Anti-tetraiodothyronine antibody and application thereof in immunochromatography detection

Abstract

The invention belongs to the technical field of biological medicine and in-vitro diagnosis, and particularly relates to an anti-tetraiodothyronine (T4) antibody and application thereof in immunochromatography detection. The invention provides a first antibody (2C1) specifically binding to T4 and a second antibody (125C5) specifically recognizing a compound of T4 and the first antibody. By using the antibody pair, the invention establishes a double-antibody sandwich fluorescence immunochromatography detection method aiming at the small molecule antigen T4. The method overcomes the defects of low sensitivity and poor specificity of a traditional competition method, and avoids the limitation that a chemiluminescence method depends on large-scale equipment. Experiments show that the kit provided by the invention is high in detection sensitivity and good in specificity, the correlation with a clinical mainstream Rogowski reagent reaches 0.985, the detection range covers 1.0-100pmol / L, and the kit is very suitable for primary medical treatment and bedside rapid detection (POCT).

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and immunoassay, specifically relating to anti-tetraiodothyronine antibody and its application in immunochromatographic assay reagents, and particularly to a T4 detection scheme based on a small molecule double antibody sandwich method. Background Technology

[0002] Thyroxine (T4) is a tyrosine derivative containing four iodine atoms, synthesized by thyroid follicular cells, and is the main storage form of circulating thyroid hormones. Serum T4 levels directly reflect thyroid function. In peripheral tissues, T4 is converted by deiodinases to form the active triiodothyronine (T3).

[0003] Currently, most immunoassay methods for detecting total T4 (TT4) and free T4 (FT4) in serum are based on competitive assays. Due to the limitations of the competitive assay principle, they suffer from low specificity and insufficient sensitivity, especially in the low concentration range of FT4 within the picomolar range, which presents a significant technical challenge. Current mainstream high-sensitivity detection methods are mainly chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA), but these methods rely on large-scale automated equipment, are complex to operate, and are difficult to meet the needs of primary healthcare institutions and point-of-care testing (POCT).

[0004] Fluorescence immunochromatography (FICA) combines signal amplification techniques (such as quantum dots and rare-earth fluorescent microspheres), offering advantages such as speed (<15 minutes), small sample volume, and strong anti-interference capabilities. However, traditional small-molecule competitive methods often struggle to achieve the ultra-high sensitivity required for clinical diagnosis on FICA platforms. Therefore, developing a reagent that can overcome the limitations of small-molecule detection and utilize the double-antibody sandwich method to detect T4 has significant clinical application value. Summary of the Invention

[0005] To address the shortcomings of the prior art, this invention provides an anti-tetraiodothyronine antibody and its application in immunochromatographic detection, enabling double-antibody sandwich detection of the small molecule antigen T4.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a first antibody (2C1) against tetraiodothyronine (T4) or its antigen-binding fragment, wherein the antibody comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises HCDR1 with the amino acid sequence shown in SEQ ID NO:1, HCDR2 with the amino acid sequence shown in SEQ ID NO:2, and HCDR3 with the amino acid sequence shown in SEQ ID NO:3; and the light chain variable region comprises LCDR1 with the amino acid sequence shown in SEQ ID NO:6, LCDR2 with the amino acid sequence shown in SEQ ID NO:7, and LCDR3 with the amino acid sequence shown in SEQ ID NO:8.

[0007] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:9.

[0008] This invention provides a second antibody (125C5) or its antigen-binding fragment that specifically binds to a complex formed by tetraiodothyronine and a first antibody, wherein the first antibody is the aforementioned antibody; the second antibody comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:12, and HCDR3 as shown in SEQ ID NO:13; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18. This second antibody can specifically bind to the complex formed by T4 and the first antibody (2C1) and does not recognize the first antibody alone.

[0009] In some embodiments, the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID NO:19.

[0010] The present invention provides an isolated nucleic acid molecule that encodes the aforementioned antibody or its antigen-binding fragment.

[0011] The present invention provides an expression vector comprising the above-described nucleic acid molecule.

[0012] The present invention provides a host cell comprising the above-described expression vector.

[0013] This invention provides a kit for detecting tetraiodothyronine (T4), comprising: (1) the first antibody described above; and (2) the second antibody described above. The kit utilizes the first antibody (2C1) to recognize and bind to small molecule T4, and the second antibody (125C5) to recognize and bind to the "T4-2C1 complex," thereby constructing a "sandwich" detection mode for small molecule T4.

[0014] In some embodiments, the kit is a fluorescent immunochromatographic kit; the first antibody is labeled with fluorescent microspheres and coated on the conjugate pad; the second antibody is coated on the detection line (T line) of the chromatographic membrane.

[0015] This invention provides the use of the above-described antibody in the preparation of a reagent for detecting tetraiodothyronine.

[0016] The present invention has the following beneficial effects: In this invention, the first antibody binds to T4 to form an immune complex, and the second antibody specifically binds to this immune complex, thereby achieving small molecule sandwich detection. This invention combines the small molecule double antibody sandwich method with fluorescence immunochromatography, breaking through the traditional technical bottleneck that small molecules can only be detected by competitive methods, and realizing an innovative detection mode.

[0017] The first antibody 2C1 of this invention has a high affinity for T4 (0.237 nM), and the second antibody 125C5 can specifically recognize immune complexes, effectively improving the signal-to-noise ratio and sensitivity of the detection.

[0018] The POCT kit prepared by the antibody of this invention has a detection time of less than 15 minutes, requires only 10 μL of sample, and has a high correlation of 0.985 with Roche chemiluminescence reagent. It is convenient to use and suitable for promotion in primary healthcare. Attached Figure Description

[0019] Figure 1 Affinity test spectrum of the first antibody 2C1 and the T4 antigen.

[0020] Figure 2 The ELISA results of the specific recognition of the "2C1+ small molecule complex" by the second antibody 125C5 are shown.

[0021] Figure 3 The conformity curve of the detection results of the T4 reagent card and Roche reagent of this invention. Detailed Implementation

[0022] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0023] Example 1: Preparation of the first antibody (2C1) Antibody acquisition: Balb / c mice were immunized with the T4-BSA antigen, and the hybridoma cells obtained were screened. The screening criteria were: cell lines that secreted high-affinity T4 antibodies and excluded those that cross-reacted with the carrier protein BSA.

[0024] Gene sequence acquisition: RNA was extracted from the selected hybridoma cells, reverse transcribed to obtain cDNA, and then cloned and sequenced.

[0025] Sequence analysis: The sequence of the 2C1 antibody obtained from the analysis is as follows: The heavy chain CDR1-3 sequences are: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3.

[0026] The light chain CDR1-3 sequences are: SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8.

[0027] The amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO:4.

[0028] The amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO:9.

[0029] Example 2: Affinity determination of 2C1 antibody The assay was performed using the GatorPrime system. The 2C1 antibody was diluted to 5 μg / mL with QBuffer (Gatorbio, Catalog No. 20-5203) and fixed using MouseFc (MFC) Probes (Gatorbio, Catalog No. 160004). T4-BSA antigen was serially diluted from 80 nM. The test results are shown in Table 1 and... Figure 1 As shown.

[0030] Table 1. Results of T4-2C1 antibody affinity test

[0031] From Table 1 and Figure 1 It can be seen that the affinity of the 2C1 antibody for the T4 antigen reaches 0.237 nM.

[0032] Example 3: Preparation of the second antibody (125C5) Immunogen preparation: Natural T4 small molecules and recombinant 2C1 antibody were mixed in PBS buffer (137 mmol / L NaCl, 2.7 mmol / L KCl, 4.3 mmol / L Na2HPO4, 1.4 mmol / L KH2PO4) at a molar ratio of 30:1, stirred at 4°C for 12 h, and dialyzed for 48 h to prepare immune complexes.

[0033] Antibody acquisition: Mice were immunized with the above-described immunogen, and the hybridoma cells obtained after immunization were then screened. The screening criteria were: specific binding to the "2C1-T4 complex" and no recognition of the 2C1 antibody alone.

[0034] The specific steps for obtaining antibodies are as follows: Balb / c mice were immunized with the above immunogen, and single spleen cells were isolated from the spleens of mice that had been immunized three times. These cells were then fused with mouse myeloma cells, plated into 96-well plates, and cultured at 37°C for one week. The culture supernatant was then used for ELISA testing.

[0035] Using 2C1-scFv as the coating antibody (10 μg / mL concentration), 50 μL of PBS diluted with 10 μg / mL T4 molecule was added to the test wells, and 50 μL of PBS was added to the control wells. After incubation for 30 min, 50 μL of cell supernatant was added to both the test and control wells, while PBS was used instead of cell supernatant in the blank wells. After incubation for 1 h, goat anti-mouse IgG-HPR was added for color development. Cell wells with color development values ​​close to those of the control wells but showing strong positivity in the test wells were selected for subcloning until strongly positive monoclonal cell lines meeting this standard were obtained (results are shown in Table 2).

[0036] Table 2. Screening results of cell supernatant for anti-2C1+ small molecule complex antibodies

[0037] As shown in Table 2, the 125C5 antibody meets the screening criteria.

[0038] Sequence analysis: RNA was extracted from the selected hybridoma cells, and cDNA was obtained by reverse transcription using the RNA as a template. This cDNA was then cloned into a vector and sequenced. Analysis of the variable regions of the antibody's light and heavy chains was performed: Sequence analysis was conducted on the VH, VL, and constant regions based on the sequencing results. The sequence of the 125C5 antibody was obtained as follows: The heavy chain CDR1-3 sequences are: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13.

[0039] The light chain CDR1-3 sequences are: SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18.

[0040] The amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO:14.

[0041] The amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO:19.

[0042] Example 4: ELISA detection of the recognition effect of the second antibody 125C5 Coating: The Fab fragment of the recombinant 2C1 antibody was coated with coating buffer (10mM phosphate buffer, pH 7.4, pH 9.4) at a concentration of 100 ng / well and incubated overnight at 2–8°C.

[0043] Blocking: Remove the blocking buffer and wash once with PBST (20 mM phosphate buffer containing 0.05% Tween). Then add 200 µL of blocking buffer (10% skim milk) to each well and incubate at 37°C for 2 hours. After blocking, remove the blocking buffer and set aside for later use.

[0044] Antibody incubation: The 125C5 assay was serially diluted 5-fold from 10 μg / mL using PBS, while the T4 molecule was diluted to 100 ng / mL. 50 μL each of the T4 molecule and the serially diluted 125C5 assay were added to the wells of the plate, with 50 μL of PBS added to the control group (no molecule). The plates were incubated at 37°C for 1 hour, followed by 5 washes.

[0045] Secondary antibody incubation: Add 100uL / well of 10000-fold diluted GoatAnti-MouseIgGFc(HRP) (catalog number ab97265, Abcom), incubate at 37℃ for 30 min, and wash 5 times after incubation.

[0046] Color development: Add 100 μL of freshly prepared substrate chromogenic solution (purchased from Sangon Biotech, catalog number D110098) to each well, incubate at 37°C for 30 min, then add 50 μL of 2 mol / L H2SO4 to terminate the reaction, and read the OD using an ELISA reader. 450 The absorbance values ​​are shown in Table 3 below. Figure 2 .

[0047] Table 3. Specific recognition of 2C1+ small molecule complexes by T4-125C5 antibody.

[0048] As shown in Table 2 and Figure 2 As shown, in the absence of T4, 125C5 does not bind to 2C1 in the solid phase; however, in the presence of T4, 125C5 specifically recognizes the complex formed by 2C1 and T4, and the signal increases with the increase of 125C5 concentration, demonstrating its unique immune complex recognition ability.

[0049] Example 5: Preparation of T4 fluorescence immunochromatographic assay kit 1. Preparation of fluorescently labeled binding pads: 1) Microsphere weighing: Take 20 μL of microspheres (label the fluorescent microspheres (Bangs Laboratories, Catalog No. FCEU003) and the first antibody 2C1 at a ratio of 0.2 mg microspheres: 0.02 mg 2C1 (mass ratio 10:1).

[0050] 2) Microsphere activation: Add 200 μL of 0.05 M MES and 20 μL of 1 mg / mL EDC to the microspheres, sonicate for 30 s, and shake at room temperature for 5 min in a constant temperature shaker; 3) Conjugation: Add 20ug of the antibody to be labeled 2C1 to the system, sonicate for 30s, and shake at room temperature in an insulated shaker for 15min; 4) Separation: Add 10 μL of 20% BSA to the system, sonicate for 30 seconds, and shake at room temperature in a constant temperature shaker for 10 minutes; 5) Centrifugation: After the reaction is complete, centrifuge at 15,000 rpm and 4°C for 15 min; 6) Resuspension: After centrifugation, carefully remove the supernatant, then add 100 μL of microsphere resuspension solution (50 mM Tris-HCl, 0.5% BSA, 0.2% Tween-20, 0.05% Proclin300) and resuspend to 5 times the volume of microspheres.

[0051] 7) Laying fiberglass pads: Then, dilute the 2C1-labeled fluorescent microspheres 50 times with microsphere resuspension solution and spray them onto a 10 mm wide fiberglass pad at a rate of 60 uL / cm. Then, place the sprayed labeling pads in a 37°C oven to dry for more than 2 hours. After drying, put them into an aluminum foil bag pre-filled with desiccant, seal and store for later use. 2. Detect the coating of the T-line and the quality control C-line: Dilute 125C5 antibody to 0.8 mg / mL using 10 mM PBS buffer as the detection T line; dilute T4-BSA antigen to 0.35 mg / mL as the control C line. Coat the detection T line and control C line onto the nitrocellulose membrane of the backing plate and bake in a 37°C oven for 24-36 hours. After baking, seal and store in an aluminum foil bag pre-filled with desiccant for later use. 3. Assembly and cutting of test kit cards At humidity levels below 25%, take absorbent paper and a blank fiberglass pad, and cut them into 30mm widths. Remove the prepared coated plate and conjugate pad, and arrange the components from top to bottom in the order of absorbent paper - nitrocellulose membrane - conjugate pad - blank fiberglass, with a 1-2mm overlap between each layer. Cut the assembled plate into 4mm widths, insert them into the cartridge case, and press them firmly to create a T4 fluorescence immunochromatographic assay card.

[0052] Example 6: Differences in the recognition of T4, T3, and rT3 small molecules using the T4 sandwich reagent card. Using T4, T3, and rT3 small molecules, serial dilutions were performed with PBS buffer starting at 500 nmol / L. The different recognition effects of the reagent card on the three small molecules were observed. It is evident that the reagent card exhibits a good gradient in the recognition of T4 small molecule, but shows no specific recognition for T3 small molecule. The reagent card only shows a certain gradient in the recognition of rT3 small molecule at concentrations far exceeding physiological concentrations, and the recognition level is much lower than that of T4 small molecule, so it will not interfere with the detection. Furthermore, TSH is a large molecule with significant structural differences from T3, T4, and rT3 small molecules; therefore, cross-contamination issues do not need to be considered.

[0053] Table 4 shows the differences in the recognition of T4, T3, and rT3 small molecules using the FT4 reagent card.

[0054] Example 7: Performance Testing of T4 Sandwich Reagent Cards The test was performed using Roche-assigned samples. 10 μL of sample was mixed with 90 μL of sample buffer (20 mM Tris-HCl, 0.3% casein, 0.2% Tween-20, 0.2% trehalose, 0.05% Proclin 300), and 80 μL of the mixture was added. The reaction was allowed to proceed for 15 minutes before detection. Results are shown in Table 3. Figure 3 As shown.

[0055] Table 5. FT4 reagent card test samples with Roche assignment values.

[0056] From Table 5 and Figure 3 It is known that the correlation (R²) between the reagent card of this invention and Roche reagent reaches 0.985, and the detection range covers 1.0-100 pmol / L, achieving highly sensitive quantitative detection.

[0057] The sequence of embodiments of the present invention is as follows: SEQ ID NO:1SSYAMS SEQ ID NO:2SISGSGGSTYYADSVKG SEQ ID NO:3DYGYYGMDV SEQ ID NO:4 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYGYYGMDVWGQGTTVTVSS SEQ ID NO:5 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYGYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:6RASQDISNYLN SEQ ID NO:7GASTLQS SEQ ID NO:8QQYNTSPPT SEQ ID NO:9 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYGASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTSPPTFGQGTKVEIK SEQ ID NO:10 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYGASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:11SDYAW SEQ ID NO:12YIYPSDSYTNYNPSLKS SEQ ID NO:13HGGFAY SEQ ID NO:14 QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQFPGNKLEWMGYIYPSDSYTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHGGFAYWGQGTLVTVSS SEQ ID NO:15 QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQFPGNKLEWMGYIYPSDSYTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHGGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:16 RASQSVSNDLA SEQ ID NO:17DASNRAT SEQ ID NO:18QQRSNWPLT SEQ ID NO:19 EIVLTQSPATLSLSPGERATLSCRASQSVSNDLAWYQQKPGQAPRLLIYDASNRATGVPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK SEQ ID NO:20 EIVLTQSPATLSLSPGERATLSCRASQSVSNDLAWYQQKPGQAPRLLIYDASNRATGVPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A first antibody or an antigen-binding fragment thereof against tetraiodothyronine (T4), characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises HCDR1 of the amino acid sequence as shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence as shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence as shown in SEQ ID NO: 3; and the light chain variable region comprises LCDR1 of the amino acid sequence as shown in SEQ ID NO: 6, LCDR2 of the amino acid sequence as shown in SEQ ID NO: 7, and LCDR3 of the amino acid sequence as shown in SEQ ID NO:

8.

2. The first antibody or antigen binding fragment thereof of claim 1, wherein, The heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO: 4, and the light chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO:

9.

3. A second antibody, or an antigen-binding fragment thereof, that specifically binds a complex of tetraiodothyronine and a first antibody, characterized in that, The first antibody is the antibody of claim 1 or 2; the second antibody comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises HCDR1 of the amino acid sequence as shown in SEQ ID NO: 11, HCDR2 of the amino acid sequence as shown in SEQ ID NO: 12, and HCDR3 of the amino acid sequence as shown in SEQ ID NO: 13; and the light chain variable region comprises LCDR1 of the amino acid sequence as shown in SEQ ID NO: 16, LCDR2 of the amino acid sequence as shown in SEQ ID NO: 17, and LCDR3 of the amino acid sequence as shown in SEQ ID NO:

18.

4. The second antibody or antigen-binding fragment thereof of claim 3, wherein, The heavy chain variable region amino acid sequence of the second antibody is as shown in SEQ ID NO: 14, and the light chain variable region amino acid sequence of the second antibody is as shown in SEQ ID NO:

19.

5. An isolated nucleic acid molecule, comprising, The nucleic acid molecule encodes the antibody or antigen binding fragment thereof of any one of claims 1-4.

6. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule of claim 5.

7. A host cell, characterized in that, The host cell comprises the expression vector of claim 6.

8. A kit for detecting tetraiodothyronine (T4), characterized by, The kit comprises: (1) the first antibody of claim 1 or 2; and (2) the second antibody of claim 3 or 4.

9. The kit of claim 8, wherein The kit is a fluorescent immunochromatographic kit; the first antibody is labeled with fluorescent microspheres and coated on a conjugate pad; and the second antibody is coated on a test line (T line) of a chromatographic membrane.

10. Use of the antibody of any one of claims 1-4 in the preparation of a reagent for detecting tetraiodothyronine.

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