of the tested samples was in the range of 4.21% to 7.27%.

[0265] Table 29: Repeatability Test Data Results

[0266]

[0267]

[0268] The samples with concentrations of 5 pmol / L, 15 pmol / L, and 50 pmol / L were tested using three different batches of this kit, as shown in Table 30. The inter-batch coefficients of variation (CV) were 5.75% and 6.25%, respectively.

[0269] Table 30 Results of inter-batch precision testing data

[0270]

[0271]

[0272] The test results above show that the precision meets the above design requirements. Therefore, the intra-batch coefficient of variation (CV) should be ≤15% and the inter-batch coefficient of variation (CV) should be ≤20% as the precision performance indicators of this product.

[0273] Example 14: Assessment of the HAMA effect

[0274] The prepared concentrations were tested using reagent cards, and the test data were statistically analyzed. The results met the acceptable criteria for the experimental design and were thus used as the HAMA effect performance index for FT3. The test results are shown in Table 31.

[0275] Table 31: Results of HAMA effect test

[0276]

[0277]

[0278] In summary, this invention provides a fluorescent immunochromatographic assay kit for free triiodothyronine (TTI), which uses a TTI capture antibody and chicken IgY as the detection line and control line, respectively, and a mixture of TTI antibody and goat anti-chicken IgY as the fluorescent microsphere labeling antibody. Compared with commonly used methods for detecting TTI, such as enzyme-linked immunosorbent assay (ELISA) / immunoturbidimetric assay, this kit significantly shortens the detection time, reduces detection costs, and improves detection sensitivity. Compared with similar methods, it has advantages such as simple labeling process, high sensitivity, accurate and stable results.

[0279] Furthermore, the present invention provides a method for preparing a free triiodothyronine fluorescence immunochromatographic assay kit. By selecting appropriate raw materials, rationally designing the dosage of each material, and selecting the optimal reaction conditions, the preparation process is simple, the product labeling process is simple, the sensitivity is high, the results are accurate and stable, the detection cost is low, and it is suitable for widespread application.

[0280] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A reagent card for the fluorescence immunochromatographic assay of free triiodothyronine, characterized in that, The reagent card includes a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper sequentially placed on the base plate, wherein: The binding pad is coated with fluorescent microsphere-labeled FT3 monoclonal antibody and fluorescent microsphere-labeled goat anti-chicken IgY antibody, and the mass ratio of the fluorescent microsphere-labeled FT3 monoclonal antibody to the fluorescent microsphere-labeled goat anti-chicken IgY antibody is 10:15-35. The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with FT3 capture antibody, and the control line is coated with chicken IgY.

2. The reagent card according to claim 1, characterized in that, The sample pad is prepared by soaking it in a sample pad treatment solution, wherein the sample pad treatment solution contains anti-human RBC antibody, blocking agent, bovine serum albumin, Tween-20 and borate-borax buffer.

3. The reagent card according to claim 1, characterized in that, The conjugate pad is obtained by spraying a fluorescent microsphere-labeled antibody solution; wherein the fluorescent microsphere-labeled antibody solution contains the fluorescent microsphere-labeled FT3 monoclonal antibody, the fluorescent microsphere-labeled goat anti-chicken IgY antibody, and a labeling preservation solution; Preferably, the labeling preservation solution comprises bovine serum albumin, sucrose, sodium caseinate, alginate, Proclin 300, and Tris buffer.

4. The reagent card according to claim 1, characterized in that, The raw materials for preparing the fluorescent microsphere-labeled FT3 monoclonal antibody include fluorescent microspheres and FT3 monoclonal antibody; And / or, the raw materials for preparing the fluorescent microsphere-labeled goat anti-chicken IgY antibody include fluorescent microspheres and goat anti-chicken IgY antibody.

5. The reagent card according to claim 4, characterized in that, The ratio of the FT3 monoclonal antibody to the goat anti-chicken IgY antibody is 5-20:8-32; Preferably, the fluorescent microspheres have a particle size of 150–350 nm; Preferably, the fluorescent microspheres include a first fluorescent microsphere and a second fluorescent microsphere; wherein the particle size of the first fluorescent microsphere is 180-220 nm; and the particle size of the second fluorescent microsphere is 280-320 nm. Preferably, the mass ratio of the first fluorescent microsphere to the second fluorescent microsphere is 1.5 to 2.5:

1.

6. The reagent card according to claim 1, characterized in that, The coating concentration of the FT3 capture antibody on the detection line is 0.8–1.0 mg / mL; And / or, the coating concentration of chicken IgY on the quality control line is 1.0 to 1.5 mg / mL.

7. The method for preparing the reagent card according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Spray the antibody solution labeled with the fluorescent microspheres onto a glass cellulose membrane, and dry it to obtain a conjugate pad; S2. Spray the first coating working solution containing the FT3 capture antibody and the second coating working solution containing the chicken IgY onto the detection line and the quality control line respectively, and dry them to obtain a nitrocellulose membrane. S3. Assemble the nitrocellulose membrane, the sample pad, the conjugation pad, and the absorbent paper sequentially on the base plate to obtain the final product.

8. The preparation method according to claim 7, characterized in that, In step S1, the amount of the fluorescent microsphere-labeled antibody solution sprayed is 3-5 μL / cm. 2 ; And / or, in step S2, the spraying amount of the first coating working fluid and the second coating working fluid is independently 1-1.5 μL / cm. 2 .

9. A kit for the fluorescence immunochromatographic assay of free triiodothyronine, characterized in that, Includes the reagent card and diluent as described in any one of claims 1 to 6.

10. The use of the reagent card according to any one of claims 1 to 6 or the free triiodothyronine fluorescence immunochromatographic assay kit according to claim 9 in the detection of free triiodothyronine.

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