Thyroxin fluorescent conjugate, its preparation method and application

Abstract

This invention provides a thyroxine fluorescent conjugate, its preparation method, and its application in a kit for detecting thyroxine. It also provides a kit for detecting thyroxine and its preparation method. Using the thyroxine fluorescent conjugate of this invention in a kit for detecting thyroxine can improve the stability of the kit. This invention proposes that during the labeling of thyroxine with acridine derivatives, a carbon chain structure with imino groups at both ends is inserted between the thyroxine and the acridine derivative as a linker arm, forming a thyroxine-linker arm-acrimidine derivative coupling structure. Using this thyroxine fluorescent conjugate as a reagent component in a kit for detecting thyroxine can improve the stability of the kit.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically a thyroxine fluorescent conjugate, its preparation method, and its application. Background Technology

[0002] Thyroxine (3,5,3',5'-tetraiodothyronine, T4) is a hormone secreted by the follicular epithelial cells of the thyroid gland, with a molecular weight of approximately 777 kDa. After being released into the bloodstream via thyroid secretion, T4 exists in both bound and free forms. The total T4 in serum is called total T4 (TT4), and the free portion is called free T4 (FT4). After entering the bloodstream, approximately 99.7% of T4 binds to thyroxine-binding proteins in the blood, with about 60% binding to TBG, 30% to TBPA, and the remainder to albumin. Only about 0.05% of T4 exists in the blood in a free state (FT4). Under normal circumstances, a dynamic balance is maintained between the two forms; only FT4 can enter target cells and bind to receptors to exert its physiological function. FT4 is the active fraction of circulating thyroid hormones and possesses biological activity. Increased or decreased secretion of thyroid hormones can lead to thyroid dysfunction and endocrine metabolic disorders. Therefore, in the diagnosis and treatment of thyroid diseases, the functional status of the thyroid gland is often reflected by detecting the levels of FT4 and / or TT4 in the serum.

[0003] Chemiluminescence immunoassay (CLIA) is a technique that combines highly sensitive chemiluminescence assays with highly specific immunoreactions for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. It is a cutting-edge immunoassay technique developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and time-resolved fluorescence immunoassay. Commonly used acridine compounds (AEs) as chemiluminescent labels include acridine esters (NSP-DMAE-NHS) and acridine sulfonamides (NSP-SA-NHS), both of which contain an acridine ring in their structure. Acridine compounds emit light through the action of luminescent initiating reagents (NaOH, H2O2), and their application in chemiluminescence detection has many advantages, such as: ① low background luminescence and high signal-to-noise ratio; ② few interfering factors in the luminescence reaction; ③ rapid and concentrated light release, high luminescence efficiency, and high luminescence intensity; ④ easy to bind to proteins without reducing photon yield after binding; ⑤ stable label (can be stored for several months at 2-8℃).

[0004] Existing research discloses the use of chemiluminescent immunoassay to detect thyroxine after labeling it with chemiluminescent markers. For example, the preparation method of acridinium ester-FT4 conjugate described in the following Chinese patent application and journal article:

[0005] Chinese patent application CN 110988368 A discloses a luminescent immunoassay kit for free thyroxine, comprising the following reagents: solid-phase reagent R1: a suspension containing streptavidin magnetic particles; liquid-phase reagent R2: a suspension containing acridinium ester-labeled T4 antibody; and biotin reagent R3: a suspension containing biotin-labeled thyroxine derivatives. The preparation method of liquid-phase reagent R2 is as follows: T4 antibody is diluted with phosphate buffer, acridinium ester is added, the molar ratio of antibody to acridinium ester labeling is 1:3, the mixture is gently shaken, and the reaction is carried out overnight in the dark; the mixture is purified by dextran gel G-25 to obtain acridinium ester-labeled T4 antibody suspension, and the labeled acridinium ester-labeled T4 antibody suspension is resuspended in phosphate buffer to obtain liquid-phase reagent R2.

[0006] Zhang Jianfeng used acridinium ester as a signaling molecule to label small molecule thyroxine. Employing a small molecule competitive assay, he introduced magnetic microspheres as a solid-phase carrier and combined this with the streptavidin-biotin system to develop a high-performance FT4 detection reagent. This reagent has significant clinical application value and provides valuable reference for related research. The preparation method of the acridinium ester-T4 conjugate is as follows: Weigh an appropriate amount of pure T4 and dissolve it in DMSO to prepare a 0.1 mmol / L solution A. Then weigh an appropriate amount of NSP–DMAE-NHS and dissolve it in anhydrous DMF to prepare a 10 mmol / L solution B. Take 5.0 mL of solution B, add 2 mg of triethylamine (TEA), then add another 5.0 mL of solution B, and finally dilute with 0.05 mol / L carbonate buffer at pH 9.6 to a total volume of 10 mL. Stir the reaction at room temperature for 16 h. The product is purified by preparative chromatography to obtain acridinium ester-T4 conjugate (Development and application of magnetic microparticle-acridinium ester chemiluminescent free thyroxine detection reagent [J], Zhang Jianfeng, International Journal of Laboratory Medicine, 2016, No. 19).

[0007] However, when the applicant used existing free / total thyroxine assay kits for thyroxine detection, they found that the existing conjugates of thyroxine and acridine esters had drawbacks such as low immunoreactivity, susceptibility to bilirubin interference, and unstable results. By embedding a linker arm between thyroxine and an acridine derivative to form a conjugate, and then using this conjugate to replace the existing acridine-labeled thyroxine in the preparation of a thyroxine assay kit, the stability of the kit can be improved. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a thyroxine fluorescent conjugate, its preparation method, and its application. The present invention also provides a kit for detecting thyroxine and its preparation method. Replacing acridine-labeled thyroxine in existing thyroxine detection kits with the thyroxine fluorescent conjugate of the present invention can improve the stability of the kit.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] In a first aspect, the present invention provides a thyroxine fluorescent conjugate having a coupling structure as shown in Formula I:

[0011]

[0012] In Formula I, AE represents an acridine derivative (preferably a group formed by removing the succinimide group from an acridine derivative); L represents a linker arm, which is a carbon chain structure with imino groups at both ends; R is H or a group containing a carbonyl group, preferably a group containing a carbonyl group.

[0013] When R is H, the structure connected to the right of L represents thyroxine; when R is a group containing a carbonyl group, the structure connected to the right of L is the structure of thyroxine substituted with a group containing a carbonyl group.

[0014] More preferably, the carbonyl group is R. 1 -CO- or R 1 -COO-, where R 1 Selected from C1-C 10 Alkyl, C3-C 10 alkenyl, C3-C 10 alkynyl group, C6-C 10 Aryl, C6-C 10 heteroaryl, C4-C 10 Silyl or C4-C 10 Siloxanes, etc.

[0015] R 1 Preferably, it is derived from methyl, ethyl, tert-butyl, allyl, or benzyl; more preferably, R 1 It is a methyl group.

[0016] According to the thyroxine fluorescent conjugate provided by the present invention, the acridine derivative is acridine sulfonamide or acridine ester, preferably acridine sulfonamide.

[0017] When the acridine derivative is an acridine sulfonamide, the preferred structure of AE in Formula I is:

[0018]

[0019] Among them, T S It refers to p-toluenesulfonyl group.

[0020] When the acridine derivative is an acridine ester, the preferred structure of AE in Formula I is:

[0021]

[0022] According to the thyroxine fluorescent conjugate provided by the present invention, the carbon chain structure of the connecting arm has less than 11 carbon atoms.

[0023] According to the thyroxine fluorescent conjugate provided by the present invention, the connecting arm has a structure shown in formula II or III:

[0024]

[0025] In Equation II, n is selected from integers 1-9;

[0026]

[0027] In Equation III, n is selected from integers 1 to 7.

[0028] Further optimization is made in Formula II, where n is selected from integers 1, 3, or 9.

[0029] Further preferably, in Formula III, n is selected from integers 1, 3, 6 or 7; even more preferably, n is selected from integers 1 or 7.

[0030] More preferably, the thyroxine fluorescent conjugate has a structure shown in formulas IV, V, VI, VII, VIII, IX, X, XI, XII, or XIII:

[0031]

[0032]

[0033]

[0034] T in equations IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII S It refers to p-toluenesulfonyl group, and AC in formulas V, VII, IX, XI, and XIII refers to acetyl group.

[0035] Among them, the kits prepared using thyroxine fluorescent conjugates having structures of formulas IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII exhibit good stability. Furthermore, the kits prepared using thyroxine fluorescent conjugates having structures of formulas V, VII, IX, XI, and XIII also demonstrate resistance to bilirubin interference.

[0036] Secondly, the present invention provides a method for preparing a thyroxine fluorescent conjugate, comprising coupling thyroxine with an acridine derivative, wherein during the coupling process, a carbon chain structure with imino groups at both ends is inserted between the thyroxine and the acridine derivative as a linker arm, thereby forming a compound having a coupling structure of thyroxine-linker arm-acridine derivative. The obtained compound is the thyroxine fluorescent conjugate described in the first aspect when R is H.

[0037] According to the preparation method of the thyroxine fluorescent conjugate provided by the present invention, in the coupling structure of the thyroxine-connector-acridine derivative, the thyroxine and the connecting arm, and the connecting arm and the acridine derivative are connected by amide bonds.

[0038] Preferably, the amide bond between the thyroxine and the linker arm is formed by the removal of a hydroxyl group from the carboxyl group of the thyroxine structure and its combination with an imino group at one end of the linker arm. The amide bond between the acridine derivative and the linker arm is formed by the breaking of a nitrogen-oxygen bond in the acridine derivative structure and its combination with an imino group at the other end of the linker arm.

[0039] According to the method for preparing thyroxine fluorescent conjugates provided by the present invention, the acridine derivative is acridine sulfonamide or acridine ester.

[0040] According to the preparation method of the thyroxine fluorescent conjugate provided by the present invention, the number of carbon atoms in the carbon chain structure of the connecting arm is less than 11.

[0041] According to the method for preparing thyroxine fluorescent conjugates provided by the present invention, the connecting arm has the structure shown in Formula II or III as described in the first aspect.

[0042] According to the method for preparing thyroxine fluorescent conjugates provided by the present invention, the method further includes amino protection treatment of the compound having a coupling structure of thyroxine-linker-acridine derivative. The thyroxine fluorescent conjugate formed after amino protection treatment is the thyroxine fluorescent conjugate described in the first aspect when R is a carbonyl group.

[0043] Preferably, the amino protection treatment involves amidation of the coupling structure of the formed thyroxine-linker-acridine derivative using an amino protecting agent. This is essentially a process of passivating the active amino group on the thyroxine structure to form a -NH-CO- group.

[0044] More preferably, the amino protecting agent may be selected from succinimide acetate, benzooxycarbonyl succinimide, allyl succinimide carbonate, di-tert-butyl dicarbonate, or N-[2-(trimethylsilyl)ethoxycarbonyloxy]succinimide, etc.

[0045] More preferably, the amino protecting agent is succinimide acetate.

[0046] Thirdly, the present invention provides the application of a thyroxine fluorescent conjugate prepared by the method described in the first aspect or the second aspect in a kit for detecting thyroxine, particularly in a free thyroxine detection kit or a total thyroxine detection kit.

[0047] Fourthly, the present invention provides a kit for detecting thyroxine, wherein the reagents of the kit contain the thyroxine fluorescent conjugate described in the first aspect or the thyroxine fluorescent conjugate prepared by the method described in the second aspect.

[0048] Preferably, the kit for detecting thyroxine includes a free thyroxine detection kit or a total thyroxine detection kit.

[0049] Further preferably, the kits for detecting thyroxine, free thyroxine detection kits, or total thyroxine detection kits mentioned in this invention all refer to kits for detecting thyroxine based on the magnetic microparticle chemiluminescence-competitive method in chemiluminescence immunoassay technology.

[0050] In some embodiments of the present invention, the free thyroxine detection kit includes the following reagents:

[0051] Reagent R1: A suspension of immunomagnetic microparticles coated with mouse anti-thyroxine monoclonal antibody;

[0052] Reagent R2: A suspension containing a quantitative fluorescent conjugate of thyroxine;

[0053] Calibrator: A series of suspensions containing thyroxine antigens at known concentration gradients.

[0054] In some embodiments of the present invention, the total thyroxine assay kit includes the following reagents:

[0055] Reagent R1: A suspension of immunomagnetic microparticles coated with mouse anti-thyroxine monoclonal antibody;

[0056] Reagent R2: A suspension containing a quantitative fluorescent conjugate of thyroxine;

[0057] Reagent R3: A suspension containing a dissociating agent;

[0058] Calibrator: A series of suspensions containing thyroxine antigens at known concentration gradients.

[0059] Fifthly, the present invention provides a method for preparing a kit for detecting thyroxine, which uses the method for preparing thyroxine fluorescent conjugates as described in the second aspect when preparing acridine-labeled thyroxine by labeling thyroxine with acridine derivatives.

[0060] Preferably, the kit for detecting thyroxine includes a free thyroxine detection kit or a total thyroxine detection kit.

[0061] In some embodiments of the present invention, the preparation method of the free thyroxine detection kit includes the following steps:

[0062] The thyroxine fluorescent conjugate was prepared using the method for preparing the thyroxine fluorescent conjugate provided in the second aspect of this invention, and reagent R2 was prepared using the thyroxine fluorescent conjugate. Reagent R1 and calibrators were then prepared using conventional methods.

[0063] In some embodiments of the present invention, the reagents R1, R2, and calibrators of the free thyroxine detection kit can be selected from the components and amounts shown in Table 1 below:

[0064] Table 1. Reagent components and dosage of the free thyroxine assay kit

[0065]

[0066] The method for preparing the free thyroxine assay kit according to the reagent components and dosages in Table 1 includes the following steps:

[0067] (1) According to the dosage of reagent R1 in Table 1, add the immunomagnetic microparticles coated with mouse anti-thyroxine monoclonal antibody, buffer, inorganic salt ions, surfactant, stabilizer, preservative and defoamer to purified water and mix evenly to obtain reagent R1.

[0068] (2) First, prepare thyroxine fluorescent conjugate using the preparation method of thyroxine fluorescent conjugate provided by the present invention. Then, add thyroxine fluorescent conjugate, buffer, inorganic salt ions, surfactant, stabilizer and preservative to purified water according to the amount of reagent R2 in Table 1 and mix evenly to obtain reagent R2.

[0069] (3) Set at least four gradient concentrations of thyroxine antigen, and mix the thyroxine antigen with buffer, inorganic salt ions, surfactant, stabilizer and preservative according to the set concentrations. Control the pH with a pH adjuster to obtain the calibrator. For example, the concentration of thyroxine antigen in the calibrator can be set to 0, 2.50, 8.00, 25.00 and 124.00 pmol / mL.

[0070] The preparation method of the total thyroxine assay kit includes the following steps:

[0071] The thyroxine fluorescent conjugate was prepared using the method for preparing the thyroxine fluorescent conjugate provided in the second aspect of this invention, and reagent R2 was prepared using the thyroxine fluorescent conjugate. Reagents R1, R3, and calibrators were then prepared using conventional methods.

[0072] In some embodiments of the present invention, the reagents R1, R2, R3, and calibrators of the total thyroxine assay kit can be selected from the components and amounts shown in Table 2 below:

[0073] Table 2. Reagent components and dosage of the total thyroxine assay kit The preparation method of the total thyroxine assay kit includes the following steps:

[0074] (1) According to the dosage of reagent R1 in Table 2, add the immunomagnetic microparticles coated with mouse anti-thyroxine monoclonal antibody, buffer, inorganic salt ions, surfactant, stabilizer, preservative and defoamer to purified water and mix evenly to obtain reagent R1.

[0075] (2) Prepare thyroxine fluorescent conjugate using the preparation method of thyroxine fluorescent conjugate provided by the present invention, and then add thyroxine fluorescent conjugate, buffer, inorganic salt ions, surfactant, stabilizer and preservative to purified water according to the amount of reagent R2 in Table 2 and mix evenly to obtain reagent R2.

[0076] (3) Add the buffer, inorganic salt ions, surfactant, dissociation agent and preservative to purified water according to the amount of reagent R3 in Table 2 and stir evenly to obtain reagent R3.

[0077] (4) Set up four or more gradient concentrations of thyroxine antigen, and mix the thyroxine antigen with buffer, inorganic salt ions, surfactant, stabilizer and preservative according to the set concentrations. Control the pH with a pH adjuster to obtain the calibrator. For example, the concentration of thyroxine antigen in the calibrator can be set to 0, 14, 37, 112 and 283 nmol / mL.

[0078] The thyroxine fluorescent conjugates mentioned in Tables 1 and 2 above refer to the thyroxine fluorescent conjugates of the first aspect or the thyroxine fluorescent conjugates prepared in the second aspect in this invention. Furthermore, the free thyroxine detection kit provided by this invention is not limited to the components and amounts shown in Table 1. Similarly, the total thyroxine detection kit is not limited to the components and amounts shown in Table 2. The selection and dosage range of conventional reagents (such as buffers, inorganic salt ions, surfactants, stabilizers, preservatives, defoamers, and pH adjusters) in the kit can be any other selection that conforms to the requirements of a thyroxine detection kit.

[0079] The beneficial effects of this invention are as follows:

[0080] This invention provides a fluorescent conjugate for thyroxine. The linker and linker arm structures of this fluorescent conjugate are relatively stable, and the coupling structure can maintain the coupling relationship for a period of time. Replacing the acridine-labeled thyroxine in existing thyroxine detection kits with this fluorescent conjugate can improve the stability of the kits; kits prepared with some thyroxine fluorescent conjugates also have strong anti-interference capabilities.

[0081] Furthermore, this invention provides a method for preparing a thyroxine fluorescent conjugate. The method proposes using an acridine derivative to conjugate thyroxine. During the conjugation process, a carbon chain structure with imino groups at both ends is inserted between the thyroxine and the acridine derivative as a linker arm, forming a compound with a thyroxine-linker arm-acridine derivative conjugate structure. The compound prepared using this method is used to formulate a reagent for thyroxine detection kits. The resulting reagent exhibits good stability and effectively improves the accuracy of the kit's detection results. The method for preparing the thyroxine fluorescent conjugate further enhances the stability of the thyroxine-linker arm-acridine derivative conjugate structure by subjecting it to amino protection. An amino protecting agent passivates the active amino groups on the conjugate structure, forming more stable -NH-CO- groups, thereby further improving the stability of the conjugate structure and the anti-interference ability of the kit. Detailed Implementation

[0082] The following examples further illustrate the technology of the present invention. These examples are illustrative and exemplary of the present invention and do not limit the scope of the invention in any way.

[0083] The coupling process of the thyroxine fluorescent conjugate used in Examples 2-5 of this invention specifically includes the following steps:

[0084] (1) Thyroxine and 9-fluorenylmethyl-N-succinimide carbonate were dissolved in a solvent and reacted at room temperature in the dark for 20-30 h. The solvent was removed and the mixture was separated by column chromatography to obtain compound 1.

[0085] (2) Dissolve the acridine derivative and the coupling agent that provides the linker structure in a solvent, mix them at room temperature on a roller mixer, and react in the dark to obtain a reaction solution.

[0086] (3) The compound 1 obtained in step (1) was dissolved in a solvent along with dicyclohexylcarbodiimide and N-hydroxysuccinimide. After reacting at room temperature in the dark for 20-30 h, the reaction solution obtained in step (2) was added and the reaction was continued in the dark for 20-30 h. The solvent was removed and the mixture was separated by column chromatography to obtain compound 2.

[0087] (4) Dissolve compound 2 and piperidine obtained in step (3) in a solvent, react at room temperature in the dark for 2-4 hours, remove the solvent, separate by column chromatography, and obtain thyroxine fluorescent conjugate.

[0088] Steps (1) and (2) can be performed in different orders or simultaneously.

[0089] The coupling agent mentioned in step (2) can be one or more compounds that can provide the coupling structure of thyroxine-linker arm-acridine derivatives with a structure as shown in formula II or III, such as ethylenediamine, butylenediamine, succinic dihydrazide, etc.

[0090] More preferably, the method for amidation treatment of the formed thyroxine fluorescent conjugate is as follows: dissolve succinimide acetate and the thyroxine fluorescent conjugate obtained above in a solvent, react at room temperature in the dark for 20-30 hours, remove the solvent, and separate by column chromatography to obtain the amidated thyroxine fluorescent conjugate.

[0091] The preferred solvent is dimethylformamide (DMF) solution.

[0092] Thyroxine comes in DL, L, and D types. The applicant used L-type thyroxine and NSP-SA-NHS as raw materials to synthesize thyroxine fluorescent conjugates and investigated their stability and anti-interference properties.

[0093] Example 1

[0094] Structural formula of T4-AE coupling:

[0095]

[0096] Synthetic route of T4-AE conjugates:

[0097]

[0098] Synthesis of T4-AE conjugates:

[0099] 50 mg NSP-SA-NHS and 84.7 mg L-thyroxine were dissolved in a 10 mL brown borosilicate glass bottle containing 5 mL DMF solution. The mixture was stirred at room temperature on a roller mixer and reacted in the dark for 24 h. DMF was removed under reduced pressure, and the product was separated by column chromatography (dichloromethane:methanol = 5:1) to obtain 59 mg of T4-AE conjugate, with a yield of 60%. HRMS (ESI) results showed C... 43 H 37 I4N3O 11 S2 m / z 1343.8108(M+H).

[0100] Example 2

[0101] Structural formula of T4-L-AE coupling a:

[0102]

[0103] Synthetic route of T4-L-AE conjugate a:

[0104]

[0105] Synthesis method of T4-L-AE conjugate a:

[0106] (1) 100 mg L-thyroxine and 52.3 mg 9-fluorenylmethyl-N-succinimide carbonate were dissolved in a thick-walled oval flask containing 15 mL of DMF solution and reacted at room temperature in the dark for 24 h. DMF was removed under reduced pressure, and the mixture was separated by column chromatography (dichloromethane:methanol = 10:1) to obtain 109.6 mg of compound 1, with a reaction yield of 85%. HRMS (ESI) showed C1... 30 H 21 I4NO6m / z 999.7620(M+H).

[0107] (2) Dissolve 61.3 mg NSP-SA-NHS and 6.6 mg butanediamine in a 10 mL brown borosilicate glass bottle containing 3 mL DMF solution, mix at room temperature on a roller mixer, and react in the dark for 24 h to obtain reaction solution A.

[0108] (3) 72.9 mg of compound 1, 30.1 mg of dicyclohexylcarbodiimide, and 16.8 mg of N-hydroxysuccinimide were dissolved in a 10 mL brown borosilicate glass bottle containing 3 mL of DMF solution. The mixture was reacted at room temperature in the dark for 24 h. Then, reaction solution A was added, and the reaction was continued in the dark for another 24 h. DMF was removed under reduced pressure, and the mixture was separated by column chromatography (dichloromethane:methanol = 10:1) to obtain 54 mg of compound 2, with a reaction yield of 45%. HRMS (ESI) results showed that C 62 H 57 I4N5O 12 S2 m / z 1635.9750(M+H).

[0109] (4) 50 mg of compound 2 and 0.6 mL of piperidine were dissolved in a 7 mL brown borosilicate glass bottle containing 3 mL of DMF solution. The mixture was reacted at room temperature in the dark for 3 h. After removing the DMF under reduced pressure, the product was separated by column chromatography (dichloromethane:methanol = 3:1) to obtain 32.9 mg of T4-L-AE conjugate a, with a reaction yield of 75%. HRMS (ESI) results showed C 47 H 48 I4N5O 10S2 m / z 1413.9213(M+H).

[0110] Example 3

[0111] Structural formula of T4-L-AE coupling a':

[0112]

[0113] The route to obtain T4-L-AE coupling a' from T4-L-AE coupling a:

[0114]

[0115] Method for obtaining T4-L-AE coupling a' from T4-L-AE coupling a:

[0116] 20 mg of T4-L-AE conjugate a and 2.7 mg of succinimide acetate were dissolved in a 7 mL brown borosilicate glass bottle containing 2 mL of DMF solution. The mixture was reacted at room temperature in the dark for 24 h. After removing the DMF under reduced pressure, the mixture was separated by column chromatography (dichloromethane:methanol = 4:1) to obtain 14 mg of T4-L-AE conjugate a', with a yield of 69%. HRMS (ESI) results showed C... 49 H 49 I4N5O 12 S2 m / z 1471.8987(M+H).

[0117] Example 4

[0118] Structural formula of T4-L-AE coupling b:

[0119]

[0120] Synthetic route of T4-L-AE conjugate b:

[0121]

[0122] Synthesis method of T4-L-AE conjugate b:

[0123] (1) 100 mg L-thyroxine and 52.3 mg 9-fluorenylmethyl-N-succinimide carbonate were dissolved in a thick-walled oval flask containing 15 mL of DMF and reacted at room temperature in the dark for 24 h. DMF was removed under reduced pressure, and the mixture was separated by column chromatography (dichloromethane:methanol = 10:1) to obtain 109.6 mg of compound 1, with a reaction yield of 85%. HRMS (ESI) results showed C 30 H 21 I4NO6 m / z 999.7620(M+H).

[0124] (2) Dissolve 61.3 mg NSP-SA-NHS and 11.0 mg succinic dihydrazide in a 10 mL brown borosilicate glass bottle containing 3 mL DMF solution, mix at room temperature on a roller mixer, and react in the dark for 24 h to obtain reaction solution B.

[0125] (3) 72.9 mg of compound 1, 30.1 mg of dicyclohexylcarbodiimide, and 16.8 mg of N-hydroxysuccinimide were dissolved in a 10 mL brown borosilicate glass bottle containing 3 mL of DMF solution. The mixture was reacted at room temperature in the dark for 24 h. Then, the above reaction solution B was added, and the reaction was continued in the dark for another 24 h. DMF was removed under reduced pressure, and the mixture was separated by column chromatography (dichloromethane:methanol = 8:1) to obtain 54 mg of compound 3, with a reaction yield of 44%. HRMS (ESI) results showed that C 62 H 55 I4N7O 14 S2 m / z 1693.9439(M+H).

[0126] (4) 40 mg of compound 3 and 0.6 mL of piperidine were dissolved in a 7 mL brown borosilicate glass bottle containing 3 mL of DMF solution. The mixture was reacted at room temperature in the dark for 3 h. After removing the DMF under reduced pressure, the product was separated by column chromatography (dichloromethane:methanol = 3:1) to obtain 27.5 mg of T4-L-AE conjugate b, with a reaction yield of 78%. HRMS (ESI) results showed C 47 H 45 I4N7O 12 S2 m / z 1471.8917(M+H).

[0127] Example 5

[0128] Structural formula of T4-L-AE coupling b':

[0129]

[0130] The route to obtain T4-L-AE coupling b' from T4-L-AE coupling b:

[0131]

[0132] Method for obtaining T4-L-AE coupling b' from T4-L-AE coupling b:

[0133] 18 mg of T4-L-AE conjugate b and 2.2 mg of succinimide acetate were dissolved in a 7 mL brown borosilicate glass bottle containing 2 mL of DMF solution. The mixture was reacted at room temperature in the dark for 24 h. After removing the DMF under reduced pressure, the product was separated by column chromatography (dichloromethane:methanol = 4:1) to obtain 13.2 mg of T4-L-AE conjugate b', with a yield of 72%. HRMS (ESI) results showed C... 49 H 47 I4N7O 14 S2 m / z 1529.9001(M+H).

[0134] Performance evaluation of couplings

[0135] To verify the performance of the T4-L-AE conjugate described in this application, the applicant used the T4-AE conjugate as a control and conducted the following reactivity studies, stability studies, and studies on interference from different concentrations of bilirubin:

[0136] (I) Reactivity Assessment

[0137] The following reagents R1, R2, and calibrators were prepared according to the reagent composition and dosage of the free thyroxine assay kit:

[0138] Reagent R1: Immunomagnetic microparticles coated with mouse anti-thyroxine monoclonal antibody 0.4 mg / mL, phosphate 3 g / L, inorganic salt ions 9 g / L, surfactant 1 mL / L, stabilizer 10 g / L, preservative 3 mL / L, solvent is purified water.

[0139] Reagent R2: 0.1 μg / mL acridine-labeled thyroxine, 3 g / L phosphate, 9 g / L inorganic salt ions, 1 mL / L surfactant, 20 g / L stabilizer, 3 mL / L preservative, and purified water as solvent. The acridine-labeled thyroxine was selected from the T4-AE conjugate of Example 1, the T4-L-AE conjugate a of Example 2, and the T4-L-AE conjugate a' of Example 3, respectively.

[0140] Calibrators: Thyroxine antigens (CalA: 0 pmol / mL, CalB: 2.56 pmol / mL, CalC: 0 pmol / mL)

[0141] 8.01 pmol / mL, CalD: 25.27 pmol / mL, CalE: 123.89 pmol / mL), phosphate 3 g / L, inorganic salt ions 9 g / L, surfactant 1 mL / L, stabilizer 50 g / L, preservative 3 mL / L.

[0142] The i3000 fully automated chemiluminescence immunoassay analyzer from Mindray Bio-Medical Electronics Co., Ltd. was used for detection, and the relative luminescence values ​​are shown in Table 3 below:

[0143] Table 3 Results of Reactivity Study

[0144]

[0145] The reactivity test results show that the kits prepared using the T4-L-AE conjugates of Examples 2 or 3 and the kits prepared using the T4-AE conjugates of Example 1 both have good reactivity; that is, using the thyroxine fluorescent conjugates provided by the present invention to prepare reagent R2 does not affect the reactivity of the kits.

[0146] (II) Stability Assessment

[0147] (1) Stability of emission signal over 7 days:

[0148] The following reagents R1, R2, and calibrators were prepared according to the reagent composition and dosage of the free thyroxine assay kit:

[0149] Reagent R1: Immunomagnetic microparticles coated with mouse anti-thyroxine monoclonal antibody 0.4 mg / mL, phosphate 3 g / L, inorganic salt ions 9 g / L, surfactant 1 mL / L, stabilizer 10 g / L, preservative 3 mL / L, solvent is purified water.

[0150] Reagent R2: 0.1 μg / mL acridine-labeled thyroxine, 3 g / L phosphate, 9 g / L inorganic salt ions, 1 mL / L surfactant, 20 g / L stabilizer, 3 mL / L preservative, and purified water as solvent. The acridine-labeled thyroxine was selected from the T4-AE conjugate of Example 1, T4-L-AE conjugate a of Example 2, T4-L-AE conjugate a' of Example 3, T4-L-AE conjugate b of Example 4, and T4-L-AE conjugate b' of Example 5, respectively.

[0151] Calibrators: phosphate 3g / L, inorganic salt ions 9g / L, surfactant 1mL / L, stabilizer 50g / L, preservative 3mL / L.

[0152] The reagent kits were placed at 37℃ for 7 days. Relative luminescence values ​​were measured using the i3000 fully automated chemiluminescence immunoassay analyzer from Mindray Bio-Medical Electronics Co., Ltd. before and after placement, yielding the first and second relative luminescence values. Then, the signal retention rate of each reagent R2 after 7 days was calculated using the formula: Signal retention rate = Second relative luminescence value / First relative luminescence value * 100%, as shown in Table 4 below.

[0153] Table 4 Results of 7-day stability study of emission signal

[0154]

[0155] Table 4 shows that the 7-day signal retention rate of the kit prepared using the T4-L-AE conjugate is significantly higher than that of the kit prepared using the T4-AE conjugate alone. Furthermore, the 7-day signal retention rate of the kit prepared using T4-L-AE conjugates a' or b' is significantly higher than that of the kit prepared using T4-L-AE conjugates a or b. This indicates that replacing the acridine-labeled thyroxine in existing thyroxine detection kits with the thyroxine fluorescent conjugate of this invention can improve the thermostability of the kit.

[0156] (2) Stability of luminescence signal under different ambient temperatures:

[0157] The following reagents R1, R2, and calibrators were prepared according to the reagent composition and dosage of the free thyroxine assay kit:

[0158] Reagent R1: Immunomagnetic microparticles coated with mouse anti-thyroxine monoclonal antibody 0.4 mg / mL, phosphate 3 g / L, inorganic salt ions 9 g / L, surfactant 1 mL / L, stabilizer 10 g / L, preservative 3 mL / L, solvent is purified water.

[0159] Reagent R2: 0.1 μg / mL acridine-labeled thyroxine, 3 g / L phosphate, 9 g / L inorganic salt ions, 1 mL / L surfactant, 20 g / L stabilizer, 3 mL / L preservative, and purified water as solvent. The acridine-labeled thyroxine used were T4-L-AE conjugate a' from Example 3 and T4-L-AE conjugate b' from Example 5, respectively.

[0160] Calibrators: phosphate 3g / L, inorganic salt ions 9g / L, surfactant 1mL / L, stabilizer 50g / L, preservative 3mL / L.

[0161] Using the i3000 fully automated chemiluminescence immunoassay analyzer from Mindray Bio-Medical Electronics Co., Ltd., the relative luminescence values ​​of the test reagents were read at ambient temperatures of 20℃, 25℃, and 30℃. With the relative luminescence value at 20℃ as 100%, the percentage of the relative luminescence values ​​at 25℃ and 30℃ relative to the relative luminescence value at 20℃ was calculated. The details are shown in Table 5 below.

[0162] Table 5 Results of stability evaluation of luminescence signal under different ambient temperatures

[0163]

[0164] As can be seen from Table 5, the kit prepared using T4-L-AE conjugate b' from Example 5 has better environmental temperature stability than the kit prepared using T4-L-AE conjugate a' from Example 3.

[0165] (III) Investigation of bilirubin interference

[0166] The following reagents R1, R2, and calibrators were prepared according to the reagent composition and dosage of the free thyroxine assay kit:

[0167] Reagent R1: Immunomagnetic microparticles coated with mouse anti-thyroxine monoclonal antibody 0.4 mg / mL, phosphate 3 g / L, inorganic salt ions 9 g / L, surfactant 1 mL / L, stabilizer 10 g / L, preservative 3 mL / L, solvent is purified water.

[0168] Reagent R2: 0.1 μg / mL acridine-labeled thyroxine, 3 g / L phosphate, 9 g / L inorganic salt ions, 1 mL / L surfactant, 20 g / L stabilizer, 3 mL / L preservative, and purified water as solvent. The acridine-labeled thyroxine used were T4-L-AE conjugate a' from Example 3, T4-L-AE conjugate b from Example 4, and T4-L-AE conjugate b' from Example 5, respectively.

[0169] Calibrators: phosphate 3g / L, inorganic salt ions 9g / L, surfactant 1mL / L, stabilizer 50g / L, preservative 3mL / L.

[0170] Different concentrations of bilirubin (1000 μmol / L, 100 μmol / L, and 10 μmol / L) were added to the calibration solution. Relative luminescence values ​​were measured using a Maccura Biotechnology i3000 fully automated chemiluminescence immunoassay analyzer before and after the addition of bilirubin. The deviations between the relative luminescence values ​​after adding different concentrations of bilirubin and the relative luminescence values ​​before adding bilirubin were calculated. The results are shown in Table 6 below.

[0171] Table 6 Results of the investigation into bilirubin interference

[0172]

[0173] When the interference of the measured value (i.e., 1 / 2Tea) is less than 12.5%, the requirements for thyroxine detection are met.

[0174] As shown in Table 6, the kit prepared using T4-L-AE conjugate b from Example 4 meets the relative luminescence deviation requirement when the bilirubin concentration is less than 100 μmol / L. The kits prepared using T4-L-AE conjugate a' from Example 3 and T4-L-AE conjugate b' from Example 5 also meet the relative luminescence deviation requirement when the bilirubin concentration is 1000 μmol / L. The kits prepared using T4-L-AE conjugate a' from Example 3 and T4-L-AE conjugate b' from Example 5 exhibit stronger resistance to bilirubin interference than the kit prepared using T4-L-AE conjugate b from Example 4.

[0175] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The embodiments describe the present invention, and it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A thyroxine fluorescent conjugate, characterized in that, The thyroxine fluorescent conjugate has the following coupling structure as shown in Formula I: Ⅰ In formula I, AE represents an acridine derivative, which is an acridine sulfonamide or an acridine ester; R is H or a group containing a carbonyl group, which is R 1 -CO- or R 1 -COO-, wherein R 1 is selected from the group consisting of C1-C 10 alkyl, C3-C 10 alkenyl, C3-C 10 alkynyl, C6-C 10 aryl, C6-C 10 heteroaryl, C4-C 10 silyl or C4-C 10 siloxy; L represents a linking arm having the structure shown in formula II or III: Ⅱ In Equation II, n is selected from integers 1-9; Ⅲ In Equation III, n is selected from integers 1-7.

2. The thyroxine fluorescent conjugate according to claim 1, characterized in that, The acridine derivative is acridine sulfonamide.

3. The thyroxine fluorescent conjugate according to claim 1, characterized in that, In Formula II, n is selected from integers 1, 3, or 9.

4. The thyroxine fluorescent conjugate according to claim 1, characterized in that, In Formula III, n is selected from integers 1, 3, 6, or 7.

5. The thyroxine fluorescent conjugate according to claim 1, characterized in that, In Equation III, n is selected from the integers 1 or 7.

6. The thyroxine fluorescent conjugate according to claim 1, characterized in that, R 1 Selected from methyl, ethyl, tert-butyl, allyl, and benzyl.

7. The thyroxine fluorescent conjugate according to claim 1, characterized in that, R 1 It is a methyl group.

8. The thyroxine fluorescent conjugate according to any one of claims 1-7, characterized in that, The structure of AE in Equation I is as follows: Wherein, Ts refers to p-toluenesulfonyl group.

9. The thyroxine fluorescent conjugate according to any one of claims 1-7, characterized in that, The structure of AE in Equation I is as follows: 。 10. The thyroxine fluorescent conjugate according to claim 1, characterized in that, The thyroxine fluorescent conjugate has a structure shown in formulas IV, V, VI, VII, VIII, IX, X, XI, XII, or XIII: Ⅳ Ⅴ Ⅵ Ⅶ Ⅷ Ⅸ Ⅹ Ⅺ Ⅻ XIII T in equations IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII S It refers to p-toluenesulfonyl group, and AC in formulas V, VII, IX, XI, and XIII refers to acetyl group.

11. A method for preparing a thyroxine fluorescent conjugate as described in any one of claims 1-10, characterized in that, The preparation method includes coupling thyroxine with an acridine derivative. During the coupling process, a carbon chain structure with imino groups at both ends is inserted between the thyroxine and the acridine derivative as a linker arm, thereby forming a compound with a coupling structure of thyroxine-linker arm-acridine derivative.

12. The preparation method according to claim 11, characterized in that, The preparation method further includes amino protection treatment of the compound having a coupling structure of thyroxine-linker-acridine derivative.

13. The preparation method according to claim 12, characterized in that, The amino protection treatment involves using an amino protectant to amidate the coupling structure of the formed thyroxine-linker-acridine derivative.

14. The preparation method according to claim 13, characterized in that, The amino protecting agent is selected from succinimide acetate, benzooxycarbonyl succinimide, allyl succinimide carbonate, ditert-butyl dicarbonate, or N-[2-(trimethylsilyl)ethoxycarbonyl]succinimide.

15. The use of a thyroxine fluorescent conjugate as described in any one of claims 1-10 or a thyroxine fluorescent conjugate prepared by the preparation method of the thyroxine fluorescent conjugate as described in any one of claims 11-14 in a kit for detecting thyroxine.

16. The application according to claim 15, characterized in that, The application is in the use of free thyroxine assay kits or total thyroxine assay kits.

17. A kit for detecting thyroxine, characterized in that, The reagents in the kit contain the thyroxine fluorescent conjugate as described in any one of claims 1-10 or the thyroxine fluorescent conjugate prepared by the preparation method described in any one of claims 11-14.

18. A method for preparing a reagent kit for detecting thyroxine, characterized in that, The preparation method is to use the preparation method of thyroxine fluorescent conjugate as described in any one of claims 11-14 when preparing acridine-labeled thyroxine by labeling thyroxine with acridine derivatives.

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