A stable complete thyroid hormone antigen, its preparation and application

Abstract

This invention discloses a stability-improved complete thyroid hormone antigen, its preparation, and its application. The free amino group of the thyroid hormone or its deiodinated derivative moiety of this complete antigen is protected by a tert-butoxycarbonyl (Boc) group, and its carboxyl group is coupled to a carrier protein via an amide bond. The preparation method includes: protecting the amino group of a thyroid hormone molecule (such as T4, T3, etc.) with Boc2O, followed by activating its carboxyl group using the EDC / NHS method and coupling it to a carrier protein. This invention effectively overcomes the chemical instability problems of traditional complete antigens, such as easy photo-oxidative deiodination, easy racemization, and easy deamination reactions. In vitro detection reagents coated with the complete antigen of this invention exhibit longer shelf life, lower batch-to-batch variability, and extremely stable detection performance.

Description

Technical Field

[0001] This invention relates to a thyroid hormone complete antigen with improved stability, its preparation and application, and belongs to the field of immunoassay technology. Background Technology

[0002] Thyroid hormones (T4, T3, rT3) are key endocrine hormones in the human body, and their accurate detection is crucial for thyroid function assessment, diagnosis, and treatment monitoring of related diseases. In the field of clinical in vitro diagnostics (IVD), current methods for detecting T4 and T3 include chemiluminescence immunoassay, fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). Chemiluminescence and immunofluorescence are the most widely used mainstream methods.

[0003] T4, T3, and rT3 are all small molecule haptens, and their detection typically employs competitive immunoassay. The detection reagents use protein conjugates of haptens such as T4, T3, and T2, immobilized on microspheres, magnetic beads, or NC membranes—the complete antigens—as competing antigens. During detection, the amount of antibody used must be strictly limited so that the target antigen (T3 / T4 in the sample) and the labeled competing antigen compete for binding with a limited amount of specific antibody. The signal intensity of the bound label is detected, and this signal is inversely proportional to the concentration of the target antigen.

[0004] Currently, there are also double-antibody sandwich immunoassays. A labeled primary antibody that recognizes a small molecule hapten binds to the antigen to be detected in the sample, forming an immune complex. This complex is then sandwiched with a secondary antibody that recognizes the immune complex, forming a double-antibody sandwich and generating a detection signal. The signal is directly proportional to the concentration of the antigen to be detected. In these methods, small molecule antigen-protein conjugates such as T4 and T3 are often used as the C-line to jointly form the signal system.

[0005] Existing complete antigen-conjugation processes for thyroid hormones (T4, T3, rT3) and their deiodinated derivatives (T2, T1, T0) generally use the EDC method. This involves EDC and NHS activating the carboxyl group of the alanine side chain in T4, T3, T2, and their deiodinated derivative molecules to form an NHS ester, which then forms an amide bond with the amino group of the lysine side chain of carrier proteins such as BSA. Alternatively, a linker arm of a certain length can be added. However, thyroid hormone molecules such as T4 exhibit inherent chemical instability: 1. Photo-oxidative deiodination: T4 / T3 / T2 molecules are extremely sensitive to light. Under ultraviolet or visible light irradiation, the iodine atom on the benzene ring easily undergoes photo-oxidation and falls off, leading to the destruction of the antigenic epitope structure. 2. Racemization: The biologically active form of T4 / T3 is the L-configuration. Its chiral center (α-carbon) is prone to racemization under heat, light, or alkaline conditions, transforming into the inactive D-configuration, directly reducing the effective active concentration of the antigen. 3. Deamination reaction: The amino group on the α-carbon is prone to deamination under heating conditions, forming an aldehyde structure. These molecular-level instabilities directly lead to the performance degradation of traditional T4-BSA and other conjugates in the following application scenarios: 1. Short liquid shelf life: As a raw material reagent, it requires strict low-temperature and light-protected storage, increasing storage and transportation costs and risks. 2. Solid-phase performance degradation: After drying and fixing on microspheres, magnetic beads, or NC membranes of test strips, the antigen activity gradually decreases during the shelf life (usually requiring 1-2 years of storage at room temperature), resulting in large batch-to-batch differences, detection signal attenuation, and sensitivity drift, ultimately affecting the long-term reliability and diagnostic accuracy of the reagent.

[0006] Therefore, developing a thyroid hormone complete antigen with enhanced stability has become an urgent industry need to improve the performance of thyroid hormone detection reagents such as T4 and T3, extend shelf life, and ensure the consistency of test results. Summary of the Invention

[0007] This invention provides a thyroid hormone complete antigen with improved stability, its preparation and application, which can effectively solve the above-mentioned problems.

[0008] This invention provides a thyroid hormone or its deiodinated derivative complete antigen with improved stability, the general structural formula of which is as follows:

[0009] Wherein, the R group is independently selected from I or H; the carboxyl group of the thyroid hormone or its deiodinated derivative is directly connected to the amino group on the carrier protein via an amide bond or via a linker arm.

[0010] In some embodiments, the carrier protein is selected from bovine serum albumin (BSA), chicken ovalbumin (OVA), keyhole hemocyanin (KLH), or bovine immunoglobulin. In one specific embodiment, the carrier protein is bovine serum albumin (BSA).

[0011] In some embodiments, the connecting arm is a linear connecting arm or a ring-shaped connecting arm, and the connecting arm is selected from fatty acid chains or polyethylene glycol (PEG) hydrophilic chains.

[0012] In some embodiments, the thyroid hormone or its deiodinated derivative is selected from one of T4, T3, rT3, T2, T1 or T0.

[0013] This invention provides a method for preparing a complete antigen of a thyroid hormone or its deiodinated derivative with improved stability as described above, comprising the following steps: Step S1: Protection of the amino group of thyroid hormone or its deiodinated derivative: The thyroid hormone or its deiodinated derivative is reacted with tert-butyloxycarbonyl anhydride (Boc2O) in an organic solvent to obtain N-α-Boc protected thyroid hormone or its deiodinated derivative. Step S2: Coupling of the protected thyroid hormone or its deiodinated derivative with the carrier protein: The product obtained in step S1 is activated with a carbodiimide coupling agent to activate the carboxyl group, and then coupled with the carrier protein in a buffer solution. After purification, the complete antigen is obtained.

[0014] In some embodiments, in step S1, the organic solvent is anhydrous N,N-dimethylformamide (DMF); the reaction is carried out under inert gas protection and light-protected conditions, and an organic base is added as a catalyst; in a specific embodiment, the organic base is 4-dimethylaminopyridine (DMAP); the molar ratio of Boc2O to thyroid hormone or its deiodinated derivative is 1.2~2.5:1, and in a specific embodiment it is 1.5:1; the reaction temperature is 0~30℃, and the reaction time is 2~12h.

[0015] In some embodiments, in step S2, the carbodiimide coupling agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS); during the activation process, the molar ratio is N-α-Boc protected product:EDC:NHS = 1:1.5~3:1~2, and in a specific embodiment it is 1:2:1.2; the activation reaction is carried out at room temperature and in the dark for 0.5~1h.

[0016] In some embodiments, in step S2, the mass ratio of the coupling reaction is N-α-Boc protected product: carrier protein = 1:0.5~2; the coupling reaction is carried out at 0~35°C under light-protected conditions for 2~6 hours; the buffer solution is a phosphate buffer solution with a concentration of 10-100mM and a pH of 6.5-7.5.

[0017] The present invention also provides the application of the complete antigen as described above or the complete antigen prepared by the above preparation method in the preparation of a thyroid hormone detection kit.

[0018] In some embodiments, the complete antigen is used as a competing antigen or a coating antigen in chemiluminescent immunoassay, fluorescence immunoassay, enzyme-linked immunosorbent assay, radioimmunoassay, or double antibody sandwich immunoassay.

[0019] The beneficial effects of this invention are: The complete antigen prepared in this invention has its free amino groups stably protected by Boc groups, which effectively improves stability during subsequent coupling, purification, drying, and storage processes. Reagents prepared using this antigen exhibit: 1. Longer shelf life and extremely high stability; 2. Significantly reduced batch-to-batch variability, ensuring the reliability of the detection products; 3. Reduced stringent storage and transportation conditions, facilitating widespread application in resource-limited areas. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a structural formula diagram of the complete antigens of thyroid hormones and their deiodinated derivatives according to the present invention. Figure 2 This is a flowchart illustrating the synthetic reaction route of the T4 complete antigen (Boc-T4-BSA) in an embodiment of the present invention. Figure 3 This is a schematic diagram of the synthetic reaction route of T4-GABA-OVA in the embodiments of the present invention; Figure 4 This is a schematic diagram of the synthesis reaction route of T3-BSA in an embodiment of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0023] Example 1: Preparation of Boc-T4-BSA (1) Dissolve 1 eq L-thyroxine (T4) in DMF. Add 0.1 eq DMAP under nitrogen protection and in the dark. Dissolve 1.5 eq Boc2O in a small amount of dichloromethane (DCM) and slowly add it dropwise to the T4 solution under ice bath cooling. Stir the reaction at 25°C under nitrogen protection for 8 hours. Monitor the reaction progress using thin-layer chromatography (TLC) (developing solvent: DCM / MeOH / EtOAc = 8:1:1).

[0024] (2) After the reaction was complete, the reaction system was added dropwise to ice water under vigorous stirring, and the solution became turbid. The mixture was extracted multiple times with ethyl acetate (EtOAc), and the combined organic phases were washed successively with saturated citric acid aqueous solution, purified water, and saturated sodium chloride solution. The organic phase was dehydrated with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at room temperature. The crude product was purified by preparative TLC with DCM / MeOH / EtOAc = 8:1:1 as the developing solvent. Silica gel containing the target product was separated and recovered, and extracted with an equal mass of DCM / MeOH = 8:2 extract. The extract was concentrated and dried under reduced pressure at room temperature to obtain the intermediate N-α-Boc-L-T4.

[0025] (3) Take 1 eq Boc-T4 and dissolve it in DMF. Take 2 eq EDC and 1.2 eq NHS and dissolve them in 20 mM pH 7.4 phosphate buffer (PB). Take the same mass of BSA as Boc-T4 and dissolve it in 20 mM pH 7.4 PBS to prepare a 10 mg / ml solution.

[0026] (4) Under room temperature and light-protected conditions, EDC and NHS solutions were added dropwise to Boc-T4 solution and the reaction was stirred for 0.5 h to activate the carboxyl group.

[0027] (5) Add the activating solution dropwise into the BSA solution and react slowly at room temperature, away from light, for 2 hours.

[0028] (6) Transfer the reaction solution to a dialysis bag and dialyze with 20 mM pH 7.4 PBS at 2-8°C in the dark for 72 h. Afterward, determine the T4-BSA concentration using the BCA method. Boc-T4-BSA is obtained. The reaction process is described in [link to reaction procedure]. Figure 2 .

[0029] Example 2: Detection of the stability of Boc-T4-BSA by immunofluorescence chromatography Comparative preparation: EDC two-step method: ① Equilibrate EDC and NHS to room temperature.

[0030] ② Dissolve BSA in MES pH 4.7 to a 5 mg / ml solution, dissolve EDC and NHS in MES pH 4.7 to a 50 mg / ml solution, and dissolve T4 in DMSO to a 5 mg / ml T4.Na solution.

[0031] ③ Mix 1 ml BSA, 0.5 ml EDC, and 0.5 ml NHS solution and react at room temperature for 30 min.

[0032] ④ After the reaction is complete, add T4.Na in twice the molar amount of activated protein BSA, adjust the pH to 7-8, and react at room temperature for 3 hours.

[0033] ⑤ The reaction was terminated by adding two molar amounts of ethanolamine, T4.Na.

[0034] ⑥ Remove unreacted small molecules using a desalting column to obtain T4-BSA coated antigen.

[0035] Preparation of immunofluorescence reagents: The Boc-T4-BSA prepared in Example 1 and the T4-BSA prepared in the comparative example were diluted to a working concentration of 0.06 mg / ml and coated onto an NC membrane using a gold spray coating apparatus (0.066 μg / cm). The detection labeling antibody was mouse anti-FT4 monoclonal antibody (Kangji Biotechnology, CAT No.: KA0149), and fluorescent microspheres were purchased from Bangs (CAT No.: 21960). The labeled microspheres were sprayed onto a sample pad (0.026 μg / cm), dried, and assembled into reagent strips.

[0036] Test method: Place the test strip in an accelerated aging environment at 37°C for 4 weeks, and test weekly. Add different concentrations of clinical samples in an environment of 25°C and 50-60% relative humidity, and read the fluorescence values.

[0037] Table 1 Stability test data

[0038] The results showed that the Boc-T4-BSA prepared in Example 1 had significantly better stability than the conventional T4-BSA in the comparative example. After accelerated aging at 37°C for 4 weeks, the decrease in fluorescence value of the reagent in the example was much smaller than that in the comparative example, which fully demonstrates that the complete antigen obtained by the technical solution of this invention can greatly extend the shelf life of the reagent and maintain stable detection performance.

[0039] Example 3: T4-OVA with γ-aminobutyric acid linker (T4-GABA-OVA) Dissolve 1 eq of L-thyroxine (T4) in DMF. Add 0.1 eq of DMAP under nitrogen protection and in the dark. Dissolve 1.5 eq of Boc₂O in a small amount of DCM and slowly add it dropwise to the T4 solution under ice bath cooling. React at 25°C under nitrogen protection and in the dark for 8 hours with stirring. The reaction progress was monitored using thin-layer chromatography (TLC) with a developing solvent of DCM / MeOH / EtOAc = 8:1:1.

[0040] After the reaction was complete, the reaction system was added dropwise to vigorously stirred ice water, resulting in a turbid solution. The mixture was extracted multiple times with EtOAc, and the combined organic phases were washed successively with saturated citric acid aqueous solution, purified water, and saturated sodium chloride solution. The organic phase was dehydrated with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at room temperature.

[0041] The crude product was purified by preparative TLC with DCM / MeOH / EtOAc = 8:1:1 as the developing solvent. Silica gel containing the target product was separated and recovered. The product was then extracted with an equal mass of DCM / MeOH = 8:2 extract. The extract was concentrated under reduced pressure at room temperature and dried to obtain N-α-Boc-L-T4.

[0042] Dissolve 1 eq of Boc-T4 in DMF. Dissolve 2 eq of EDC and 1.2 eq of NHS in DCM. Under light-protected conditions at room temperature, add the EDC and NHS solutions dropwise to the Boc-T4 solution and stir for 4 h to activate the carboxyl group of T4. The reaction progress is monitored using thin-layer chromatography (TLC) with DCM / MeOH / EtOAc as the developing solvent at 8:1:1. After the reaction is complete, add ice water and stir vigorously. Extract multiple times with EtOAc, combine the organic phases, and wash successively with purified water and saturated sodium chloride solution. Dehydrate the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure at room temperature. This yields N-α-Boc-L-T4-NHS.

[0043] Dissolve 1 eq of N-α-Boc-L-T4-NHS in DMF. Dissolve 1.5 eq of γ-aminobutyric acid (GABA) in 20 mM pH 7.4 PB. Add the GABA solution dropwise to the N-α-Boc-L-T4-NHS solution and stir for 4 h at room temperature in the dark. Monitor the reaction progress using thin-layer chromatography (TLC) with DCM / MeOH / EtOAc as the developing solvent (8:1:1). After the reaction is complete, add ice water and stir vigorously. Extract multiple times with EtOAc, combine the organic phases, and wash successively with purified water and saturated sodium chloride solution. Dehydrate the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure at room temperature. This yields N-α-Boc-L-T4-GABA.

[0044] Dissolve 1 eq of N-α-Boc-L-T4-GABA in DMF. Dissolve 2 eq of EDC and 1.2 eq of NHS in DCM. Under light-protected conditions at room temperature, add the EDC and NHS solutions dropwise to the Boc-T4 solution and stir for 4 h to activate the carboxyl group of GABA. The reaction progress is monitored using thin-layer chromatography (TLC) with DCM / MeOH / EtOAc as the developing solvent at 8:1:1. After the reaction is complete, add ice water and stir vigorously. Extract multiple times with EtOAc, combine the organic phases, and wash successively with purified water and saturated sodium chloride solution. Dehydrate the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure at room temperature. This yields N-α-Boc-L-T4-GABA-NHS.

[0045] Dissolve 1 eq of N-α-Boc-L-T4-GABA-NHS in DMF. Dissolve an equal mass of OVA (oxygenated vitamin A) in 20 mM pH 7.4 PB to prepare a 10 mg / mL solution. Add the activated Boc-T4-GABA solution dropwise to the OVA solution and react at room temperature, protected from light, with slow stirring for 2 h.

[0046] After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed for 72 hours at 2-8°C in the dark using 20mM pH7.4 PB to remove unreacted small molecules, byproducts, etc.

[0047] After dialysis, the concentration of T4-GABA-OVA was determined using the BCA method. The UV absorbance at 260 nm and 280 nm was detected using UV spectrophotometry, and the ratio of the two was calculated. If the ratio was higher than that of OVA, the coupling was considered successful.

[0048] Freeze-dry by vacuum centrifugation or freeze-drying, and store at -20°C.

[0049] The synthesis reaction process is as follows Figure 3 As shown.

[0050] Example 4: Preparation of Boc-T3-BSA Dissolve 1 eq of 3,3',5-triiodo-L-methylformamide (T3) in DMF. Add 0.1 eq of DMAP under nitrogen protection and in the dark. Dissolve 1.5 eq of Boc₂O in a small amount of DCM and slowly add it dropwise to the T3 solution under ice bath cooling. Stir the reaction at 25°C under nitrogen protection and in the dark for 8 hours. The reaction progress was monitored by thin-layer chromatography (TLC) with a developing solvent of DCM / MeOH / EtOAc = 8:1:1.

[0051] After the reaction was complete, the reaction system was added dropwise to vigorously stirred ice water, resulting in a turbid solution. The mixture was extracted multiple times with EtOAc, and the combined organic phases were washed successively with saturated citric acid aqueous solution, purified water, and saturated sodium chloride solution. The organic phase was dehydrated with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at room temperature.

[0052] The crude product was purified by preparative TLC using DCM / MeOH / EtOAc = 8:1:1 as the developing solvent. Silica gel containing the target product was separated and recovered. The product was then extracted with an equal mass of DCM / MeOH = 8:2 extract. The extract was concentrated under reduced pressure at room temperature and dried to obtain N-α-Boc-L-T3.

[0053] Dissolve 1 eq of Boc-T3 in DMF. Dissolve 2 eq of EDC and 1.2 eq of NHS in 20 mM pH 7.4 PB. Dissolve an equal mass of BSA (Boc-T3) in 20 mM pH 7.4 PB to prepare a 10 mg / mL solution.

[0054] Under room temperature and light-protected conditions, EDC and NHS solutions were added dropwise to Boc-T3 solution and the mixture was stirred for 0.5 h to activate the carboxyl group of T3.

[0055] The activated Boc-T3 solution was added dropwise to the BSA solution and reacted at room temperature, in the dark, with slow stirring for 2 h.

[0056] After the reaction was completed, the reaction solution was transferred to a dialysis bag and dialyzed for 72 hours at 2-8°C in the dark using 20mM pH7.4 PB to remove unreacted small molecules, byproducts, etc.

[0057] After dialysis, the concentration of T3-BSA was determined using the BCA method. The UV absorbance at 260 nm and 280 nm was detected using UV spectrophotometry, and the ratio of the two was calculated. If the ratio was higher than that of BSA, the coupling was considered successful.

[0058] Freeze-dry by vacuum centrifugation or freeze-drying, and store at -20°C.

[0059] The synthesis reaction process is as follows Figure 4 As shown.

[0060] Example 5: Detection of the stability of Boc-T3-BSA by immunofluorescence chromatography Comparative preparation T3-BSA was prepared using the EDC two-step method described in Example 2, following conventional processes. It was then refrigerated and vacuum centrifuged or lyophilized, and stored at -20°C.

[0061] Thermal acceleration experiment Take Boc-T3-BSA and comparative T3-BSA powders, place them at 37℃, and take samples at 0 days, 1 day, 2 days, 4 days, and 7 days.

[0062] Preparation of immunofluorescence reagents The heat-accelerated Boc-T3-BSA and comparative T3-BSA were diluted to working concentrations and coated onto NC membranes using a gold-spraying membrane coating system. The antigen dilution buffer was 20 mM pH 7.4 phosphate buffer, the working coating concentration was 0.06 mg / ml, and the volume was 0.066 μg / cm. The coating method is well-known to those skilled in the art. The labeled antibody used for detection was mouse anti-FT3 monoclonal antibody (Kangji Biotechnology, CAT No.: KA0148), and the fluorescent microspheres were purchased from Bangs (CAT No.: 21960). The antibody labeling method is well-known to those skilled in the art. The labeled microspheres were sprayed onto the sample pad at a volume of 0.026 μg / cm, vacuum dried for 3 hours, and assembled into test strips. During detection, clinical samples of different concentrations were added in an environment of 25℃ and 50-60% relative humidity, and the fluorescence values ​​were read.

[0063] The stability test data are shown in Table 2:

[0064] The stability results show that the T3-BSA in the example is significantly more stable than the T3-BSA in the comparative example. During a one-week storage period at 37°C, the change in fluorescence value after preparation as a reagent was significantly smaller than that in the comparative example. This indicates that the detection reagent prepared using the T3-BSA in the example has the potential for longer shelf life, lower batch-to-batch variability, and more stable detection performance.

[0065] In summary, it is generally believed in the art that introducing a large and hydrophobic protecting group (such as Boc) onto a small molecule hapten can easily cause steric hindrance or alter the conformation of the antigen epitope, thereby severely affecting the recognition and binding of specific antibodies. The technical solution of this invention overcomes this technical bias, not only without disrupting the binding between antigen and antibody, but also significantly improving the thermal stability and shelf life of the reagent, resulting in unexpected technical effects.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A complete antigen of a thyroid hormone or its deiodinated derivative with improved stability, characterized in that, Its general structural formula is as follows: Wherein, the R group is independently selected from I or H; the carboxyl group of the thyroid hormone or its deiodinated derivative is directly connected to the amino group on the carrier protein via an amide bond or via a linker arm.

2. The complete antigen according to claim 1, characterized in that, The carrier protein is selected from one of bovine serum albumin (BSA), ovalbumin (OVA), keyhole hemocyanin (KLH), or bovine immunoglobulin.

3. The complete antigen according to claim 1, characterized in that, The connecting arm is a straight-chain connecting arm or a ring-shaped connecting arm, and the connecting arm is selected from fatty acid chains or polyethylene glycol (PEG) hydrophilic chains.

4. The complete antigen according to claim 1 or 2, characterized in that, The thyroid hormone or its deiodinated derivative is selected from one of T4, T3, rT3, T2, T1 or T0.

5. A method for preparing a complete antigen of a thyroid hormone or its deiodinated derivative with improved stability as described in any one of claims 1-4, characterized in that, Includes the following steps: Step S1: Protection of the amino group of thyroid hormone or its deiodinated derivative: The thyroid hormone or its deiodinated derivative is reacted with tert-butyloxycarbonyl anhydride (Boc2O) in an organic solvent to obtain N-α-Boc protected thyroid hormone or its deiodinated derivative. Step S2: Coupling of the protected thyroid hormone or its deiodinated derivative with the carrier protein: The product obtained in step S1 is activated with a carbodiimide coupling agent to activate the carboxyl group, and then coupled with the carrier protein in a buffer solution. After purification, the complete antigen is obtained.

6. The preparation method according to claim 5, characterized in that, In step S1, the organic solvent is anhydrous N,N-dimethylformamide (DMF); the reaction is carried out under inert gas protection and light-protected conditions, and an organic base is added as a catalyst; the molar ratio of Boc2O to thyroid hormone or its deiodinated derivative is 1.2~2.5:1; the reaction temperature is 0~30℃, and the reaction time is 2~12h.

7. The preparation method according to claim 5, characterized in that, In step S2, the carbodiimide coupling agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS); during the activation process, the molar ratio is N-α-Boc protected product:EDC:NHS = 1:1.5~3:1~2; the activation reaction is carried out at room temperature and in the dark for 0.5~1h.

8. The preparation method according to claim 5 or 7, characterized in that, In step S2, the mass ratio of the coupling reaction is N-α-Boc protected product: carrier protein = 1:0.5~2; the coupling reaction is carried out at 0~35℃ under light-protected conditions for 2~6 hours; the buffer solution is a phosphate buffer solution with a concentration of 10-100mM and a pH of 6.5-7.

5.

9. The use of a complete antigen as described in any one of claims 1-4 or a complete antigen prepared by any one of claims 5-8 in the preparation of a thyroid hormone detection kit.

10. The application according to claim 9, characterized in that, The complete antigen is used as a competing antigen or a coating antigen in chemiluminescent immunoassay, fluorescence immunoassay, enzyme-linked immunosorbent assay, radioimmunoassay, or double antibody sandwich immunoassay.

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