A freeze-dried preparation of horseradish peroxidase-labeled antibody and a method for its preparation

Abstract

The application discloses a horseradish peroxidase-labeled antibody freeze-drying preparation and a preparation method thereof, relates to the technical field of biological detection, and the freeze-drying preparation comprises a horseradish peroxidase-labeled antibody, a protein protective agent and a buffer; the protein protective agent comprises the following preparation raw materials in parts by weight: 30-40 parts of trehalose, 0.5-2 parts of oxidized sodium alginate, 0.1-0.4 parts of 2, 4, 6-tris (4-aminophenyl) -1, 3, 5-triazine, 8-12 parts of betaine, 3-6 parts of proline, 0.5-0.7 parts of bovine serum albumin, 2-2.5 parts of glycine, 0.25-0.35 parts of beta-cyclodextrin, 0.5-0.7 parts of a reducing agent and 0.25-0.4 parts of a surfactant. The application has the effects of improving the storage stability and activity recovery rate of the horseradish peroxidase-labeled antibody.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and in particular to a horseradish peroxidase-labeled antibody freeze-dried preparation and its preparation method. Background Technology

[0002] Horseradish peroxidase (HRP)-labeled antibodies are among the most widely used enzyme-labeled antibodies in the field of immunoassay, playing a crucial role in applications such as enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, Western blotting, and biosensors. HRP's advantages, including high catalytic efficiency, a wide variety of substrates, and high detection sensitivity, make it an indispensable core raw material in in vitro diagnostics, food safety testing, life science research, and clinical testing.

[0003] Currently, commercially available HRP-labeled antibodies are mainly supplied in liquid form. However, liquid enzyme-labeled antibodies face several technical bottlenecks in practical applications. First, their stability is poor; the active site of the HRP enzyme is prone to auto-oxidation, and the antibody protein is also susceptible to conformational changes or aggregation in a liquid environment, leading to activity degradation. Typically, the shelf life of liquid HRP-labeled antibodies under 4°C refrigeration conditions is no more than 6 months, and at room temperature, they can only maintain activity for 1-2 weeks. Repeated freeze-thaw cycles result in even more severe activity loss. Second, liquid reagents are highly dependent on cold chain transportation and storage. The entire process from production to end-use requires maintaining a low-temperature environment of 2-8°C, resulting in high logistics costs and making normal use difficult in grassroots units, remote areas, or when the cold chain is interrupted. In addition, long-term storage of liquid systems also carries the risk of microbial contamination, further limiting their application scope.

[0004] Preparing HRP-labeled antibodies into lyophilized formulations is an effective way to solve the above problems. Lyophilization technology removes moisture under low-temperature vacuum conditions, allowing the product to remain stable at room temperature for a long time and significantly reducing dependence on the cold chain. However, during the freeze-drying process, factors such as ice crystal formation, gas-liquid interface, and ice-liquid interface stress can easily cause irreversible conformational damage to HRP and antibody proteins, resulting in a significant decrease in activity. Summary of the Invention

[0005] To improve activity and stability, this application provides a horseradish peroxidase-labeled antibody lyophilized formulation and its preparation method.

[0006] In a first aspect, this application provides a horseradish peroxidase-labeled antibody lyophilized formulation, which adopts the following technical solution: A horseradish peroxidase-labeled antibody lyophilized formulation comprises a horseradish peroxidase-labeled antibody, a protein protectant, and a buffer solution; the protein protectant comprises the following raw materials in parts by weight: trehalose 30-40 parts, sodium alginate 0.5-2 parts, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine 0.1-0.4 parts, betaine 8-12 parts, proline 3-6 parts, bovine serum albumin 0.5-0.7 parts, glycine 2-2.5 parts, β-cyclodextrin 0.25-0.35 parts, reducing agent 0.5-0.7 parts, and surfactant 0.25-0.4 parts.

[0007] By adopting the above technical solution, this application constructs a rigid-flexible three-dimensional network structure. This network works synergistically with the trehalose-betaine-proline ternary glassy system to form a multi-dimensional protective barrier, thereby significantly improving the storage stability of horseradish peroxidase-labeled antibodies before and after freeze-drying, and thus improving the activity recovery rate. Specifically, the aldehyde group of oxidized sodium alginate and the amino group of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine undergo a mild Schiff base cross-linking reaction during freeze-drying to form an interpenetrating network structure, providing mechanical support and molecular confinement. Betaine maintains the hydration layer on the surface of horseradish peroxidase through preferential hydration, and proline acts as a plasticizer to regulate the glassy flexibility. Both can work synergistically with trehalose to reduce the damage to protein conformation caused by ice crystals and interfacial stress during freeze-drying. In addition, the triazine ring of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine can form π-π stacking and hydrogen bond interactions with amino acid residues on the surface of horseradish peroxidase, further synergistically stabilizing the protein conformation; bovine serum albumin, polysorbate-20, and β-cyclodextrin act as interfacial protectants, which can synergistically reduce the risk of protein denaturation at gas-liquid or solid-liquid interfaces; ascorbic acid, as a reducing agent, can scavenge reactive oxygen free radicals in the system and protect horseradish peroxidase and antibodies from oxidative damage; glycine directly participates in stabilizing the native conformation of the protein.

[0008] Optionally, the weight ratio of the oxidized sodium alginate to 2,4,6-tris(4-aminophenyl)-1,3,5-triazine is (3-5):1.

[0009] By employing the above-mentioned technical solution, this application utilizes a specific weight ratio of oxidized sodium alginate and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine to achieve synergistic optimization of cross-linking network rigidity and antibody activity. Specifically, the aldehyde group of oxidized sodium alginate and the amino group of the triazine compound form a moderately cross-linked three-dimensional network during freeze-drying. This network provides sufficient mechanical support for horseradish peroxidase-labeled antibodies through molecular confinement effects, inhibiting ice crystal growth and interfacial stress-induced protein conformational damage. Furthermore, it avoids excessively dense networks or over-anchoring of antibodies due to excessive cross-linking, ensuring rapid antibody release after reconstitution and maintaining the integrity and accessibility of the antigen-binding site and the horseradish peroxidase active site. Simultaneously, at this ratio, the triazine ring can form stable π-π stacking and hydrogen bond interactions with amino acid residues on the antibody surface, further synergistically stabilizing the protein conformation, thereby significantly improving freeze-drying activity recovery and long-term storage stability.

[0010] Optionally, the weight ratio of betaine to proline is (2-4):1.

[0011] By adopting the above technical solution, this application uses a specific weight ratio of betaine and proline to achieve a synergistic effect between "preferential hydration stability" and "glassy flexibility regulation." Specifically, betaine maintains an intact hydration layer on the surface of the horseradish peroxidase-labeled antibody through a preferential hydration effect, effectively resisting the damage to the protein's native conformation caused by ice crystal formation and dehydration stress during freeze-drying; proline, as a plasticizer, moderately reduces the brittleness of the glassy matrix, preventing microcracks from forming in the glassy state during storage and thus damaging the protein microenvironment.

[0012] Optionally, the preparation steps of the oxidized sodium alginate include: mixing sodium alginate and phosphate buffer, adding sodium periodate solution, stirring evenly, reacting in the dark, adding ethylene glycol to terminate the reaction, stirring evenly, dialyzing, and freeze-drying to obtain oxidized sodium alginate.

[0013] By adopting the above technical solution, sodium alginate is selectively oxidized using sodium periodate, and the ortho-dihydroxyl groups on the sodium alginate sugar units are controllably cleaved into aldehyde groups under light-protected conditions, thereby providing reactive sites for subsequent cross-linking reactions.

[0014] Optionally, the reducing agent is ascorbic acid.

[0015] By adopting the above technical solution and selecting ascorbic acid as a reducing agent, reactive oxygen free radicals generated in the system by sodium alginate residue, metal ion catalysis or photothermal induction can be efficiently removed during the freeze-drying process and long-term storage. This effectively inhibits the oxidative inactivation of the iron porphyrin group in the active center of horseradish peroxidase, while preventing the side chain modification and conformational changes of easily oxidized residues such as methionine and cysteine ​​in antibody proteins.

[0016] Optionally, the surfactant is at least one of polysorbate-20, polysorbate-60, and polysorbate-80.

[0017] By adopting the above technical solution, this application selects at least one of polysorbate-20, polysorbate-60, and polysorbate-80 as a surfactant. The unique structural characteristics of these nonionic surfactants can play a synergistic interfacial protection role throughout the freeze-drying process and after reconstitution. These surfactants have suitable hydrophilic-lipophilic balance values ​​(HLB values ​​of 14-17). Through competitive adsorption of hydrophobic tail chains at gas-liquid, ice-liquid, and solid-liquid interfaces, they significantly reduce interfacial tension and effectively prevent horseradish peroxidase-labeled antibodies from being directly exposed to high-stress interfaces and undergoing conformational unfolding or irreversible aggregation. At the same time, their polyoxyethylene hydrophilic head chains can form a steric barrier around antibody molecules, inhibiting the mutual approach and aggregation of protein molecules during freeze-drying concentration.

[0018] Secondly, this application provides a method for preparing a horseradish peroxidase-labeled antibody lyophilized formulation, using the following technical solution: A method for preparing a horseradish peroxidase-labeled antibody lyophilized formulation includes the following steps: Trehalose, betaine, proline, bovine serum albumin, glycine, β-cyclodextrin, reducing agent, and surfactant were mixed, buffer was added, and the mixture was stirred evenly. Horseradish peroxidase-labeled antibody was added, and the mixture was stirred evenly. Then, sodium alginate and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine were added, and the mixture was ultrasonically dispersed, pre-frozen, annealed, and dried to obtain the lyophilized horseradish peroxidase-labeled antibody preparation.

[0019] Optionally, the stirring speed is 300-500 rpm, the ultrasonic power is 400W-600W, the pre-freezing temperature is -60~-40℃, and the annealing temperature is -10~-30℃.

[0020] Thirdly, this application provides the application of a horseradish peroxidase-labeled antibody lyophilized preparation in the preparation of an immunoassay kit.

[0021] In summary, this application includes at least one of the following beneficial technical effects: 1. This application constructs a rigid-flexible three-dimensional network structure. This network, in synergy with the trehalose-betaine-proline ternary glassy system, forms a multi-dimensional protective barrier, thereby significantly improving the activity recovery rate and long-term storage stability of horseradish peroxidase-labeled antibodies before and after freeze-drying. Specifically, the aldehyde group of oxidized sodium alginate undergoes a mild Schiff base cross-linking reaction with the amino group of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine during freeze-drying, forming an interpenetrating network structure that provides mechanical support and molecular confinement. Betaine maintains the hydration layer on the surface of horseradish peroxidase through preferential hydration, while proline acts as a plasticizer to regulate the glassy flexibility. Both of these components synergistically work with trehalose to reduce the damage to protein conformation caused by ice crystals and interfacial stress during freeze-drying. Furthermore, the triazine ring of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine can form π-π stacking and hydrogen bond interactions with amino acid residues on the surface of horseradish peroxidase, further synergistically stabilizing the protein conformation; bovine serum albumin, polysorbate-20, and β-cyclodextrin act as interfacial protectants, synergistically reducing the risk of protein denaturation at gas-liquid or solid-liquid interfaces; ascorbic acid, as a reducing agent, can scavenge reactive oxygen free radicals in the system, protecting horseradish peroxidase and antibodies from oxidative damage; and glycine directly participates in stabilizing the native conformation of the protein. 2. This application utilizes a specific weight ratio of oxidized sodium alginate and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine to achieve synergistic optimization of cross-linking network rigidity and antibody activity. Specifically, the aldehyde group of oxidized sodium alginate and the amino group of the triazine compound form a moderately cross-linked three-dimensional network during freeze-drying. This network provides sufficient mechanical support for horseradish peroxidase-labeled antibodies through molecular confinement effects, inhibiting ice crystal growth and interfacial stress-induced protein conformational damage. It also avoids excessively dense networks or over-anchoring of the antibody due to excessive cross-linking, ensuring rapid antibody release after reconstitution and maintaining the integrity and accessibility of the antigen-binding site and the horseradish peroxidase active site. Simultaneously, at this ratio, the triazine ring can form stable π-π stacking and hydrogen bond interactions with amino acid residues on the antibody surface, further synergistically stabilizing the protein conformation, thereby significantly improving freeze-drying activity recovery and long-term storage stability. 3. This application employs a specific weight ratio of betaine and proline, which can achieve a synergistic effect between "preferential hydration stability" and "glassy flexibility regulation." Specifically, betaine maintains an intact hydration layer on the surface of the horseradish peroxidase-labeled antibody through a preferential hydration effect, effectively resisting the damage to the protein's native conformation caused by ice crystal formation and dehydration stress during freeze-drying; proline, acting as a plasticizer, moderately reduces the brittleness of the glassy matrix, preventing microcracks from forming in the glassy state during storage and thus damaging the protein microenvironment. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] This application discloses a horseradish peroxidase-labeled antibody lyophilized formulation, comprising a horseradish peroxidase-labeled antibody, a protein protectant, and a buffer solution; the protein protectant comprises the following raw materials in parts by weight: trehalose 30-40 parts, sodium alginate 0.5-2 parts, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine 0.1-0.4 parts, betaine 8-12 parts, proline 3-6 parts, bovine serum albumin 0.5-0.7 parts, glycine 2-2.5 parts, β-cyclodextrin 0.25-0.35 parts, reducing agent 0.5-0.7 parts, and surfactant 0.25-0.4 parts.

[0024] This application discloses a method for preparing a horseradish peroxidase-labeled antibody lyophilized formulation, comprising the following steps: Trehalose, betaine, proline, bovine serum albumin, glycine, β-cyclodextrin, reducing agent, and surfactant are mixed, and solvent is added. The mixture is stirred at 300-500 rpm for 10-30 min. Horseradish peroxidase-labeled antibody is added, and the mixture is stirred at 300-500 rpm for 5-10 min. Then, sodium alginate and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine are added. The mixture is ultrasonically dispersed at 400-600 W for 30-60 min, pre-frozen at -60~-40℃ for 3-5 h, annealed to -10~-30℃ and held for 12-15 h, and then dried once at -60~-40℃ in a freeze dryer for 4-6 h. The refrigeration system is then turned off, and the mixture is vacuum dried at 0.2-0.3 mbar for 12-15 h. Finally, the mixture is heated to 10-15℃ at 0.2-0.3 mbar and vacuum dried for 2-4 h to obtain the horseradish peroxidase-labeled antibody lyophilized formulation.

[0025] All raw materials used in the embodiments of this application are commercially available, wherein: Horseradish peroxidase-labeled secondary antibody, Wuhan Boster Biological Engineering Co., Ltd. Bovine serum albumin, Sigma-Aldrich (Germany); Trehalose, Shanghai Aladdin Biochemical Technology Co., Ltd.; Polysorbate-20, Beijing Wokai Biotechnology Co., Ltd.; Sucrose, Shanghai Aladdin Biochemical Technology Co., Ltd.; Glycine, purity ≥99%, Shanghai Aladdin Biochemical Technology Co., Ltd.; β-Cyclodextrin, purity ≥98%, Shanghai Aladdin Biochemical Technology Co., Ltd. Tris(hydroxymethyl)aminomethane buffer, pH 7.4, Hubei Xindesheng Materials Technology Co., Ltd.; Sodium alginate, Shanghai Aladdin Biochemical Technology Co., Ltd.; Phosphate buffer, Shanghai Coleman Reagent Co., Ltd.; Sodium periodate, Shanghai Aladdin Biochemical Technology Co., Ltd.; Ethylene glycol, Shanghai Aladdin Biochemical Technology Co., Ltd.; 2,4,6-Tris(4-aminophenyl)-1,3,5-triazine, Shanghai Coleman Reagent Co., Ltd.; Betaine, Beijing Solarbio Technology Co., Ltd.; Proline, Shanghai Coleman Reagent Co., Ltd. Ascorbic acid, purity ≥97%, Shanghai Aladdin Biochemical Technology Co., Ltd.

[0026] Preparation Example 1 Preparation of oxidized sodium alginate: Mix 5g of sodium alginate with 200mL of phosphate buffer, add 20mL of sodium periodate solution (made by mixing 2g of sodium periodate and 20mL of deionized water), stir at 300rpm for 10min, react in the dark for 8h, add 2mL of ethylene glycol to terminate the reaction, stir at 300rpm for 30min, put into a dialysis bag with a molecular weight cutoff of 10-15kDa, change the water every day, dialyze for 3 days, take it out, vacuum dry at -80℃ for 4h, vacuum dry at -40℃ for 24h, and then desorb and dry at 20℃ for 8h to obtain oxidized sodium alginate.

[0027] Example 1 Mix 30g trehalose, 8g betaine, 4g proline, 0.5g bovine serum albumin, 2g glycine, 0.25g β-cyclodextrin, 0.5g ascorbic acid, and 0.25g polysorbate-60. Add 100mL of tris(hydroxymethyl)aminomethane buffer (a mixture of 0.121g tris(hydroxymethyl)aminomethane and 100mL ultrapure water), and stir at 300rpm for 10min. Add 50μL of horseradish peroxidase-labeled antibody, and stir at 300rpm for 5min. Then add 0.5g of sodium alginate obtained in Preparation Example 1 and 0.1g... 2,4,6-Tris(4-aminophenyl)-1,3,5-triazine was ultrasonically dispersed at 400W for 60 min, pre-frozen at -40℃ for 5 h, annealed to -10℃ and held for 15 h, dried once at -40℃ in a freeze dryer for 6 h, the refrigeration system was turned off, and vacuum drying was maintained at 0.2 mbar for 15 h, and then vacuum dried at 0.2 mbar and 10℃ for 4 h to obtain the horseradish peroxidase-labeled antibody lyophilized preparation.

[0028] Example 2 Mix 35g trehalose, 10g betaine, 5g proline, 0.6g bovine serum albumin, 2.25g glycine, 0.3g β-cyclodextrin, 0.6g ascorbic acid, and 0.3g polysorbate-20. Add 100mL of tris(hydroxymethyl)aminomethane buffer (a mixture of 0.121g tris(hydroxymethyl)aminomethane and 100mL ultrapure water), and stir at 400rpm for 8 minutes. Add 50μL of horseradish peroxidase-labeled antibody, and stir at 400rpm for 8 minutes. Then add 1g of sodium alginate obtained in Preparation Example 1 and 0.2g... 2,4,6-Tris(4-aminophenyl)-1,3,5-triazine was ultrasonically dispersed at 500W for 45 min, pre-frozen at -50℃ for 4 h, annealed to -20℃ and held for 13 h, dried once at -50℃ in a freeze dryer for 5 h, the refrigeration system was turned off, and vacuum drying was maintained at 0.25 mbar for 13 h, and then vacuum dried at 0.25 mbar and 13℃ for 3 h to obtain the horseradish peroxidase-labeled antibody lyophilized preparation.

[0029] Example 3 Mix 40g trehalose, 12g betaine, 6g proline, 0.7g bovine serum albumin, 2.5g glycine, 0.35g β-cyclodextrin, 0.7g ascorbic acid, and 0.4g polysorbate-80. Add 100mL of tris(hydroxymethyl)aminomethane buffer (a mixture of 0.121g tris(hydroxymethyl)aminomethane and 100mL ultrapure water), and stir at 400rpm for 8min. Add 50μL of horseradish peroxidase-labeled antibody, and stir at 500rpm for 5min. Then add 2g of sodium alginate obtained in Preparation Example 1 and 0.4g... 2,4,6-Tris(4-aminophenyl)-1,3,5-triazine was ultrasonically dispersed at 600W for 30 min, pre-frozen at -60℃ for 3 h, annealed to -30℃ and held for 12 h, dried once at -60℃ in a freeze dryer for 3 h, the refrigeration system was turned off, and vacuum drying was maintained at 0.3 mbar for 12 h, and then vacuum dried at 0.3 mbar and 15℃ for 2 h to obtain the horseradish peroxidase-labeled antibody lyophilized preparation.

[0030] Examples 4-5 Based on Example 2, except for the weight ratio of sodium alginate and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine, the other components and preparation methods are the same as in Example 2, and the total weight of sodium alginate and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine remains unchanged.

[0031] Example 4 The difference between this embodiment and Embodiment 2 is that the weight ratio of sodium alginate oxide to 2,4,6-tris(4-aminophenyl)-1,3,5-triazine in this embodiment is 4:1. Specifically, the weight of sodium alginate oxide is 0.96g and the weight of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine is 0.24g.

[0032] Example 5 The difference between this embodiment and Embodiment 2 is that the weight ratio of sodium alginate oxide to 2,4,6-tris(4-aminophenyl)-1,3,5-triazine in this embodiment is 3:1. Specifically, the weight of sodium alginate oxide is 0.9g and the weight of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine is 0.3g.

[0033] Examples 6-7 Based on Example 4, except for the weight ratio of betaine and proline, the other components and preparation methods are the same as in Example 4, and the total weight of betaine and proline remains unchanged.

[0034] Example 6 The difference between this embodiment and embodiment 4 is that the weight ratio of betaine to proline in this embodiment is 3:1. Specifically, the weight of betaine is 11.25g and the weight of proline is 3.75g.

[0035] Example 7 The difference between this embodiment and embodiment 4 is that the weight ratio of betaine to proline in this embodiment is 4:1. Specifically, the weight of betaine is 12g and the weight of proline is 3g.

[0036] Comparative Example 1 The difference between this comparative example and Example 2 is that the sodium oxidized alginate in Example 2 is replaced by 2,4,6-tris(4-aminophenyl)-1,3,5-triazine.

[0037] Comparative Example 2 The difference between this comparative example and Example 2 is that in this comparative example, the 2,4,6-tris(4-aminophenyl)-1,3,5-triazine in Example 2 is replaced by sodium alginate.

[0038] Comparative Example 3 The difference between this comparative example and Example 2 is that in this comparative example, betaine and other substances in Example 2 are replaced with proline.

[0039] Comparative Example 4 The difference between this comparative example and Example 2 is that the mass of proline in Example 2 is replaced with betaine.

[0040] Performance Test 1: Activity Recovery Rate Test The activity recovery rate of the samples prepared in Examples 1-7 and Comparative Examples 1-4 was tested, and the results are shown in Table 1.

[0041] Activity recovery test method: The horseradish peroxidase-labeled antibody lyophilized preparations obtained in Examples 1-3 and Comparative Examples 1-4 were reconstituted using 10 mL of reconstitution solution (phosphate buffer containing 1% bovine serum albumin). After reconstitution, the samples were subjected to ELISA plate experiments together with chloramphenicol antigen ELISA plates, chloramphenicol monoclonal antibody, and chloramphenicol standards, with 4 replicates for each. ELISA plate experiment: 50 μL of sample was added to each of the 4 wells of the chloramphenicol antigen ELISA plate, followed by 50 μL / well of chloramphenicol standard (chloramphenicol concentration of 0 ng / mL) and 50 μL / well of chloramphenicol monoclonal antibody, and reacted at 25°C for 30 min. After washing the plates according to the procedure, 100 μL / well of 3,3',5,5'-tetramethylbenzidine chromogenic solution was added to the microplates, and reacted at 25°C for 15 min. The reaction was terminated by adding stop solution, and the OD value was read at a wavelength of 450 nm on the ELISA reader. The average OD value of each sample was calculated. m A control group consisting of samples that were not lyophilized was also set up. ELISA plate tests were performed according to the above protocol, and the average OD value (OD0) of the control group was obtained. Activity recovery rate = (OD0 - OD0) m / OD0)×100%. The specific preparation steps of the control group are as follows: 35g trehalose, 10g betaine, 5g proline, 0.6g bovine serum albumin, 2.25g glycine, 0.3g β-cyclodextrin, 0.6g ascorbic acid, and 0.3g polysorbate-20 were mixed and 100mL of tris(hydroxymethyl)aminomethane buffer (0.121g tris(hydroxymethyl)aminomethane and 100mL ultrapure water were mixed). The mixture was stirred at 400rpm for 8min. 50μL of horseradish peroxidase-labeled antibody was added and stirred at 400rpm for 8min. Then, 1g of sodium alginate obtained in Preparation Example 1 and 0.2g of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine were added and the mixture was ultrasonically dispersed at 500W for 45min to obtain the horseradish peroxidase-labeled antibody solution.

[0042] Table 1. Activity recovery rates of lyophilized formulations in Examples 1-7 and Comparative Examples 1-4

[0043] As can be seen from Examples 1-3 and Table 1, the activity recovery rate of the horseradish peroxidase-labeled antibody lyophilized formulation of this application is above 97.07%, indicating that this application can significantly improve the activity recovery rate of horseradish peroxidase-labeled antibody before and after lyophilization.

[0044] As shown in Example 2, Comparative Examples 1-2, and Table 1, the activity recovery rate of the horseradish peroxidase-labeled antibody lyophilized formulation of Example 2 of this application was 98.46%, significantly higher than that of Comparative Examples 1-2. This indicates that the present application utilizes the oxidation of sodium alginate to form a three-dimensional network structure with the amino group of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine during lyophilization, providing mechanical support and molecular confinement, thereby effectively inhibiting the growth of ice crystals and the destruction of the conformation of the horseradish peroxidase-labeled antibody by interfacial stress, and significantly improving the lyophilization activity recovery rate.

[0045] As shown in Example 2, Comparative Examples 3-4, and Table 1, the activity recovery rate of the horseradish peroxidase-labeled antibody lyophilized formulation in Example 2 of this application was 98.46%, significantly higher than that in Comparative Examples 3-4. This indicates that this application utilizes the synergistic effect of betaine and trehalose to form a stable glassy protective system, significantly reducing the risk of protein denaturation during lyophilization and reconstitution, thereby significantly improving the lyophilization activity recovery rate.

[0046] As shown in Examples 2, 4-5, and Table 1, the activity recovery rate of the horseradish peroxidase-labeled antibody lyophilized formulation in Example 4 of this application was 99.21%, significantly higher than that in Examples 2 and 5. This indicates that the use of a specific weight ratio of oxidized sodium alginate and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine in this application can significantly improve the lyophilized activity recovery rate. While the excessively high proportion of oxidized sodium alginate in Example 2 can form a denser network structure, it may lead to incomplete release of the antibody after reconstitution, resulting in a decrease in activity recovery. Similarly, while the excessively high proportion of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine in Example 5 can enhance π-π stacking and hydrogen bond interactions, insufficient cross-linking weakens the molecular confinement effect and reduces the protective effect on protein conformation.

[0047] As shown in Examples 4, 6-7, and Table 1, the activity recovery rate of the horseradish peroxidase-labeled antibody lyophilized formulation in Example 6 of this application was 99.95%, significantly higher than that in Examples 4 and 7. This indicates that the use of a specific weight ratio of betaine and proline in this application can significantly improve the lyophilized activity recovery rate. While the excessively high proportion of proline in Example 4 can maintain a strong preferential hydration effect, it increases glassy brittleness, making it prone to microcracks and affecting the long-term stability of the protein microenvironment. Similarly, while the excessively high proportion of betaine in Example 7 can provide sufficient hydration layer protection, it may excessively restrict the flexible adjustment of protein conformation, leading to a decrease in activity recovery rate.

[0048] Performance Test 2: Acceleration Stability Test The horseradish peroxidase-labeled antibody lyophilized formulation prepared in Example 7 was divided into 6 groups (Experiment 1, Experiment 2, Experiment 3, Experiment 4, Experiment 5, and Experiment 6). Accelerated aging tests were conducted at -20℃, 4℃, 25℃, 37℃, 50℃, and 70℃, respectively. A control group (not lyophilized in Performance Test 1) was also included and subjected to accelerated aging at 4℃. All 7 groups were stored at the above temperatures for 7 days, 15 days, 22 days, and 30 days, and then subjected to ELISA plate tests (using the same method as Performance Test 1). The average OD value of each group was obtained. m The residual activity rate was calculated based on the average OD0 of the control group in performance test 1, and the results are shown in Table 2. Residual activity rate = (OD0 - OD0) / ... m / OD0)×100%.

[0049] Table 2 Accelerated stability of the lyophilized formulation in Example 7

[0050] As shown in Table 2, the freeze-drying of the horseradish peroxidase-labeled antibody preparation of this application has no effect on the activity of the enzyme when stored at -20℃ and freeze-thawed. After storage at 50℃ for 30 days, the activity residual rate is still more than 90%, and after storage at 70℃ for 7 days, the activity residual rate is still 51.05%.

[0051] Performance Test 3: Accelerated Aging Performance Test The horseradish peroxidase-labeled antibody lyophilized formulation prepared in Example 7 was divided into 6 groups (Experiment 1, Experiment 2, Experiment 3, Experiment 4, Experiment 5, and Experiment 6). Accelerated aging tests were conducted at -20℃, 4℃, 25℃, 37℃, 50℃, and 70℃, respectively. A control group (not lyophilized in Performance Test 1) was also included and subjected to accelerated aging at 4℃. All 7 groups were stored at the above temperatures for 7, 15, 22, and 30 days. ELISA plate tests were performed using chloramphenicol standards (0 ng / mL, 0.05 ng / mL, 0.15 ng / mL, 0.5 ng / mL, 1.5 ng / mL, 4.5 ng / mL) and accuracy references (0.1 ng / mL, 1 ng / mL), with two replicates for each spot. The ELISA plate test method was the same as in Performance Test 1, and the OD values ​​for each group were obtained. m Perform linear fitting (R) 2 The concentration was calculated, and the results are shown in Table 3-5.

[0052] Table 3. OD value results of the lyophilized formulation of Example 7 after 7 days of accelerated aging.

[0053] Table 4. Test concentrations of the accelerated aging reference sample for the lyophilized formulation of Example 7 (7 days).

[0054] As shown in Table 3, the R-value of the standard curve fitted by the horseradish peroxidase-labeled antibody lyophilized formulation of this application after accelerated aging for 7 days is [missing information]. 2 All values ​​were greater than 0.99, indicating good linearity. Table 4 shows that the detection concentrations of the accuracy reference samples in all experiments were close to the actual concentrations of the reference samples, indicating that the accuracy measurements of the horseradish peroxidase-labeled antibody lyophilized formulations after accelerated aging for 7 days are highly reliable.

[0055] Table 5. OD value results of the lyophilized formulation of Example 7 after 15 days of accelerated aging.

[0056] Table 6. Detection concentrations of the reference sample for accelerated aging of the lyophilized formulation in Example 7 (15 days).

[0057] As shown in Table 5, the R-value of the standard curve fitted by the horseradish peroxidase-labeled antibody lyophilized formulation of this application after 15 days of accelerated aging is [missing value]. 2 All values ​​were greater than 0.99, indicating good linearity. As shown in Table 6, the detection concentrations of the accuracy reference samples in all experiments were close to the actual concentrations of the reference samples, indicating that the accuracy measurements of the horseradish peroxidase-labeled antibody lyophilized formulations after accelerated aging for 15 days are highly reliable.

[0058] Table 7. OD value results of the lyophilized formulation of Example 7 after 22 days of accelerated aging.

[0059] Table 8. Detection concentrations of the reference sample for accelerated aging of the lyophilized formulation of Example 7 (22 days).

[0060] As shown in Table 7, the R-value of the standard curve fitted by the horseradish peroxidase-labeled antibody lyophilized formulation of this application after 22 days of accelerated aging is [missing value]. 2 All values ​​were greater than 0.99, indicating good linearity. As shown in Table 8, the measured values ​​of the accuracy reference in all experiments were close to the prepared concentration of the reference, indicating that the accuracy measurement values ​​of the horseradish peroxidase-labeled antibody lyophilized preparation after accelerated aging for 22 days are highly reliable.

[0061] Table 9. OD value results after 30 days of accelerated aging

[0062] Table 10 Detection concentration of reference material after 30 days of accelerated aging

[0063] As shown in Table 9, the R-value of the standard curve fitted by the horseradish peroxidase-labeled antibody lyophilized formulation of this application after 30 days of accelerated aging is [missing information]. 2 All values ​​were greater than 0.99, indicating good linearity. As shown in Table 10, the measured values ​​of the accuracy reference in all experiments were close to the prepared concentration of the reference, indicating that the accuracy measurement values ​​of the horseradish peroxidase-labeled antibody lyophilized preparation after accelerated aging for 30 days are highly reliable.

[0064] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A horseradish peroxidase-labeled antibody lyophilized formulation, characterized in that, The preparation includes horseradish peroxidase-labeled antibody, protein protectant, and buffer solution; the protein protectant comprises the following raw materials in parts by weight: trehalose 30-40 parts, sodium alginate 0.5-2 parts, 2,4,6-tris(4-aminophenyl)-1,3,5-triazine 0.1-0.4 parts, betaine 8-12 parts, proline 3-6 parts, bovine serum albumin 0.5-0.7 parts, glycine 2-2.5 parts, β-cyclodextrin 0.25-0.35 parts, reducing agent 0.5-0.7 parts, and surfactant 0.25-0.4 parts.

2. The horseradish peroxidase-labeled antibody lyophilized formulation according to claim 1, characterized in that, The weight ratio of the oxidized sodium alginate to 2,4,6-tris(4-aminophenyl)-1,3,5-triazine is (3-5):

1.

3. The horseradish peroxidase-labeled antibody lyophilized formulation according to claim 1, characterized in that, The weight ratio of betaine to proline is (2-4):

1.

4. The horseradish peroxidase-labeled antibody lyophilized formulation according to claim 1, characterized in that, The preparation steps of the oxidized sodium alginate include: mixing sodium alginate and phosphate buffer, adding sodium periodate solution, stirring evenly, reacting in the dark, adding ethylene glycol to terminate the reaction, stirring evenly, dialyzing, and freeze-drying to obtain oxidized sodium alginate.

5. The horseradish peroxidase-labeled antibody lyophilized formulation according to claim 1, characterized in that, The buffer solution is tris(hydroxymethyl)aminomethane buffer.

6. The horseradish peroxidase-labeled antibody lyophilized formulation according to claim 1, characterized in that, The reducing agent is ascorbic acid.

7. The horseradish peroxidase-labeled antibody lyophilized formulation according to claim 1, characterized in that, The surfactant is at least one of polysorbate-20, polysorbate-60, and polysorbate-80.

8. A method for preparing a lyophilized formulation of horseradish peroxidase-labeled antibody according to any one of claims 1-7, characterized in that, Includes the following steps: Trehalose, betaine, proline, bovine serum albumin, glycine, β-cyclodextrin, reducing agent, and surfactant were mixed, buffer was added, and the mixture was stirred evenly. Horseradish peroxidase-labeled antibody was added, and the mixture was stirred evenly. Then, sodium alginate and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine were added, and the mixture was ultrasonically dispersed, pre-frozen, annealed, and dried to obtain the lyophilized horseradish peroxidase-labeled antibody preparation.

9. The method for preparing the horseradish peroxidase-labeled antibody lyophilized formulation according to claim 8, characterized in that, The pre-freezing temperature is -60 to -40°C.

10. The use of a horseradish peroxidase-labeled antibody lyophilized preparation according to any one of claims 1-7 in the preparation of an immunoassay kit.

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