Enzyme-labeled antibody stabilizer based on plant-derived polysaccharide as well as preparation method and application of enzyme-labeled antibody stabilizer

Abstract

The invention provides an enzyme-labeled antibody stabilizer based on plant-derived polysaccharide as well as a preparation method and application of the enzyme-labeled antibody stabilizer. The enzyme-labeled antibody stabilizer comprises 1% w / v-3% w / v of konjac glucomannan, 2% w / v-5% w / v of astragalus polysaccharide, 0.1% w / v-0. 5% w / v of tea polyphenol, 0.05% w / v-0. 2% w / v of N-acetylcysteine, 3% w / v-5% w / v of betaine and a Tris-HCl solution. Compared with a common buffer solution without the stabilizer, the stability of the enzyme-labeled antibody stabilizer provided by the invention is remarkably improved, and the enzyme-labeled antibody can be stably stored for 6 months at 25 DEG C; storing for 15 months under the condition of 2-8 DEG C; and the protective agent can be stably preserved for 4 weeks at 37 DEG C, and can be compatible with stable preservation of different types of enzyme-labeled antibodies.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme-linked immunoassay reagent technology, in particular to a plant source polysaccharide-based enzyme-labeled antibody stabilizer and a preparation method and application thereof. BACKGROUND

[0002] Enzyme-linked immunosorbent assay (ELISA) has become the most widely used technology in the current immunological detection field due to its high sensitivity, high specificity, and simple operation. At present, many biotechnology companies at home and abroad have developed various commercial ELISA detection kits, which are widely used in disease prevention and control, drug residue, hormone level evaluation, and other fields. In the development of commercial ELISA detection kits with good detection performance, in addition to high-quality core raw materials, auxiliary raw materials are also needed to ensure the stability of the kit detection. These raw materials are added to the blocking solution, diluent, control, enzyme-labeled antibody, and other components, which are expected to ensure the detection performance indicators of the kit do not decline during long-term storage without affecting the normal immunological reaction. However, in actual application, there is still an inevitable problem that the longer the storage time, the worse the detection effect.

[0003] For example, the protective agent formulations disclosed in Chinese patents CN113567661B and CN110187098A contain animal-derived components such as gelatin and casein. These components may not effectively maintain the detection performance of the kit during long-term storage, and may even have adverse effects on ELISA detection results, thereby exacerbating the problem of "the longer the storage time, the worse the detection effect". This leads to the problem of rapid decline in ELISA detection performance with longer storage time, for example, gelatin may bind to anti-collagen antibodies in the sample, causing false positives; casein phosphopeptide in casein may block target antigen epitopes, causing low detection OD values; animal-derived protein residues may cause cross-reactions with antigens, resulting in false positives. These situations cause the kit to significantly decrease in detection accuracy, reliability, and other performance indicators as the storage time increases. SUMMARY

[0004] The present application aims to provide a plant source polysaccharide-based enzyme-labeled antibody stabilizer and a preparation method and application thereof, to solve the problem of rapid decline in ELISA detection performance with longer storage time in traditional ELISA detection kits.

[0005] In a first aspect, the present application provides a plant source polysaccharide-based enzyme-labeled antibody stabilizer, which comprises: 1% w / v~3% w / v konjac glucomannan, 2% w / v~5% w / v astragalus polysaccharide, 0.1% w / v~0.5% w / v tea polyphenols, 0.05% w / v~0.2% w / v N-acetylcysteine, 3% w / v~5% w / v betaine, Tris-HCl solution.

[0006] In summary, the enzyme-labeled antibody stabilizer described above, formulated using a plant-derived stabilization system combining konjac glucomannan and astragalus polysaccharide, along with an antioxidant system of tea polyphenols, N-acetylcysteine, and betaine, can significantly reduce the tendency for enzyme-labeled antibodies to decrease in activity over storage time. Two enzyme-labeled antibodies (rabbit anti-porcine HRP-IgG and HRP-Protein A) in a classical swine fever and porcine circovirus type 2 indirect ELISA detection kit, preserved with this stabilizer, maintained essentially unchanged activity after 15 months of storage at 2–8°C. This enzyme-labeled antibody stabilizer allows enzyme-labeled antibodies to maintain their activity for an extended period.

[0007] In some embodiments, the enzyme-labeled antibody stabilizer comprises: 3% w / v konjac glucomannan, 2% w / v astragalus polysaccharide, 0.5% w / v tea polyphenols, 0.1% w / v N-acetylcysteine, 5% w / v betaine, Tris-HCl solution.

[0008] In some embodiments, the enzyme-labeled antibody stabilizer comprises: 1% w / v konjac glucomannan, 5% w / v astragalus polysaccharide, 0.1% w / v tea polyphenols, 0.1% w / v N-acetylcysteine, 3% w / v betaine, Tris-HCl solution.

[0009] In some embodiments, the enzyme-labeled antibody stabilizer further includes 1% w / v to 3% w / v polyethylene glycol and 0.05% w / v to 0.2% w / v Proclin 300.

[0010] In some embodiments, the pH value of the enzyme-labeled antibody stabilizer is 7.6-7.8.

[0011] Secondly, the present invention also provides a method for preparing an enzyme-labeled antibody stabilizer based on plant-derived polysaccharides, used to prepare the above-mentioned enzyme-labeled antibody stabilizer based on plant-derived polysaccharides, the preparation method comprising: Dissolve each component thoroughly in Tris-HCl solution according to mass-volume fraction, mix evenly, filter to remove bacteria, and adjust the pH to 7.2-7.6 using concentrated ammonia or concentrated saline.

[0012] Thirdly, the present invention also proposes the application of the above-mentioned plant-derived polysaccharide-based enzyme-labeled antibody stabilizer in the cryopreservation, dilution and preparation of ELISA detection kits of enzyme-labeled antibodies.

[0013] In some embodiments, the enzyme-labeled antibody is at least one of rabbit anti-pig HRP-IgG, HRP-Protein A, mouse anti-pig HRP-IgG, goat anti-mouse HRP-IgG, and rabbit anti-chicken HRP-IgY.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The enzyme-labeled antibody stabilizer based on plant-derived polysaccharides of this invention contains plant-derived polysaccharides that, through their abundant hydrophilic hydroxyl and carboxyl groups, can form a stable three-dimensional network structure to encapsulate the enzyme-labeled antibody, effectively isolating it from various destructive components in the external environment. Simultaneously, the natural biocompatibility and low reactivity of the plant-derived polysaccharides enable effective protection while avoiding the introduction of exogenous interfering substances, thus improving the accuracy and reliability of immunoassay reactions. Specifically, konjac glucomannan, through its high molecular weight and unique acetyl-modified main chain structure, achieves a high-strength gel network protection system, effectively preventing mechanical damage from the enzyme-labeled antibody. Astragalus polysaccharides, through their rich α-1,4-glucan branch structure and active hydroxyl groups, achieve [the desired effect] with the antibody. The hydrophobic regions of the enzyme form directional hydrogen bonds, stabilizing the spatial conformation of the antigen binding site (Fab segment) and reducing the antibody denaturation rate in high-temperature accelerated assays. The complex antioxidants it contains, through the addition of specialized antioxidants targeting different oxidation mechanisms, achieve effects such as scavenging free radicals in solution, reducing reactions, and dynamically repairing oxidatively broken chemical bonds. Specifically, tea polyphenols, through their catechol structure and high reduction potential, achieve efficient scavenging of dissolved oxygen and peroxidation free radicals; N-acetylcysteine, through its free sulfhydryl groups and strong reducing ability, achieves dynamic repair of oxidatively broken disulfide bonds in enzyme-labeled antibodies; and betaine, through its zwitterionic properties and osmotic pressure regulation function, provides a glassy protective layer for enzyme-labeled antibodies.

[0015] 2. Compared with general buffer solutions without stabilizers, the plant-derived polysaccharide-based enzyme-labeled antibody stabilizer of the present invention has significantly improved stability, enabling enzyme-labeled antibodies to be stably stored at 25°C for 6 months; at 2-8°C for 15 months; and at 37°C for 4 weeks. Moreover, this protectant is compatible with the stable storage of different types of enzyme-labeled antibodies. Attached Figure Description

[0016] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 Figure 1 shows the stability test results of HRP-Protein A and rabbit anti-porcine HRP-IgG diluted with different protective agents at 37℃ (using a commercial kit for classical swine fever virus and porcine circovirus type 2 as the test product). Figure 2 Figure 1 shows the stability test results of HRP-Protein A and rabbit anti-porcine HRP-IgG diluted with different protective agents at 25℃ (using a commercial kit for classical swine fever virus and porcine circovirus type 2 as the test product). Figure 3 The results of stability tests of HRP-Protein A and rabbit anti-porcine HRP-IgG diluted with different protective agents at 2~8℃ are shown in the figure (using commercial kits for classical swine fever virus and porcine circovirus type 2 as test products). Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0018] The following detailed description is exemplary and intended to provide further detailed explanation of the invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this invention is for describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention.

[0019] Example 1: Preparation of HRP-protein A enzyme-labeled antibody solution containing a protective agent Preparation and detection methods: Prepare enzyme-labeled antibody protectant according to the concentrations of each component in the table below.

[0020] Table 1. Composition and content of enzyme-labeled antibody protective agent (for 1000ml).

[0021] The above components are fully dissolved and mixed evenly, and finally filtered through a 0.22μm filter for sterilization.

[0022] HRP-Protein A was diluted 5000-fold with the prepared protective agent described above. Positive controls were tested using both a classical swine fever virus ELISA antibody detection kit and a porcine circovirus type 2 ELISA antibody detection kit. The testing conditions were as follows: the prepared enzyme-labeled antibody was tested monthly at 25℃ for 15 months; the prepared enzyme-labeled antibody was tested monthly at 2–8℃ for 18 months; and the prepared enzyme-labeled antibody was tested at 37℃ at 0, 7, 14, 21, 28, and 35 days for 5 weeks. The changes in the OD value of the HRP-Protein A enzyme-labeled antibody against the positive controls in the kits under different storage conditions were analyzed.

[0023] Table 2. Stability test results of HRP-protein A enzyme-labeled antibody containing protective agent at 37℃

[0024] Note: "d" represents day.

[0025] Table 3. Stability test results of HRP-protein A enzyme-labeled antibody containing protective agent at 25℃

[0026] Note: "m" represents the month.

[0027] Table 4. Stability test results of HRP-protein A enzyme-labeled antibody containing protective agent at 2-8℃

[0028] Note: "m" represents the month.

[0029] Example 2: Preparation of rabbit anti-porcine HRP-IgG enzyme-labeled antibody solution containing a protective agent Preparation and detection methods: Prepare enzyme-labeled antibody protectant according to the concentrations of each component in the table below.

[0030] Table 5. Composition and content of enzyme-labeled antibody protective agent (for 1000ml).

[0031] The above components are fully dissolved and mixed evenly, and finally filtered through a 0.22μm filter for sterilization.

[0032] Rabbit anti-porcine HRP-IgG was diluted 5000-fold with the prepared protective agent. Positive controls were tested using both a classical swine fever virus ELISA antibody detection kit and a porcine circovirus type 2 ELISA antibody detection kit. The testing conditions were as follows: the prepared enzyme-labeled antibody was tested monthly at 25℃ for 15 months; the prepared enzyme-labeled antibody was tested monthly at 2–8℃ for 18 months; and the prepared enzyme-labeled antibody was tested at 37℃ at 0, 7, 14, 21, 28, and 35 days for 5 weeks. The changes in the OD value of the rabbit anti-porcine HRP-IgG enzyme-labeled antibody against the positive controls in the kits under different storage conditions were analyzed.

[0033] Table 6. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing protective agent at 37℃

[0034] Note: "d" represents day.

[0035] Table 7. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing protective agent at 25℃

[0036] Note: "m" represents the month.

[0037] Table 8. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing protective agent at 2-8℃

[0038] Note: "m" represents the month.

[0039] Comparative Example 1: Preparation of HRP-protein A enzyme-labeled antibody solution without protective agent. Preparation and detection methods: HRP-Protein was prepared using commercially available PBS buffer (Biosharp, catalog number: BL2218A). Horseradish peroxidase-labeled protein A (HRP-protein A, purchased from Thermo Fisher Scientific, catalog number: 200-103-077S) was diluted 5000-fold and used to test the positive control in the kits with a classical swine fever virus ELISA antibody detection kit (purchased from IDS Biotechnology Co., Ltd., catalog number: 99-43220) and a porcine circovirus type 2 ELISA antibody detection kit (purchased from Beijing Jinno Biotech Co., Ltd., catalog number: JN60315). The testing conditions were as follows: the prepared enzyme-labeled antibody was tested monthly at 25℃ for 15 months; the prepared enzyme-labeled antibody was tested monthly at 2–8℃ for 18 months; and the prepared enzyme-labeled antibody was tested at 37℃ at 0, 7, 14, 21, 28, and 35 days for 5 weeks. The changes in the OD value of the HRP-protein A enzyme-labeled antibody against the positive control in the kits under different storage conditions were analyzed.

[0040] Table 9. Stability test results of HRP-protein A enzyme-labeled antibody without protective agent at 37℃

[0041] Note: "d" represents day.

[0042] Table 10. Stability test results of HRP-protein A enzyme-labeled antibody without protective agent at 25℃

[0043] Note: "m" represents the month.

[0044] Table 11. Stability test results of HRP-protein A enzyme-labeled antibody without protective agent at 2-8℃

[0045] Note: "m" represents the month.

[0046] Comparative Example 2: Preparation of rabbit anti-porcine HRP-IgG enzyme-labeled antibody solution without protective agent. Preparation and detection methods: Rabbit anti-porcine HRP-IgG (SIGMA, A5670) was diluted 5000-fold with commercial PBS buffer (Biosharp, catalog number: ). Positive controls were tested using a classical swine fever virus ELISA antibody detection kit (IDEX Biotech, catalog number: 99-43220) and a porcine circovirus type 2 ELISA antibody detection kit (Beijing Jinno Biotech Co., Ltd., catalog number: JN60315). The testing conditions were as follows: the prepared enzyme-labeled antibody was tested monthly at 25℃ for 15 months; the prepared enzyme-labeled antibody was tested monthly at 2–8℃ for 18 months; and the prepared enzyme-labeled antibody was tested at 37℃ at 0, 7, 14, 21, 28, and 35 days for 5 weeks. The changes in the OD value of the rabbit anti-porcine HRP-IgG enzyme-labeled antibody against the positive controls in the kits under different storage conditions were analyzed.

[0047] Table 12. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody without protective agent at 37℃

[0048] Note: "d" represents day.

[0049] Table 13. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody without protective agent at 25℃

[0050] Note: "m" represents the month.

[0051] Table 14. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody without protective agent at 2-8℃

[0052] Note: "m" represents the month.

[0053] Comparative Example 3: Preparation of HRP-protein A enzyme-labeled antibody solution containing common polysaccharide protectant. Preparation and detection methods: Prepare enzyme-labeled antibody protectant according to the concentrations of each component in the table below.

[0054] Table 15 Composition and content of enzyme-labeled antibody protective agent (for 1000ml)

[0055] The above components are fully dissolved and mixed evenly, and finally filtered through a 0.22μm filter for sterilization.

[0056] The HRP-protein A enzyme-labeled antibody was diluted 5000-fold using the prepared protective agent. Positive controls were tested using both a classical swine fever virus ELISA antibody detection kit and a porcine circovirus type 2 ELISA antibody detection kit. The testing conditions were as follows: the prepared enzyme-labeled antibody was tested monthly at 25℃ for 15 months; the prepared enzyme-labeled antibody was tested monthly at 2–8℃ for 18 months; and the prepared enzyme-labeled antibody was tested at 37℃ at 0, 7, 14, 21, 28, and 35 days for 5 weeks. The changes in the OD value of the HRP-protein A enzyme-labeled antibody against the positive controls in the kits under different storage conditions were analyzed.

[0057] Table 16. Stability test results of HRP-protein A enzyme-labeled antibody containing common polysaccharide protectant at 37℃

[0058] Note: "d" represents day.

[0059] Table 17. Stability test results of HRP-protein A enzyme-labeled antibody containing common polysaccharide protectant at 25℃

[0060] Note: "m" represents the month.

[0061] Table 18. Stability test results of HRP-protein A enzyme-labeled antibody containing common polysaccharide protectant at 2-8℃

[0062] Note: "m" represents the month.

[0063] Comparative Example 4: Preparation of rabbit anti-porcine HRP-IgG enzyme-labeled antibody solution containing common polysaccharide protectant. Preparation and detection methods: Prepare enzyme-labeled antibody protectant according to the concentrations of each component in the table below.

[0064] Table 19 Composition and content of enzyme-labeled antibody protective agent (for 1000ml)

[0065] The above components are fully dissolved and mixed evenly, and finally filtered through a 0.22μm filter for sterilization.

[0066] The rabbit anti-porcine HRP-IgG enzyme-labeled antibody was diluted 5000-fold using the prepared protective agent. Positive controls were tested using both a classical swine fever virus ELISA antibody detection kit and a porcine circovirus type 2 ELISA antibody detection kit. The testing conditions were as follows: the prepared enzyme-labeled antibody was tested monthly at 25℃ for 15 months; the prepared enzyme-labeled antibody was tested monthly at 2–8℃ for 18 months; and the prepared enzyme-labeled antibody was tested at 37℃ at 0, 7, 14, 21, 28, and 35 days for 5 weeks. The changes in the OD value of the rabbit anti-porcine HRP-IgG enzyme-labeled antibody against the positive controls in the kits under different storage conditions were analyzed.

[0067] Table 20. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing common polysaccharide protectant at 37℃.

[0068] Note: "d" represents day.

[0069] Table 21. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing common polysaccharide protectant at 25℃.

[0070] Note: "m" represents the month.

[0071] Table 22. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing common polysaccharide protectant at 2-8℃

[0072] Note: "m" represents the month.

[0073] Comparative Example 5: Preparation of HRP-protein A enzyme-labeled antibody solution containing a single antioxidant protectant. Preparation and detection methods: Prepare enzyme-labeled antibody protectant according to the concentrations of each component in the table below.

[0074] Table 23 Composition and content of enzyme-labeled antibody protective agent (for 1000ml)

[0075] The above components are fully dissolved and mixed evenly, and finally filtered through a 0.22μm filter for sterilization.

[0076] The HRP-protein A enzyme-labeled antibody was diluted 5000-fold using the prepared protective agent. Positive controls were tested using both a classical swine fever virus ELISA antibody detection kit and a porcine circovirus type 2 ELISA antibody detection kit. The testing conditions were as follows: the prepared enzyme-labeled antibody was tested monthly at 25℃ for 15 months; the prepared enzyme-labeled antibody was tested monthly at 2–8℃ for 18 months; and the prepared enzyme-labeled antibody was tested at 37℃ at 0, 7, 14, 21, 28, and 35 days for 5 weeks. The changes in the OD value of the HRP-protein A enzyme-labeled antibody against the positive controls in the kits under different storage conditions were analyzed.

[0077] Table 24. Stability test results of HRP-protein A enzyme-labeled antibody containing a single antioxidant protectant at 37°C.

[0078] Note: "d" represents day.

[0079] Table 25. Stability test results of HRP-protein A enzyme-labeled antibody containing a single antioxidant protectant at 25°C.

[0080] Note: "m" represents the month.

[0081] Table 26. Stability test results of HRP-protein A enzyme-labeled antibody containing a single antioxidant protectant at 2-8℃

[0082] Note: "m" represents the month.

[0083] Comparative Example 6: Preparation of rabbit anti-porcine HRP-IgG enzyme-labeled antibody solution containing a single antioxidant protectant. Preparation and detection methods: Prepare enzyme-labeled antibody protectant according to the concentrations of each component in the table below.

[0084] Table 27 Composition and content of enzyme-labeled antibody protective agent (for 1000ml)

[0085] The above components are fully dissolved and mixed evenly, and finally filtered through a 0.22μm filter for sterilization.

[0086] The rabbit anti-porcine HRP-IgG enzyme-labeled antibody was diluted 5000-fold using the prepared protective agent. Positive controls were tested using both a classical swine fever virus ELISA antibody detection kit and a porcine circovirus type 2 ELISA antibody detection kit. The testing conditions were as follows: the prepared enzyme-labeled antibody was tested monthly at 25℃ for 15 months; the prepared enzyme-labeled antibody was tested monthly at 2–8℃ for 18 months; and the prepared enzyme-labeled antibody was tested at 37℃ at 0, 7, 14, 21, 28, and 35 days for 5 weeks. The changes in the OD value of the rabbit anti-porcine HRP-IgG enzyme-labeled antibody against the positive controls in the kits under different storage conditions were analyzed.

[0087] Table 28. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing a single antioxidant protectant at 37℃.

[0088] Note: "d" represents day.

[0089] Table 29. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing a single antioxidant protectant at 25°C.

[0090] Note: "m" represents the month.

[0091] Table 30. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing a single antioxidant protectant at 2-8℃.

[0092] Note: "m" represents the month.

[0093] Comparative Example 7: Preparation of HRP-protein A enzyme-labeled antibody solution containing a single plant-derived polysaccharide protectant. Preparation and detection methods: Prepare enzyme-labeled antibody protectant according to the concentrations of each component in the table below.

[0094] Table 31 Composition and content of enzyme-labeled antibody protective agent (for 1000ml)

[0095] The above components are fully dissolved and mixed evenly, and finally filtered through a 0.22μm filter for sterilization.

[0096] The HRP-protein A enzyme-labeled antibody was diluted 5000-fold using the prepared protective agent. Positive controls were tested using both a classical swine fever virus ELISA antibody detection kit and a porcine circovirus type 2 ELISA antibody detection kit. The testing conditions were as follows: the prepared enzyme-labeled antibody was tested monthly at 25℃ for 15 months; the prepared enzyme-labeled antibody was tested monthly at 2–8℃ for 18 months; and the prepared enzyme-labeled antibody was tested at 37℃ at 0, 7, 14, 21, 28, and 35 days for 5 weeks. The changes in the OD value of the HRP-protein A enzyme-labeled antibody against the positive controls in the kits under different storage conditions were analyzed.

[0097] Table 32. Stability test results of HRP-protein A enzyme-labeled antibody containing a single plant-derived polysaccharide protectant at 37℃.

[0098] Note: "d" represents day.

[0099] Table 33. Stability test results of HRP-protein A enzyme-labeled antibody containing a single plant-derived polysaccharide protectant at 25℃.

[0100] Note: "m" represents the month.

[0101] Table 34. Stability test results of HRP-protein A enzyme-labeled antibody containing a single plant-derived polysaccharide protectant at 2–8 °C

[0102] Note: "m" represents the month.

[0103] Comparative Example 8: Preparation of rabbit anti-porcine HRP-IgG enzyme-labeled antibody solution containing a single plant-derived polysaccharide protectant. Preparation and detection methods: Prepare enzyme-labeled antibody protectant according to the concentrations of each component in the table below.

[0104] Table 35 Composition and content of enzyme-labeled antibody protective agent (for 1000ml)

[0105] The above components are fully dissolved and mixed evenly, and finally filtered through a 0.22μm filter for sterilization.

[0106] The rabbit anti-porcine HRP-IgG enzyme-labeled antibody was diluted 5000-fold using the prepared protective agent. Positive controls were tested using both a classical swine fever virus ELISA antibody detection kit and a porcine circovirus type 2 ELISA antibody detection kit. The testing conditions were as follows: the prepared enzyme-labeled antibody was tested monthly at 25℃ for 15 months; the prepared enzyme-labeled antibody was tested monthly at 2–8℃ for 18 months; and the prepared enzyme-labeled antibody was tested at 37℃ at 0, 7, 14, 21, 28, and 35 days for 5 weeks. The changes in the OD value of the rabbit anti-porcine HRP-IgG enzyme-labeled antibody against the positive controls in the kits under different storage conditions were analyzed.

[0107] Table 36. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing a single plant-derived polysaccharide protectant at 37℃.

[0108] Note: "d" represents day.

[0109] Table 37. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing a single plant-derived polysaccharide protectant at 25℃.

[0110] Note: "m" represents the month.

[0111] Table 38. Stability test results of rabbit anti-porcine HRP-IgG enzyme-labeled antibody containing a single plant-derived polysaccharide protectant at 2-8℃.

[0112] Note: "m" represents the month.

[0113] Conclusion Analysis: In Examples 1 and 2, after using a stabilizer containing a complex formula of konjac glucomannan, astragalus polysaccharide, tea polyphenols, N-acetylcysteine, and betaine, HRP-Protein... The activity of rabbit anti-porcine HRP-IgG enzyme-labeled antibody A remained essentially unchanged after 15 months of storage at 2-8℃, with a stable period of over 6 months at 25℃ and maintaining activity for more than 4 weeks at 37℃. In contrast, Comparative Examples 1-2 without stabilizers and Comparative Examples 3-4 using common polysaccharide protectants showed a significant decrease in enzyme-labeled antibody activity over time under the same conditions, with particularly rapid inactivation at high temperatures. Comparative Examples 5-6 further confirmed that a single antioxidant could not achieve the synergistic protective effect of the composite system (tea polyphenols + N-acetylcysteine ​​+ betaine). Comparative Examples 7-8 indicated that the stability of a single plant-derived polysaccharide was significantly weaker than that of the composite system of konjac glucomannan and astragalus polysaccharide. The above comparative results collectively verify that the present invention, through the synergistic mechanism of constructing a three-dimensional network structure using plant-derived polysaccharides and scavenging free radicals and repairing oxidative damage using composite antioxidants, can effectively solve the technical problem of activity decay during long-term storage of enzyme-labeled antibodies, providing a high-performance and stable storage solution for ELISA detection kits.

[0114] also, Figure 1 The effects of different protective agents on the stability of HRP-Protein A and rabbit anti-porcine HRP-IgG enzyme-labeled antibodies were shown at 37°C. The enzyme-labeled antibody containing konjac glucomannan, astragalus polysaccharide, tea polyphenols, N-acetylcysteine, and betaine showed a significantly smaller decrease in OD value compared to the control group (without stabilizer or using ordinary protective agent) over a 35-day detection period. This indicates that the composite stabilizer of the present invention can effectively resist the degradation of enzyme-labeled antibodies by high temperature and maintain their activity. Figure 2 This indicates that the enzyme-labeled antibody formulated in the examples exhibits more stable performance at 25°C. Compared to the comparative example, the OD value of the enzyme-labeled antibody in the examples fluctuated less over a 15-month detection period, demonstrating a higher activity retention rate. This further validates the long-term protective effect of the stabilizer at room temperature, making it suitable for kit applications requiring long-term storage. Figure 3 This indicates that the enzyme-labeled antibody formulations in the examples exhibit a more significant stability advantage under low-temperature conditions of 2–8°C. 18 months of testing data showed that the OD value of the enzyme-labeled antibody in the examples showed almost no decay, while the activity of the comparative formulation decreased significantly over time. This demonstrates that the stabilizer of the present invention can maintain the activity of the enzyme-labeled antibody for a long period at low temperatures, meeting the stringent requirements of commercial kits for long-term storage performance.

[0115] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or equivalent to the scope of this invention are included in this invention.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A plant-derived polysaccharide-based enzyme-labeled antibody stabilizer, characterized in that, The enzyme-labeled antibody stabilizer includes: 1% w / v~3% w / v konjac glucomannan, 2% w / v~5% w / v astragalus polysaccharide, 0.1% w / v~0.5% w / v tea polyphenols, 0.05% w / v~0.2% w / v N-acetylcysteine, 3% w / v~5% w / v betaine, Tris-HCl solution.

2. The enzyme-labeled antibody stabilizer based on plant-derived polysaccharides according to claim 1, characterized in that, The enzyme-labeled antibody stabilizer includes: 3% w / v konjac glucomannan, 2% w / v astragalus polysaccharide, 0.5% w / v tea polyphenols, 0.1% w / v N-acetylcysteine, 5% w / v betaine, Tris-HCl solution.

3. The enzyme-labeled antibody stabilizer based on plant-derived polysaccharides according to claim 1, characterized in that, The enzyme-labeled antibody stabilizer includes: 1% w / v konjac glucomannan, 5% w / v astragalus polysaccharide, 0.1% w / v tea polyphenols, 0.1% w / v N-acetylcysteine, 3% w / v betaine, Tris-HCl solution.

4. The enzyme-labeled antibody stabilizer based on plant-derived polysaccharides according to any one of claims 1-3, characterized in that, The enzyme-labeled antibody stabilizer also includes 1% w / v~3% w / v polyethylene glycol and 0.05% w / v~0.2% w / v Proclin 300.

5. The enzyme-labeled antibody stabilizer based on plant-derived polysaccharides according to claim 4, characterized in that, The pH value of the enzyme-labeled antibody stabilizer is 7.6-7.

8.

6. A method for preparing an enzyme-labeled antibody stabilizer based on plant-derived polysaccharides, used to prepare the enzyme-labeled antibody stabilizer based on plant-derived polysaccharides according to any one of claims 1-5, characterized in that, The preparation method includes: Dissolve each component thoroughly in Tris-HCl solution according to mass-volume fraction, mix evenly, filter to remove bacteria, and adjust the pH to 7.2-7.6 using concentrated ammonia or concentrated saline.

7. The application of a plant-derived polysaccharide-based enzyme-labeled antibody stabilizer as described in any one of claims 1-5 in the cryopreservation, dilution, and preparation of ELISA detection kits for enzyme-labeled antibodies.

8. The application according to claim 7, characterized in that, The enzyme-labeled antibody is at least one of rabbit anti-pig HRP-IgG, HRP-Protein A, mouse anti-pig HRP-IgG, goat anti-mouse HRP-IgG, and rabbit anti-chicken HRP-IgY.

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