Biotin molecular labeled triiodothyronine and preparation method thereof

Abstract

The invention relates to biotin molecule labeled triiodothyronine and a preparation method thereof, triiodothyronine which is easy to obtain and low in cost is selected as an initial raw material of a competitive antigen, and triiodothyronine which is directly labeled by a chemiluminescence biotin single molecule is prepared through a simple reaction. As a key competitive antigen raw material of a free triiodothyronine (FT3) magnetic particle chemiluminescence immunodetection reagent, the magnetic particle has good specificity and stability when being matched with an antibody reagent to detect free triiodothyronine (FT3), so that the detection result is more accurate, and the large-scale detection of free triiodothyronine (FT3) is met.

Description

Technical Field

[0001] The present application relates to triiodothyronine labeled with a biotin molecule and a preparation method thereof, and belongs to the technical field of chemiluminescence immunoassay. Background Art

[0002] Chemiluminescence immunoassay (CLIA) combines highly sensitive chemiluminescence assays with highly specific immune responses. It is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. It is the latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and time-resolved fluorescence immunoassay.

[0003] Free triiodothyronine (FT3) is a small molecule hapten. It is generally detected by competitive method in chemiluminescent immunoassay. The competitive antigen in the detection reagent is a key raw material. The affinity between the competitive antigen and the antibody should be neither too large nor too small. If it is too large, the free triiodothyronine antigen in the sample will have difficulty binding to the antibody. If it is too small, it will not be able to bind to the antibody well, affecting the detection effect.

[0004] The free triiodothyronine index is one of the specific indicators of thyroid function test. Its clinical significance includes: (1) an increase in the index: hyperthyroidism, use of thyroid hormones, heparin and other drugs; (2) a decrease in the index: hypothyroidism.

[0005] Currently, free triiodothyronine magnetic microparticle chemiluminescent immunoassays on the domestic market generally use a triiodothyronine derivative coupled to a carrier protein as a competing antigen. During the antigen relabeling process, the coupling efficiency is difficult to precisely control, resulting in large batch-to-batch variability. Furthermore, the use of carrier proteins, which are susceptible to environmental influences such as temperature and pH, can easily cause conformational changes or denaturation of the protein, leading to poor product stability.

[0006] In view of this, this application is filed. Summary of the Invention

[0007] The purpose of the present application is to provide a biotin molecule-labeled triiodothyronine and a preparation method thereof. The biotin molecule-labeled triiodothyronine can be used as a key competitive antigen raw material for a free triiodothyronine (FT3) magnetic particle chemiluminescence immunoassay reagent, has good specificity and stability, makes the detection results more accurate, and meets the requirements of large-scale detection of free triiodothyronine (FT3).

[0008] In order to achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present application provides a biotin-labeled triiodothyronine having the structural formula shown in Formula 1: (Formula 1) .

[0009] In one or more possible embodiments, the biotin molecule is linked to the amino terminus of triiodothyronine via an amide bond.

[0010] In one or more feasible embodiments, Formula 1 comprises a covalent bond formed by the pentanoic acid side chain of the biotin molecule and the amino group or carboxyl group of triiodothyronine.

[0011] In a second aspect, the present application provides a method for preparing triiodothyronine labeled with a biotin molecule, comprising the following steps: In an organic solvent, biotin is subjected to a condensation reaction with an activating agent to obtain biotin-NHS; In an alkaline solution, the biotin-NHS is coupled with triiodothyronine, and the triiodothyronine labeled with the biotin molecule is obtained after purification.

[0012] In one or more feasible embodiments, the organic solvent is selected from at least one aprotic polar solvent selected from DMF, DMSO, NMP or DMAc.

[0013] In one or more feasible embodiments, the activator is selected from at least one N-hydroxy compound selected from HOSU, HOBt, HOAt or sulfo-NHS.

[0014] In one or more feasible embodiments, in the step of subjecting biotin to a condensation reaction with an activator in an organic solvent to obtain biotin-NHS, the condensation agent used in the condensation reaction is at least one carbodiimide condensation agent selected from DCC, EDC, DIC or DIPC.

[0015] In one or more feasible embodiments, in the step of coupling the biotin-NHS with triiodothyronine in an alkaline solution and purifying to obtain the biotin-labeled triiodothyronine, at least one of the following methods is used for purification: a) Reverse phase HPLC purification, mobile phase is acetonitrile-water system; b) Silica gel column chromatography, using a chloroform-methanol gradient system as the eluent; c) Acid-base dissolution-precipitation cycle purification.

[0016] In a third aspect, the present application provides a detection kit for detecting free triiodothyronine, wherein the reagents in the kit contain triiodothyronine labeled with a biotin molecule as described in the first aspect or triiodothyronine labeled with a biotin molecule prepared by the preparation method described in the second aspect.

[0017] In a fourth aspect, the present application provides a chemiluminescent immunoassay method for quantitative detection of free triiodothyronine, using the detection kit described in the third aspect.

[0018] According to one or more embodiments of the present application, the beneficial effects of the present application are: The present application provides triiodothyronine labeled with a biotin molecule and a preparation method thereof. Triiodothyronine is selected as the starting material for a competitive antigen, and triiodothyronine directly labeled with a single chemiluminescent biotin molecule is prepared through a simple reaction. Triiodothyronine is used as a key competitive antigen raw material for a magnetic particle chemiluminescent immunoassay reagent for free triiodothyronine (FT3). It can be used in combination with an antibody reagent for large-scale detection of free triiodothyronine (FT3). The method has the characteristics of readily available raw materials, simple preparation, and low cost, and has high specificity and stability, making the detection results more accurate.

[0019] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The structural formula of triiodothyronine labeled with biotin molecules in an embodiment of the present application; Figure 2 This is a structural change of the method for preparing triiodothyronine labeled with biotin molecules in an embodiment of the present application; Figure 3 This is a flow chart of a method for preparing triiodothyronine labeled with biotin molecules according to an embodiment of the present application; Figure 4 This is a mass spectrometry result diagram of triiodothyronine labeled with biotin molecules in an embodiment of the present application; Figure 5 This is another mass spectrometry result of triiodothyronine labeled with biotin molecules in an embodiment of the present application. DETAILED DESCRIPTION

[0021] The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other unless they conflict.

[0022] It should be noted that, in this application, "accuracy" refers to the degree of agreement between a measured or calculated quantity (a test report value) and its actual (or true) value. Clinical accuracy refers to the ratio of true outputs (true positives (TP) or true negatives (TN)) to misclassified outputs (false positives (FP) or false negatives (FN)) and can be expressed as, among other measures, sensitivity, specificity, positive predictive value (PPV) or negative predictive value (NPV), Matheus correlation coefficient (MCC), likelihood, odds ratio, receiver operating characteristic (ROC) curve, or area under the curve (AUC).

[0023] For the diagnostic (or prognostic) interventions of the present application, since each output (which may be TP, FP, TN, or FN in a disease classification diagnostic test) carries a different cost, a health economic utility function may be based on the clinical situation and individual output costs and values, preferably favoring sensitivity over specificity, or PPV over NPV, thereby providing another measure of health economic performance and value that may differ from more direct clinical or analytical performance measures. These different measures and relative tradeoffs will generally converge only in the case of a perfect test with zero error rate (also known as zero predicted object output misclassification or FP and FN), and all performance measures will tend to be imperfect, but to varying degrees.

[0024] "Measure," "determine," "detect," or "examine" refers to evaluating the presence, absence, amount, or quantity (which can be a significant amount) of a given substance or a derived sample from a subject (including a derivative of the qualitative or quantitative concentration level of such substance) in a clinical setting, or otherwise assessing the value or classification of a non-analyte clinical parameter or clinical-Determinant of a subject.

[0025] In the context of this application, all data meet statistical requirements (statistically significant). "Statistically significant" means a change greater than would be expected by chance alone (which may be a "false positive"). Statistical significance can be determined by any method known in the art. Commonly used measures of significance include p-values, which represent at least a limiting value the probability of obtaining a result at a given data point, assuming that the result was due to chance alone. A p-value of 0.05 or less is generally considered highly significant.

[0026] It should be noted that, in the following examples, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this field or the product instructions were used. Reagents or instruments used without manufacturer specified are all conventional products that can be purchased through regular channels.

[0027] The present application provides a biotin molecule labeled triiodothyronine having the structural formula shown in Formula 1: (Formula 1) .

[0028] Alternatively, a biotin molecule is linked to the amino terminus of triiodothyronine via an amide bond.

[0029] Optionally, Formula 1 comprises a covalent bond formed between the pentanoic acid side chain of the biotin molecule and the amino group or carboxyl group of triiodothyronine.

[0030] Based on the above situation, the present application also provides a method for preparing triiodothyronine labeled with biotin molecules, comprising the following steps: In an organic solvent, biotin is subjected to a condensation reaction with an activating agent to obtain biotin-NHS; In an alkaline solution, the biotin-NHS is coupled with triiodothyronine, and the triiodothyronine labeled with the biotin molecule is obtained after purification.

[0031] For example, the changes in the structural formula in the preparation method are as shown in the attached Figure 2 shown.

[0032] Optionally, the organic solvent is selected from at least one aprotic polar solvent selected from DMF, DMSO, NMP or DMAc.

[0033] Optionally, the activating agent is selected from at least one N-hydroxy compound selected from HOSU, HOBt, HOAt or sulfo-NHS.

[0034] Optionally, in the step of subjecting biotin to a condensation reaction with an activator in an organic solvent to obtain biotin-NHS, the condensation agent used in the condensation reaction is at least one carbodiimide condensation agent selected from DCC, EDC, DIC or DIPC.

[0035] Optionally, in the step of coupling the biotin-NHS with triiodothyronine in an alkaline solution and purifying to obtain the biotin-labeled triiodothyronine, at least one of the following methods is used for purification: a) Reverse phase HPLC purification, mobile phase is acetonitrile-water system; b) Silica gel column chromatography, using a chloroform-methanol gradient system as the eluent; c) Acid-base dissolution-precipitation cycle purification.

[0036] In one or more feasible embodiments, as shown in the attached Figure 3 As shown, the preparation method provided in this application specifically includes the following steps: S1. Add biotin to DMF, heat to 70°C to dissolve, and then cool to room temperature. Add HOSU and DCC, and react at room temperature overnight. Then filter to remove DCU, and concentrate the filtrate to obtain a white solid product. Add appropriate amount of ethanol to slurry, filter, and dry to obtain Bio-NHS as a white solid.

[0037] S2. Dissolve Bio-NHS in DMF and add dropwise to a Na2CO3 aqueous solution of T3 (white suspension). The solution will quickly become clear. React at room temperature for at least 3 hours. Add an appropriate amount of water and adjust the pH to acidic with dilute hydrochloric acid. A white solid will precipitate. Filter and purify the solid by HPLC to obtain pure Bio-T3.

[0038] Preferably, this embodiment employs method a) above for purification. Reversed-phase HPLC purification (using an acetonitrile-water mobile phase) is a highly efficient separation technique based on differences in the hydrophobicity of compounds. Its core principle is to achieve selective separation of the target product through the synergistic effect of a non-polar stationary phase (e.g., a C18 column) and a polar mobile phase. In one or more feasible embodiments, the mobile phase comprises a gradient mixture of acetonitrile (a strong elution solvent) and water (a weak elution solvent). By gradually increasing the acetonitrile percentage (e.g., 30% → 80%), the polarity difference between biotin-labeled triiodothyronine (with enhanced hydrophobicity) and unreacted starting materials and byproducts is exploited to extend the retention time of the target product in the chromatographic column, ultimately achieving high-purity separation (purity >98%). This method combines high resolution (theoretical plates >20,000 / column) with high recovery (>95%), making it particularly suitable for accurately distinguishing the labeled product from free biotin and uniodinated impurities. The low viscosity and good compatibility of the acetonitrile-water system ensure the stability and reproducibility of the separation process.

[0039] It should be noted that silica gel column chromatography (eluent: chloroform-methanol gradient system) is a normal-phase chromatographic separation technique based on compound polarity differences. It achieves fractional purification of target compounds through the synergistic effect of a polar silica gel stationary phase and a non-polar to moderately polar eluent. In one or more feasible embodiments, the eluent is eluated by gradually increasing the methanol ratio (e.g., a gradient from 20:1 to 1:1 chloroform). This exploits the polarity difference between biotinylated triiodothyronine (moderately polar) and highly polar byproducts (e.g., uniodinated impurities) or low-polarity components (e.g., free biotin derivatives) to selectively migrate the target compound during the silica gel adsorption-desorption process. Ultimately, separation with a purity exceeding 90% is achieved through fractional collection. This method offers the advantages of high sample loading (up to 5%-10% of the column volume) and low cost, making it particularly suitable for laboratory-scale initial purification of crude products. The excellent solubility of iodinated aromatic compounds in the chloroform-methanol system effectively avoids loss of column efficiency.

[0040] It should also be noted that acid-base dissolution-precipitation cyclic purification is a classic purification method based on the differential solubility of target compounds under different pH conditions. Its core approach is to selectively remove impurities by alternating the solution pH (e.g., acidic dissolution at pH 2-4 → alkaline precipitation at pH 8-10). Specifically, the crude product is dissolved in dilute hydrochloric acid (e.g., 0.1 M HCl) to form a soluble salt. Acid-insoluble impurities (e.g., inorganic salts or hydrophobic byproducts) are then removed by filtration. Subsequently, an alkaline solution (e.g., NaOH or ammonia) is slowly added dropwise to the isoelectric point (pH 4.5-5.5), causing the target compound to precipitate in a neutral form while water-soluble impurities (e.g., unreacted biotin activator) remain in the liquid phase. Two to three cycles can significantly improve product purity (>85%). Gradient washing with an ethanol / water mixture (e.g., a 1:3 volume ratio) can further remove residual polar impurities. This method has simple equipment and low cost, and is particularly suitable for the primary purification of compounds containing ionized groups (such as carboxylic acids and amino groups). By controlling the crystallization rate (such as slow precipitation at low temperature), the crystal form and particle uniformity can be optimized.

[0041] Based on the above situation, the present application also provides a detection kit for detecting free triiodothyronine, wherein the reagents in the kit contain triiodothyronine labeled with biotin molecules as described above or triiodothyronine labeled with biotin molecules prepared by the preparation method described above.

[0042] Based on the above situation, the present application also provides a chemiluminescent immunoassay method for quantitative detection of free triiodothyronine, using the detection kit as described above.

[0043] The present application provides a biotin molecule-labeled triiodothyronine and a preparation method thereof. Triiodothyronine is selected as the starting material for a competitive antigen. Through a simple reaction, triiodothyronine directly labeled with a single chemiluminescent biotin molecule is prepared. Triiodothyronine serves as a key competitive antigen raw material for a magnetic particle chemiluminescent immunoassay reagent for free triiodothyronine (FT3). The reagent can be used in conjunction with an antibody reagent for large-scale detection of free triiodothyronine (FT3). The reagent has the characteristics of readily available raw materials, simple preparation, and low cost, and has high specificity and stability, making the detection results more accurate.

[0044] The present application will be further described in detail below with reference to specific embodiments. Example

[0045] The method for preparing the key competitive antigen raw material (Bio-T3) of the free triiodothyronine (FT3) magnetic microparticle chemiluminescent immunoassay reagent comprises the following steps: Step 1: Add biotin (1 g, 1 eq) to DMF (20 ml), heat to 70°C to dissolve, and then cool to room temperature. Add HOSU (0.56 g, 1.2 eq) and DCC (0.93 g, 1.2 eq), and react at room temperature overnight. Then, filter to remove DCU and concentrate the filtrate to obtain a white solid product. Add appropriate amount of ethanol to slurry, filter, and dry to obtain Bio-NHS as a white solid with a yield of 92%.

[0046] Step 2: Dissolve Bio-NHS (85 mg, 1.3 eq) in DMF (10 ml) and add dropwise to a solution of T3 (123 mg, 1 eq) in Na₂CO₃ (41 mg, 2 eq) in water (10 ml) (white suspension). The solution quickly becomes clear and is allowed to react at room temperature for at least 3 hours. Add an appropriate amount of water and adjust the pH to acidic with dilute hydrochloric acid. A white solid precipitates, which is filtered and purified by HPLC to obtain pure Bio-T3 in an 83% yield.

[0047] Specifically, the mass spectrometry results are as follows: 877.90 (M+1) (see attached Figure 4 ); 875.70 (M-1) (as shown in the attached Figure 5 shown). Example

[0048] A method for preparing a key competitive antigen raw material (Bio-T3) for a free triiodothyronine (T3) magnetic microparticle chemiluminescent immunoassay reagent comprises the following steps: Step 1. Add biotin (2 g, 1 eq) to DMF (40 ml), heat to 70°C to dissolve, and then cool to room temperature. Add HOSU (1.12 g, 1.2 eq) and DCC (1.86 g, 1.2 eq), and react at room temperature overnight. Then filter to remove DCU and concentrate the filtrate to obtain a white solid product. Add appropriate amount of ethanol to slurry, filter, and dry to obtain Bio-NHS as a white solid with a yield of 94%.

[0049] Step 2: Dissolve Bio-NHS (170 mg, 1.3 eq) in DMF (20 ml) and add dropwise to a solution of T3 (246 mg, 1 eq) in Na₂CO₃ (82 mg, 2 eq) in water (20 ml) (white suspension). The solution quickly becomes clear and is allowed to react at room temperature for at least 3 hours. An appropriate amount of water is added and the pH is adjusted to acidic with dilute hydrochloric acid. A white solid precipitates, which is filtered and purified by HPLC to obtain pure Bio-T3 in an 81% yield. Example

[0050] A method for preparing a key competitive antigen raw material (Bio-T3) for a free triiodothyronine (T3) magnetic microparticle chemiluminescent immunoassay reagent comprises the following steps: Step 1: Add biotin (3 g, 1 eq) to DMF (60 ml), heat to 70°C to dissolve, and then cool to room temperature. Add HOSU (1.68 g, 1.2 eq) and DCC (2.79 g, 1.2 eq), and react at room temperature overnight. Then, filter to remove DCU and concentrate the filtrate to obtain a white solid product. Add appropriate amount of ethanol to slurry, filter, and dry to obtain Bio-NHS as a white solid with a yield of 92%.

[0051] Step 2: Dissolve Bio-NHS (255 mg, 1.3 eq) in DMF (30 mL) and add dropwise to a solution of T2 (369 mg, 1 eq) in Na2CO3 (123 mg, 2 eq) in water (30 mL) (white suspension). The solution quickly becomes clear and is allowed to react at room temperature for at least 3 hours. An appropriate amount of water is added and the pH is adjusted to acidic with dilute hydrochloric acid. A white solid precipitates, which is filtered and purified by HPLC to obtain pure Bio-T3 in an 82% yield.

[0052] Preparation of acridinium salt (AE) labelled triiodothyronine (T3) antibody: Dissolve AE-NHS active ester in DMSO to a concentration of 5 mg / ml. Prepare a 1 mg / ml solution of triiodothyronine (T3) antibody in 0.1 M PB buffer, pH 7.4. Based on the desired labeling ratio of AE-NHS active ester to triiodothyronine (T3) antibody, add an appropriate amount of AE-NHS active ester DMSO solution to the triiodothyronine (T3) antibody solution in PB buffer. Incubate at room temperature with shaking for 1.5 hours. Then, dialysis and purification are performed three times against 0.1 M PB, pH 7.4, to obtain the AE-labeled triiodothyronine (T3) antibody.

[0053] The target values ​​of the working calibrators S0, S1, S2, S3, S4, and S5, i.e., the concentrations of the analytes increase from low to high: 0.00 pmol / ml, 1.99 pmol / ml, 4.48 pmol / ml, 8.97 pmol / ml, 25.23 pmol / ml, and 49.62 pmol / ml.

[0054] Streptavidin magnetic beads: Dilute the streptavidin magnetic beads with magnetic bead buffer.

[0055] Reagent R1: Dilute the AE label of triiodothyronine (T3) antibody with reagent buffer to obtain reagent R1.

[0056] Reagent R2: Dissolve 1 mg of Bio-T3 in 1 ml of DMSO. Dilute once with DMSO, then again with reagent buffer, for a total dilution of 200,000-fold to obtain Reagent R2.

[0057] The results of the calibrator test using a chemiluminescence analyzer are shown in Table 1 below: Table 1: Chemiluminescence Analyzer Calibrator Test Results The results showed that there was a good correlation between concentration and luminescence value, and the luminescence value gradually decreased with the increase of the concentration of the test substance.

[0058] It can be seen from this that the biotin molecule-labeled triiodothyronine and its preparation method provided in this application select triiodothyronine as the starting material for the competitive antigen, and prepare triiodothyronine directly labeled with a single chemiluminescent biotin molecule through a simple reaction. It serves as the key competitive antigen raw material for the free triiodothyronine (FT3) magnetic particle chemiluminescent immunoassay reagent, and can be used in combination with antibody reagents to perform large-scale detection of free triiodothyronine (FT3). It has the characteristics of easy availability of raw materials, simple preparation, and low cost, and has high specificity and stability, making the detection results more accurate.

[0059] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A biotin-labeled triiodothyronine, characterized in that: It has the structural formula shown in Formula 1: (Formula 1) 。 2. The biotin-labeled triiodothyronine according to claim 1, characterized in that: The biotin molecule is attached to the amino terminus of triiodothyronine via an amide bond.

3. The biotin-labeled triiodothyronine according to claim 1, characterized in that: Formula 1 includes a covalent bond formed between the pentanoic acid side chain of the biotin molecule and the amino group or carboxyl group of triiodothyronine.

4. A method for preparing triiodothyronine labeled with biotin molecules, characterized in that: The preparation method is used to prepare triiodothyronine labeled with a biotin molecule according to any one of claims 1 to 3, comprising the following steps: In an organic solvent, biotin is subjected to a condensation reaction with an activating agent to obtain biotin-NHS; In an alkaline solution, the biotin-NHS is coupled with triiodothyronine, and the triiodothyronine labeled with the biotin molecule is obtained after purification.

5. The preparation method according to claim 4, characterized in that The organic solvent is selected from at least one aprotic polar solvent selected from DMF, DMSO, NMP or DMAc.

6. The preparation method according to claim 4, characterized in that The activating agent is selected from at least one N-hydroxy compound selected from HOSU, HOBt, HOAt or sulfo-NHS.

7. The preparation method according to claim 4, characterized in that In the step of allowing biotin to undergo a condensation reaction with an activator in an organic solvent to obtain biotin-NHS, the condensation agent used in the condensation reaction is at least one carbodiimide condensation agent selected from DCC, EDC, DIC or DIPC.

8. The preparation method according to claim 4, characterized in that In the step of coupling the biotin-NHS with triiodothyronine in an alkaline solution and obtaining the biotin-labeled triiodothyronine after purification, at least one of the following methods is used for purification: a) Reverse phase HPLC purification, mobile phase is acetonitrile-water system; b) Silica gel column chromatography, using a chloroform-methanol gradient system as the eluent; c) Acid-base dissolution-precipitation cycle purification.

9. A detection kit for detecting free triiodothyronine, characterized in that: The reagents in the kit contain the biotin molecule-labeled triiodothyronine according to any one of claims 1 to 3 or the biotin molecule-labeled triiodothyronine prepared by the preparation method according to any one of claims 4 to 8.

10. A chemiluminescent immunoassay method for quantitative detection of free triiodothyronine, characterized in that: The detection kit according to claim 9 is used.

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