A stable antibody-drug conjugate for radioimmunoassay kits and its preparation method

Abstract

This invention discloses a stable antibody-drug conjugate for radioimmunoassay kits and its preparation method. The antibody-drug conjugate is an aqueous solution system containing hydroxypropyl-β-cyclodextrin, melatonin, L-arginine, tetrahydropyrimidine, polyvinylpyrrolidone, I-125-labeled antibody, and phosphate buffer. Melatonin forms a host-guest inclusion complex within the cavity of the hydroxypropyl-β-cyclodextrin, while L-arginine is distributed at the interface of the inclusion complex. Tetrahydropyrimidine and polyvinylpyrrolidone synergistically construct a highly viscous hydrated matrix. The preparation method employs a stepwise assembly process: first, a concentrated arginine-modified inclusion complex solution and a highly viscous hydrated matrix solution are prepared, mixed and equilibrated, and then the I-125-labeled antibody stock solution is added. This invention utilizes the inclusion complex to scavenge free radicals generated by water radiation decomposition, and prevents non-specific antibody adsorption and denaturation through interface modification and hydration layer construction. This effectively solves the problems of radiation oxidation damage and physical adsorption of I-125-labeled antibodies during liquid storage, significantly extending the shelf life of the kit and ensuring the accuracy of the detection results.

Description

Technical Field

[0001] This invention relates to the field of biomedical immunoassay technology, specifically to a stable antibody-drug conjugate for radioimmunoassay kits and its preparation method. Background Technology

[0002] Radioimmunoassay (RIA) utilizes radiolabeled antigens or antibodies for trace substance detection and is widely used in clinical diagnosis and medical research. I-125-labeled antibodies serve as crucial tracer reagents, and their stability during storage and use directly impacts the sensitivity and reproducibility of the detection system. However, I-125-labeled antibodies face complex physicochemical degradation risks in liquid environments. Due to the continuous decay of I-125, the released radiation acts on the solvent water molecules, producing a radiodecomposition effect that generates highly reactive hydroxyl radicals and superoxide anions. These radicals possess extremely strong oxidizing capabilities, attacking sensitive amino acid residues such as tyrosine and tryptophan on the antibody protein surface, leading to disulfide bond breakage or misfolding, thereby destroying the antibody's three-dimensional structure and causing it to lose its immunorecognition activity.

[0003] Besides radiation-induced chemical degradation, labeled antibodies in radioimmunoassay reagents are typically at extremely low concentrations, thermodynamically tending to undergo non-specific physical adsorption onto the container's inner wall. This adsorption not only results in the loss of effective antibody components but may also cause surface denaturation of antibody molecules, leading to increased non-specific binding rates and interfering with the accuracy of detection results. Existing conventional protection methods usually rely on adding high concentrations of bovine serum albumin or gelatin as carrier proteins and sacrificial agents. However, bio-based additives suffer from problems such as large batch-to-batch quality variations, susceptibility to microbial growth, and potential introduction of exogenous impurities. While simple chemical antioxidants can scavenge some free radicals, they often have poor stability or, at high concentrations, alter the ionic environment of the solution, or even react with antibodies. Therefore, constructing a stable system that can simultaneously block the radiation oxidation pathway and inhibit physical adsorption without introducing exogenous biological proteins is a key technical challenge that needs to be addressed in the field of radioimmunoassay. Summary of the Invention

[0004] The technical problem solved by this invention is that existing I-125 labeled antibodies face oxidative damage caused by free radicals generated by the radiolysis of water molecules during liquid storage, as well as the risk of non-specific adsorption of antibody proteins on the container wall surface, which leads to a decrease in the immunobinding activity of the kit, an increase in the non-specific binding rate, and a shortened shelf life.

[0005] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a stable antibody-conjugate for a radioimmunoassay kit, employing the following technical solution: A stable antibody-drug conjugate for use in a radioimmunoassay kit, the antibody-drug conjugate being an aqueous solution system comprising, based on the total volume of the antibody-drug conjugate, the following concentration ranges: Hydroxypropyl-β-cyclodextrin: 1.0%-3.0% (w / v); Melatonin: 2.0-6.0 mM; L-arginine: 20-50 mM; Tetrahydropyrimidine: 1.0%-3.0% (w / v); Polyvinylpyrrolidone: 0.5%-1.5% (w / v); I-125 labeled antibody; and The remaining amount of phosphate buffer.

[0006] By adopting the above technical solution, the present invention constructs a physicochemical defense system through multiple components, and the specific mechanism of action is as follows: First, hydroxypropyl-β-cyclodextrin encapsulates melatonin through a hydrophobic cavity, improving melatonin's water solubility and preventing its self-oxidation. The encapsulated melatonin continuously scavenge hydroxyl radicals and superoxide anions generated by radiation in the aqueous solution, blocking the oxidation chain reaction, protecting the tyrosine residues and disulfide bonds of the antibody protein, and maintaining the antibody's three-dimensional conformation.

[0007] Secondly, L-arginine acts as an interface modifier, binding to the host-guest inclusion complex port formed by hydroxypropyl-β-cyclodextrin and melatonin through hydrogen bonding and electrostatic interactions. The steric hindrance effect formed by L-arginine at the inclusion complex interface prevents the hydrophobic cavity of hydroxypropyl-β-cyclodextrin from contacting the hydrophobic amino acids on the antibody surface, thus preventing cyclodextrin from inducing antibody unfolding or denaturation.

[0008] Furthermore, tetrahydropyrimidine, as a compatible solute, promotes the orderly arrangement of water molecules on the surface of the antibody protein to form a dense hydrated layer. This hydrated layer increases the repulsive volume of the antibody protein, inhibits intermolecular aggregation of antibody molecules and nonspecific adsorption of antibodies to the container wall, thereby reducing background count.

[0009] Finally, polyvinylpyrrolidone increases the microviscosity of the solution system, restricts the mean free path of free radical diffusion in the solution and generates a cage effect, thereby improving the efficiency of melatonin in capturing free radicals and delaying the radiation degradation of antibodies.

[0010] Preferably, the hydroxypropyl-β-cyclodextrin has an average molecular weight of 1380-1500 Da and a molar degree of substitution of 4.0-6.0; the polyvinylpyrrolidone is selected from polyvinylpyrrolidone with a K value of 13-17, polyvinylpyrrolidone with a K value of 27-32, or a combination thereof; the tetrahydropyrimidine is (S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid; and the phosphate buffer has a pH of 7.2-7.6.

[0011] By adopting the above-mentioned preferred scheme and limiting specific parameters of cyclodextrin and polyvinylpyrrolidone, the solution system can achieve a balance between viscosity and inclusion efficiency while maintaining a clear and homogeneous phase, thus preventing polymer flocculation or phase separation.

[0012] Preferably, the melatonin, hydroxypropyl-β-cyclodextrin, and L-arginine in the antibody conjugate exist in the form of a supramolecular assembly, wherein: the melatonin molecule is located in the hydrophobic cavity of the hydroxypropyl-β-cyclodextrin to form a host-guest inclusion complex, and the L-arginine is distributed at the port interface of the host-guest inclusion complex through electrostatic or hydrogen bonding.

[0013] By adopting the above-mentioned preferred scheme, the spatial distribution structure of the free radical scavenger was established, ensuring that melatonin was bound within the cyclodextrin cavity. At the same time, L-arginine was used to seal the cyclodextrin port, thus avoiding competitive adsorption of the cyclodextrin cavity between components from a microstructural perspective.

[0014] Preferably, the final concentrations of each component in the antibody conjugate satisfy any of the following ratio schemes: Option 1: Hydroxypropyl-β-cyclodextrin 1.0% (w / v), melatonin 2.0 mM, L-arginine 20 mM, tetrahydropyrimidine 1.0% (w / v), polyvinylpyrrolidone 0.5% (w / v); Option 2: Hydroxypropyl-β-cyclodextrin 2.0% (w / v), melatonin 4.0 mM, L-arginine 35 mM, tetrahydropyrimidine 2.0% (w / v), polyvinylpyrrolidone 1.0% (w / v); Option 3: Hydroxypropyl-β-cyclodextrin 3.0% (w / v), melatonin 6.0 mM, L-arginine 50 mM, tetrahydropyrimidine 3.0% (w / v), polyvinylpyrrolidone 1.5% (w / v).

[0015] By adopting the above-mentioned preferred scheme, a verified concentration gradient is provided for application scenarios with different radiation intensities or stability requirements, ensuring the physicochemical stability of the solution system under different viscosity conditions.

[0016] Preferably, the antibody conjugate further comprises a preservative, wherein the preservative is sodium azide, and the concentration of sodium azide is 0.01%-0.1% (w / v); and the radioactivity concentration of the I-125-labeled antibody is 1-10 μCi / mL.

[0017] Secondly, the present invention provides a method for preparing a stable antibody-conjugate for a radioimmunoassay kit, employing the following technical solution: A method for preparing a stable antibody-conjugate for radioimmunoassay kits, employing a stepwise assembly process, includes the following steps: Step S1: Prepare a concentrated solution of arginine-modified inclusion complex; Step S2: Dissolve tetrahydropyrimidine and polyvinylpyrrolidone in phosphate buffer to prepare a high-viscosity hydrated matrix solution; Step S3: Mix the concentrated arginine-modified inclusion complex solution with the high-viscosity hydrated matrix solution at a volume ratio to obtain a stable matrix solution; Step S4: Add I-125 labeled antibody stock solution to the stable matrix solution, mix well, and let stand at 2-8℃ to equilibrate, thus obtaining the antibody conjugate.

[0018] By adopting the above technical solution, competitive interactions during the mixing of complex components are avoided through a step-by-step assembly process: In step S1, an inclusion complex concentrate is prepared independently to allow melatonin to preferentially enter the cyclodextrin cavity. Before introducing the antibody, L-arginine is used to modify the cyclodextrin interface to avoid direct mixing that would cause the antibody to compete with melatonin for the cyclodextrin cavity or for the antibody to come into contact with unmodified cyclodextrin.

[0019] In step S3, the inclusion complex and the high-viscosity matrix are premixed to ensure that the functional components are evenly dispersed and reach thermodynamic equilibrium.

[0020] In step S4, I-125 labeled antibody is introduced. At this time, the solution system has formed an antioxidant and hydration microenvironment. After the antibody enters, it is in a protective medium, avoiding antibody damage caused by local concentration gradients or thermal effects during the dissolution process, and improving the batch consistency of the finished product.

[0021] Preferably, the specific preparation process of the arginine-modified inclusion complex concentrate in step S1 is as follows: Step 1: Dissolve melatonin in anhydrous ethanol to prepare a melatonin ethanol solution, and dissolve hydroxypropyl-β-cyclodextrin in deionized water to prepare an aqueous solution; Step 2: Under light-protected and stirring conditions, the melatonin ethanol solution is added dropwise to the aqueous solution, and the mixture is continuously stirred to carry out the inclusion reaction, wherein the molar ratio of melatonin to hydroxypropyl-β-cyclodextrin is 1:3 to 1:5; Step 3: Remove anhydrous ethanol from the system; Step 4: Add L-arginine, adjust the pH to 7.4±0.1, and incubate for 1 hour.

[0022] By adopting the above-mentioned preferred scheme, ethanol is used as a co-solvent to promote the dispersion and inclusion of melatonin in the aqueous cyclodextrin. The subsequent removal of anhydrous ethanol eliminates the risk of antibody denaturation caused by organic solvents. The molar ratio is controlled to ensure a relative excess of cyclodextrin, thereby improving the inclusion rate of melatonin and providing sufficient cyclodextrin cavities for L-arginine binding, ensuring complete interface modification.

[0023] Preferably, the method for removing anhydrous ethanol in step three is rotary evaporation at 40°C and 0.08 MPa vacuum.

[0024] Preferably, in step S3, the mixing volume ratio of the arginine-modified inclusion complex concentrate to the high-viscosity hydrated matrix solution is 1:9; and the concentration of tetrahydropyrimidine in the high-viscosity hydrated matrix solution is 1.1%-3.3% (w / v), and the concentration of polyvinylpyrrolidone is 0.55%-1.65% (w / v).

[0025] Preferably, in step S4, the settling and balancing time is 12-24 hours; the stirring method during the mixing process is magnetic stirring, and the speed is controlled at 100-200 rpm.

[0026] By adopting the above-mentioned preferred scheme, the static equilibrium period is set to allow the hydrated layer formed by tetrahydropyrimidine and the polyvinylpyrrolidone molecular chain to complete conformational adjustment around the antibody, thereby eliminating the instantaneous stress generated by the mixing shear force and enabling the solution system to reach an energy stable state.

[0027] This invention provides a stable antibody-conjugate for radioimmunoassay kits and its preparation method. It has the following beneficial effects: 1. This invention constructs a targeted scavenging system for water radiation decomposition products by encapsulating melatonin with hydroxypropyl-β-cyclodextrin and modifying the interface with L-arginine. This system utilizes the encapsulated melatonin to efficiently capture hydroxyl radicals, blocking oxidative damage pathways. Simultaneously, the steric hindrance effect of L-arginine isolates the hydrophobic cavity of cyclodextrin from the antibody surface, mitigating the potential denaturation risk of antibodies from cyclodextrin and significantly improving the radiation tolerance and immunomodulatory retention rate of I-125-labeled antibodies in a liquid environment.

[0028] 2. This invention utilizes the synergistic effect of tetrahydropyrimidine and polyvinylpyrrolidone to improve the microscopic hydration environment and solution kinetics of antibody proteins. Tetrahydropyrimidine induces water molecules to form a dense hydration layer on the antibody surface, increasing the repulsive volume. Combined with the adjustment of solution viscosity by polyvinylpyrrolidone, this effectively inhibits the nonspecific adsorption and intermolecular aggregation of low-concentration antibody molecules to the container wall, reduces the nonspecific binding background of radioimmunoassay, and ensures the accuracy of detection results.

[0029] 3. The preparation method of this invention adopts a stepwise assembly and post-addition antibody strategy, which eliminates competitive interference and thermal effects during the multi-component mixing process. By pre-constructing thermodynamically stable arginine-modified inclusion complexes and high-viscosity matrix, and then introducing sensitive labeled antibodies, it is ensured that the antibodies directly enter the already formed protective microenvironment, avoiding local concentration shocks in the initial stage of mixing, and improving batch-to-batch consistency and initial quality of the formulation. Attached Figure Description

[0030] Figure 1 This is a flowchart of the preparation process of the present invention. Detailed Implementation

[0031] The technical solutions in 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 some embodiments of the present invention, and not all embodiments. 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.

[0032] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned (such as sodium chloride, phosphate, sodium azide, etc.) are all commercially available analytical grade or higher grade products.

[0033] The mouse anti-human insulin monoclonal antibody was a protein purified by protein A affinity chromatography at a concentration of 4.5 mg / mL, with an isoelectric point (pI) range of 5.8-6.2. It was stored in 0.01 M phosphate buffer and contained no bovine serum albumin or other exogenous carrier proteins.

[0034] Iodine-125 radionuclide solution, CAS No. 14158-31-7, chemical form is carrier-free sodium iodide (NaI), dissolved in 0.04M sodium hydroxide solution, radioactivity concentration is 100 mCi / mL, radionuclide purity is greater than 99.9%.

[0035] Hydroxypropyl-β-cyclodextrin, CAS No. 128446-35-5, average molecular weight 1380-1500 Da, molar degree of substitution (MS) range 4.0-6.0, pharmaceutical injection grade.

[0036] Melatonin, CAS No. 73-31-4, chemical name N-acetyl-5-methoxytryptamine, purity ≥99.5%, store protected from light.

[0037] Tetrahydropyrimidine, CAS No. 96702-03-3, chemical name (S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, purity ≥99.0%, microbial fermentation source.

[0038] Polyvinylpyrrolidone, CAS No. 9003-39-8, N-vinyl-2-pyrrolidone homopolymer. In this example, two specifications are used: K15 specification (K value 13-17, weight average molecular weight 8000-12000 Da) and K30 specification (K value 27-32, weight average molecular weight 40000-60000 Da).

[0039] L-Arginine, CAS No. 74-79-3, free base form, purity ≥99.0%, cell culture grade.

[0040] To fully support the generalization regarding the component content range in the claims (i.e., HP-β-CD 1.0%-3.0%, melatonin 2.0-6.0 mM, L-arginine 20-50 mM, tetrahydropyrimidine 1.0%-3.0%, PVP 0.5%-1.5%), this section provides multiple preparation examples, respectively preparing "arginine-modified inclusion complex concentrate (solution A)" and "high-viscosity hydrated matrix solution (solution B)" at different concentrations.

[0041] Preparation Example 1: Preparation of Low-Concentration Solution A (Solution A1) This preparation example provides a concentrated solution A1 for subsequent formulation, and the process steps are as follows: (1) Weigh 0.465g of melatonin (about 2mmol), dissolve it in 10mL of anhydrous ethanol, and prepare a melatonin ethanol solution; (2) Weigh 10.0g of hydroxypropyl-β-cyclodextrin and dissolve it completely in about 60mL of deionized water; (3) Under the conditions of 25°C in the dark and magnetic stirring at 500 rpm, the solution obtained in step (1) is slowly added dropwise to the aqueous solution obtained in step (2), and stirring is continued for 4 hours to form an inclusion complex; (4) Place the mixed solution in a rotary evaporator and evaporate it at 40°C and 0.08MPa vacuum for 45 minutes to completely remove ethanol; (5) Add 3.48 g L-arginine (about 20 mmol) to the rotary evaporated solution, stir to dissolve, and then adjust the pH to 7.4 ± 0.1 using phosphoric acid solution or hydrochloric acid; (6) Transfer the solution to a volumetric flask, add deionized water to make up to 100 mL, and you will get solution A1.

[0042] Parameter description: The concentration of HP-β-CD in solution A1 is 10.0% (w / v), the concentration of melatonin is 20mM, and the concentration of L-arginine is 200mM.

[0043] Preparation Example 2: Preparation of Medium Concentration Solution A (Solution A2) This preparation example provides a concentrated solution A2 for subsequent formulation, and the process steps are as follows: (1) Weigh 0.930g of melatonin (about 4mmol) and dissolve it in 20mL of anhydrous ethanol; (2) Weigh 20.0g of hydroxypropyl-β-cyclodextrin and dissolve it completely in about 50mL of deionized water; (3) Mix the two under light-protected conditions at 25°C, and continue stirring for 5 hours after the addition is complete; (4) Remove ethanol by rotary evaporation, under the same conditions as in Preparation Example 1; (5) Add 6.10 g L-arginine (about 35 mmol) to the solution, stir to dissolve and adjust the pH to 7.4 ± 0.1; (6) Add deionized water to make up to 100 mL to obtain solution A2.

[0044] Parameter description: The concentration of HP-β-CD in solution A2 is 20.0% (w / v), the concentration of melatonin is 40mM, and the concentration of L-arginine is 350mM.

[0045] Preparation Example 3: Preparation of High-Concentration Solution A (Solution A3) This preparation example provides a concentrated solution A3 for subsequent formulation, and the process steps are as follows: (1) Weigh 1.40g of melatonin (about 6mmol) and dissolve it in 30mL of anhydrous ethanol; (2) Weigh 30.0g of hydroxypropyl-β-cyclodextrin and dissolve it completely in about 40mL of deionized water; (3) Mix the two under light-protected conditions at 25°C and stir continuously for 6 hours; (4) Remove ethanol by rotary evaporation, under the same conditions as in Preparation Example 1; (5) Add 8.71 g L-arginine (about 50 mmol) to the solution, stir to dissolve and adjust the pH to 7.4 ± 0.1; (6) Add deionized water to make up to 100 mL to obtain solution A3.

[0046] Parameter description: The concentration of HP-β-CD in solution A3 is 30.0% (w / v), the concentration of melatonin is 60mM, and the concentration of L-arginine is 500mM.

[0047] Preparation Example 4: Preparation of Low-Viscosity Matrix Solution (Solution B1) This preparation example provides a matrix concentrate B1 for mixing with solution A at a volume ratio of 9:1.

[0048] (1) Prepare approximately 800 mL of 0.05 M, pH 7.4 phosphate-buffered saline (PBS); (2) Add 11.1g of tetrahydropyrimidine and stir until completely dissolved; (3) Add 5.55g of polyvinylpyrrolidone K15 and stir until completely dissolved; (4) Add 0.55g of sodium azide and stir until well mixed; (5) Adjust the volume to 1000 mL with PBS buffer and filter it through a 0.22 μm filter membrane to remove bacteria, thus obtaining solution B1.

[0049] Parameter description: When 900 mL of solution B1 is mixed with 100 mL of solution A to make 1 L of final product, the final concentration of tetrahydropyrimidine provided by solution B1 is 1.0% (w / v) and the final concentration of PVPK15 is 0.5% (w / v).

[0050] Preparation Example 5: Preparation of a medium viscosity matrix solution (solution B2) This preparation example provides a matrix concentrate B2 for mixing with solution A at a volume ratio of 9:1.

[0051] (1) Prepare approximately 800 mL of 0.05 M, pH 7.4 phosphate buffer; (2) Add 22.2g of tetrahydropyrimidine and stir to dissolve; (3) Add 11.1g of polyvinylpyrrolidone K30 and stir to dissolve; (4) Add 0.55g of sodium azide and stir until well mixed; (5) Adjust the volume to 1000 mL with PBS buffer and filter it through a 0.22 μm filter membrane to obtain solution B2.

[0052] Parameter description: When 900 mL of solution B2 is mixed with 100 mL of solution A to make 1 L of final product, the final concentration of tetrahydropyrimidine provided by solution B2 is 2.0% (w / v), and the final concentration of PVPK30 is 1.0% (w / v).

[0053] Preparation Example 6: Preparation of High-Viscosity Matrix Solution (Solution B3) This preparation example provides a matrix concentrate B3 for mixing with solution A at a volume ratio of 9:1.

[0054] (1) Prepare approximately 800 mL of 0.05 M, pH 7.4 phosphate buffer; (2) Add 33.3g of tetrahydropyrimidine and stir to dissolve; (3) Add 16.7g of polyvinylpyrrolidone K30 and stir to dissolve; (4) Add 0.55g of sodium azide and stir until well mixed; (5) Adjust the volume to 1000 mL with PBS buffer and filter it through a 0.22 μm filter membrane to obtain solution B3.

[0055] Parameter description: When 900 mL of solution B3 is mixed with 100 mL of solution A to make 1 L of final product, the final concentration of tetrahydropyrimidine provided by solution B3 is 3.0% (w / v), and the final concentration of PVPK30 is 1.5% (w / v).

[0056] Example 1: This embodiment provides a basic protective antibody conjugate and its preparation method, corresponding to the lower limit of the concentration range of each component in the claims, including the following steps: (1) Measure 900 mL of the high viscosity hydrated matrix solution (solution B1) prepared in Preparation Example 4 and place it in a clean glass container. Turn on the magnetic stirrer at room temperature (20-25℃) and set the speed to 100 rpm. (2) Slowly add 100 mL of the arginine-modified inclusion complex concentrate (solution A1) prepared in Preparation Example 1 to the above-stirred solution B1, and continue stirring for 15 minutes until the solution is mixed evenly and appears colorless and transparent; (3) Add the I-125 labeled stock solution of mouse anti-human insulin monoclonal antibody to the mixed matrix. The amount added is calculated based on the specific activity of the stock solution to make the final radioactive concentration of the system reach 5 μCi / mL. Mix it by gently inverting (avoid vigorous shaking to prevent bubbles). (4) Place the prepared solution in a chromatography cabinet at 2-8℃ and let it stand for 12 hours to allow the tetrahydropyrimidine hydrated layer and PVP molecular chain to form a stable thermodynamic distribution around the antibody, thus obtaining the final product.

[0057] Parameter description: The final concentrations of each key component in the final antibody conjugate prepared in this embodiment are as follows: Hydroxypropyl-β-cyclodextrin: 1.0% (w / v) Melatonin: 2.0 mM L-arginine: 20mM Tetrahydropyrimidine: 1.0% (w / v) Polyvinylpyrrolidone (K15): 0.5% (w / v) Example 2: This embodiment provides a standard enhanced antibody-drug conjugate and its preparation method, corresponding to the intermediate preferred values ​​of the concentration range of each component in the claims, including the following steps: (1) Measure 900 mL of the high viscosity hydrated matrix solution (solution B2) prepared in Preparation Example 5, place it in a clean container, turn on the magnetic stirrer at room temperature, and set the speed to 150 rpm; (2) Slowly add 100 mL of the arginine-modified inclusion complex concentrate (solution A2) prepared in Preparation Example 2 to the above solution B2, and stir continuously for 20 minutes to ensure that the high viscosity component is fully and evenly dispersed; (3) Add the I-125 labeled stock solution of mouse anti-human insulin monoclonal antibody to the mixed matrix, and control the final radioactive concentration to 5 μCi / mL. Mix gently. (4) Place the solution in an environment of 2-8℃ and let it stand for 16 hours to achieve uniform diffusion and locking of the host and guest inclusion complex in the viscous medium, thus obtaining the final product.

[0058] Parameter description: The final concentrations of each key component in the final antibody conjugate prepared in this embodiment are as follows: Hydroxypropyl-β-cyclodextrin: 2.0% (w / v) Melatonin: 4.0 mM L-arginine: 35mM Tetrahydropyrimidine: 2.0% (w / v) Polyvinylpyrrolidone (K30): 1.0% (w / v) Example 3: This embodiment provides a highly viscous, potent protective antibody conjugate and its preparation method, corresponding to the upper limit of the concentration range of each component in the claims, and includes the following steps: (1) Measure 900 mL of the high viscosity hydrated matrix solution (solution B3) prepared in Preparation Example 6 and place it in a clean container. Since the high viscosity hydrated matrix solution has a high viscosity, set the magnetic stirring speed to 200 rpm. (2) Slowly add 100 mL of the concentrated solution of arginine-modified inclusion complex prepared in Preparation Example 3 (solution A3) to the center of the vortex. After the addition is complete, continue stirring for 30 minutes. Observe that there are no filamentous streaks in the solution to ensure complete homogeneity. (3) Add the I-125 labeled stock solution of mouse anti-human insulin monoclonal antibody to the system, adjust the final radioactivity concentration to 5 μCi / mL, and mix gently with a paddle stirrer; (4) Place the solution in an environment of 2-8℃ and let it stand for 24 hours to overcome the diffusion resistance caused by high viscosity and ensure that the microenvironment is constructed, thus obtaining the final product.

[0059] Parameter description: The final concentrations of each key component in the final antibody conjugate prepared in this embodiment are as follows: Hydroxypropyl-β-cyclodextrin: 3.0% (w / v) Melatonin: 6.0 mM L-arginine: 50mM Tetrahydropyrimidine: 3.0% (w / v) Polyvinylpyrrolidone (K30): 1.5% (w / v) Example 4: This embodiment provides a composite viscosity-type antibody conjugate for a specific antibody type and its preparation method, aiming to verify the process adaptability of PVPs with different molecular weights, including the following steps: (1) Measure 900 mL of the matrix solution (solution B1, containing PVPK15) prepared in Preparation Example 4 and place it in a container and stir. (2) Slowly add 100 mL of the concentrated solution (solution A2, medium concentration inclusion complex) prepared in Preparation Example 2 and mix well; (3) Add an additional 5.0g of solid polyvinylpyrrolidone K30 to the above mixture, adjust the stirring speed to 200rpm, and continue stirring until the solid is completely dissolved and there are no particles left. (4) Add I-125 labeled antibody stock solution to make the final radioactive concentration 5 μCi / mL, and mix well; (5) Allow to stand at 2-8℃ for 18 hours to reach equilibrium, and the final product will be obtained.

[0060] Parameter description: The final concentrations of each key component in the final antibody conjugate prepared in this embodiment are as follows: Hydroxypropyl-β-cyclodextrin: 2.0% (w / v) Melatonin: 4.0 mM L-arginine: 35mM Tetrahydropyrimidine: 1.0% (w / v) Total polyvinylpyrrolidone: 1.0% (w / v) (composed of 0.5% PVPK15 and 0.5% PVPK30).

[0061] To comprehensively demonstrate the necessity and inventiveness of each component and process step of the "multi-dimensional linkage defense system" in this invention, this section includes seven comparative examples. These comparative examples feature omissions or substitutions of the interface passivator (arginine), diffusion inhibitor (PVP), hydration stabilizer (tetrahydropyrimidine), inclusion structure (cyclodextrin), and preparation process (stepwise assembly), respectively, for comparison in subsequent test cases.

[0062] The following comparative examples all use Example 2 (standard enhanced formulation) as the baseline control.

[0063] Comparative Example 1: This comparative example provides a conventional conjugate containing an animal-derived protein protectant. The difference from Example 2 is that, instead of using solutions A and B of the present invention, the I-125-labeled antibody was directly stored in 0.05M PBS buffer (pH 7.4) containing 1.0% (w / v) bovine serum albumin (BSA) and 0.05% (w / v) sodium azide.

[0064] Comparative Example 2: This comparative example aims to verify the role of L-arginine in passivating the cyclodextrin interface and preventing antibody adsorption. The difference from Example 2 is that L-arginine was not added during the preparation of solution A2; the pH was simply adjusted to 7.4 before volume adjustment. All other raw materials and steps remained the same.

[0065] Comparative Example 3: This comparative example aims to verify the role of polyvinylpyrrolidone (PVP) in constructing a "free radical cage effect" and inhibiting diffusion. The difference from Example 2 is that polyvinylpyrrolidone K30 was not added during the preparation of solution B2; all other raw materials and steps remained the same.

[0066] Comparative Example 4: This comparative example aims to verify the role of ectoine in maintaining the stability of the protein hydration layer. The difference from Example 2 is that ectoine was not added during the preparation of solution B2; all other raw materials and steps remained the same.

[0067] Comparative Example 5: This comparative example aims to verify the necessity of the "stepwise pre-assembly-microenvironment locking" process. The difference from Example 2 is that instead of preparing and mixing solutions A and B separately, a "one-pot" mixing method is used. Specifically, hydroxypropyl-β-cyclodextrin, melatonin (dissolved in ethanol and then added), L-arginine, tetrahydropyrimidine, and PVPK30 are added sequentially to PBS buffer and stirred to dissolve. No prolonged inclusion equilibration or stepwise incubation is performed. After pH adjustment, the antibody is added directly.

[0068] Comparative Example 6: This comparative example aims to verify the advantages of the "host-guest inclusion complex" as a long-lasting free radical scavenging center, compared with traditional small molecule antioxidants. The difference from Example 2 is that melatonin and hydroxypropyl-β-cyclodextrin are not used, but are replaced with equimolar concentrations of vitamin C (ascorbic acid), while the remaining components (PVP, tetrahydropyrimidine, arginine) remain unchanged.

[0069] Comparative Example 7: This comparative example aims to verify the necessity of "encapsulating" melatonin within cyclodextrin, i.e., to verify the effectiveness of preventing hydrophobic small molecules from directly contacting the antibody. The difference from Example 2 is that hydroxypropyl-β-cyclodextrin is not used; instead, an equal amount of melatonin is directly dissolved in a solubilizer and added to the system (maintaining a final melatonin concentration of 4.0 mM). The remaining steps are the same.

[0070] Test Example 1: Verification of Process Feasibility and Basic Physicochemical Indicators Experimental steps Fresh antibody-conjugate solutions prepared in Examples 1 to 4 were used as test samples, and freshly prepared, stabilizer-free I-125-labeled mouse anti-human insulin monoclonal antibody was used as a control. Each group of samples was placed in a colorimetric tube, and the color, transparency, and presence of suspended particles were observed under natural light against a black and white background. 500 μL of sample was taken, and the pH value of the solution was measured using a calibrated micro-pH composite electrode. The measurement was performed in triplicate, and the average value was taken. Radiochemical purity (RCP) was determined using paper chromatography. 1 μL of sample was spotted on the starting line of the chromatographic filter paper, and the mixture was developed using 85% methanol solution as the developing solvent. After air drying, the sample was sheared, and the radioactivity counts at the origin (bound state) and the leading edge (free state) were measured using a gamma counter. The percentage of the origin count relative to the total count was calculated. Maximum immunobinding rate (B0 / T) was determined using the excess antibody method. 100 μL of sample was mixed with an excess of anti-human insulin antibody solid-phase secondary antibody suspension, incubated at 37°C for 2 hours, and the precipitate was separated. The radioactivity count was then measured, and the percentage of the total added count was calculated. The nonspecific binding rate (NSB) assay is performed in the same manner as above, but the specific antibody is replaced with blank buffer and non-immunoglobulin precipitant, and the radioactivity count of the nonspecific precipitate is measured.

[0071] Results Analysis and Conclusions The data in Table 1 show that the antibody conjugates prepared in Examples 1 to 4 all meet the technical requirements in terms of physicochemical properties and immunomodulatory activity.

[0072] All samples were colorless and clear liquids, and no phase separation or precipitation was observed due to the addition of polyvinylpyrrolidone or cyclodextrin. The pH value was maintained between 7.38 and 7.43, indicating good buffering capacity of the system, and the addition of L-arginine did not cause significant fluctuations in pH. The radiochemical purity of all samples was higher than 96.5%, with no statistically significant difference compared to the control, indicating that the preparation process conditions were mild and the microenvironment during the mixing of solution A and solution B did not cause oxidative loss or replacement of iodine atoms on the labeled antibody.

[0073] Regarding immunomodulatory indicators, the maximum binding rate (BO / T) of the sample examples ranged from 47.6% to 48.2%, which was very close to the 48.5% of the control. This result indicates that hydroxypropyl-β-cyclodextrin in the system did not mask or alter the conformation of the antibody active site. This is attributed to the competitive distribution of L-arginine in the system; the electrostatic or hydrogen bonding layer formed by arginine molecules at the cyclodextrin port hinders direct contact between the hydrophobic cavity of the cyclodextrin and the hydrophobic amino acid residues on the antibody surface. Furthermore, the nonspecific binding rate (NSB) of each group was controlled below 2.5%. Even in Example 3, which contained a higher concentration of PVP, no significant increase in NSB was observed, indicating that the hydration layer constructed with tetrahydropyrimidine effectively maintained the dispersibility of the antibody in the colloidal solution, avoiding nonspecific aggregation or tube wall adsorption that might be caused by polymers.

[0074] In summary, this preparation process can obtain a homogeneous and stable solution system, in which each component performs its respective physicochemical function without having an immediate negative impact on the structural integrity and immune recognition function of the antibody.

[0075] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A stable antibody-drug conjugate for use in radioimmunoassay kits, characterized in that, The antibody conjugate is an aqueous solution system, and based on the total volume of the antibody conjugate, it contains components in the following concentration ranges: Hydroxypropyl-β-cyclodextrin: 1.0%-3.0% (w / v); Melatonin: 2.0-6.0 mM; L-arginine: 20-50mM; Tetrahydropyrimidine: 1.0%-3.0% (w / v); Polyvinylpyrrolidone: 0.5%-1.5% (w / v); I-125 labeled antibody; and The remaining amount of phosphate buffer.

2. The stable antibody-drug conjugate for a radioimmunoassay kit according to claim 1, characterized in that, The specifications and characteristics of each component are as follows: The hydroxypropyl-β-cyclodextrin has an average molecular weight of 1380-1500 Da and a molar degree of substitution of 4.0-6.

0. The polyvinylpyrrolidone is selected from polyvinylpyrrolidone with a K value of 13-17, polyvinylpyrrolidone with a K value of 27-32, or a combination thereof; The tetrahydropyrimidine is (S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid; The pH value of the phosphate buffer solution is 7.2-7.

6.

3. The stable antibody-drug conjugate for a radioimmunoassay kit according to claim 1, characterized in that, Melatonin, hydroxypropyl-β-cyclodextrin, and L-arginine in the antibody-drug conjugate exist in the form of a supramolecular assembly, wherein: Melatonin molecules form a host-guest inclusion complex within the hydrophobic cavity of hydroxypropyl-β-cyclodextrin, and L-arginine is distributed at the port interface of the host-guest inclusion complex via electrostatic or hydrogen bonding.

4. A stable antibody-drug conjugate for a radioimmunoassay kit according to claim 1, characterized in that, The final concentrations of each component in the antibody conjugate meet any of the following ratio schemes: Option 1: Hydroxypropyl-β-cyclodextrin 1.0% (w / v), melatonin 2.0 mM, L-arginine 20 mM, tetrahydropyrimidine 1.0% (w / v), polyvinylpyrrolidone 0.5% (w / v); Option 2: Hydroxypropyl-β-cyclodextrin 2.0% (w / v), melatonin 4.0 mM, L-arginine 35 mM, tetrahydropyrimidine 2.0% (w / v), polyvinylpyrrolidone 1.0% (w / v); Option 3: Hydroxypropyl-β-cyclodextrin 3.0% (w / v), melatonin 6.0 mM, L-arginine 50 mM, tetrahydropyrimidine 3.0% (w / v), polyvinylpyrrolidone 1.5% (w / v).

5. A stable antibody-conjugate for a radioimmunoassay kit according to any one of claims 1-4, characterized in that, The antibody conjugate also contains a preservative, which is sodium azide at a concentration of 0.01%-0.1% (w / v); the radioactivity concentration of the I-125-labeled antibody is 1-10 μCi / mL.

6. A method for preparing a stable antibody-drug conjugate for a radioimmunoassay kit, based on a stable antibody-drug conjugate for a radioimmunoassay kit according to any one of claims 1-5, characterized in that, The process employs a step-by-step assembly method, including the following steps: Step S1: Prepare a concentrated solution of arginine-modified inclusion complex; Step S2: Dissolve tetrahydropyrimidine and polyvinylpyrrolidone in phosphate buffer to prepare a high-viscosity hydrated matrix solution; Step S3: Mix the concentrated arginine-modified inclusion complex solution with the high-viscosity hydrated matrix solution at a volume ratio to obtain a stable matrix solution; Step S4: Add I-125 labeled antibody stock solution to the stable matrix solution, mix well, and let stand at 2-8℃ to equilibrate, thus obtaining the antibody conjugate.

7. The method for preparing a stable antibody-conjugate for a radioimmunoassay kit according to claim 6, characterized in that, The specific preparation process of the arginine-modified inclusion complex concentrate in step S1 is as follows: Step 1: Dissolve melatonin in anhydrous ethanol to prepare a melatonin ethanol solution, and dissolve hydroxypropyl-β-cyclodextrin in deionized water to prepare an aqueous solution; Step 2: Under light-protected and stirring conditions, the melatonin ethanol solution is added dropwise to the aqueous solution, and the mixture is continuously stirred to carry out the inclusion reaction, wherein the molar ratio of melatonin to hydroxypropyl-β-cyclodextrin is 1:3 to 1:5; Step 3: Remove anhydrous ethanol from the system; Step 4: Add L-arginine, adjust the pH to 7.4±0.1, and incubate for 1 hour.

8. The method for preparing a stable antibody-conjugate for a radioimmunoassay kit according to claim 7, characterized in that, The method for removing anhydrous ethanol in step three is rotary evaporation at 40°C and 0.08 MPa vacuum.

9. A stable antibody-conjugate for a radioimmunoassay kit according to claim 6, and its preparation method thereof, characterized in that, In step S3, the mixing volume ratio of the arginine-modified inclusion complex concentrate to the high-viscosity hydrated matrix solution is 1:9; and the concentration of tetrahydropyrimidine in the high-viscosity hydrated matrix solution is 1.1%-3.3% (w / v), and the concentration of polyvinylpyrrolidone is 0.55%-1.65% (w / v).

10. A method for preparing a stable antibody-conjugate for a radioimmunoassay kit according to claim 6, characterized in that, In step S4, the settling and balancing time is 12-24 hours; the stirring method during the mixing process is magnetic stirring, and the speed is controlled at 100-200 rpm.

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