An antibody stabilizer composition for maintaining stability of elisa detection performance and use thereof

Abstract

The application provides an antibody stabilizer composition for maintaining the stability of ELISA detection performance and application thereof. The antibody stabilizer composition for maintaining the stability of ELISA detection performance provided by the application comprises the following components: a buffer with a final concentration of 10-100 mM, a saccharide and / or polyol with a mass percentage concentration of 1-15%, and an amino acid with a final concentration of 1-300 mM. The antibody stabilizer composition of the application is applied to the preparation of an antibody stabilizer for the preservation, dilution or use of an antibody in ELISA detection. The method for the preservation, dilution or use of an antibody in ELISA detection by the antibody stabilizer composition of the application comprises the following steps: dissolving or diluting the antibody for ELISA detection in the above-mentioned antibody stabilizer composition, so as to be used in ELISA detection. The application solves the common problem of poor stability of domestic animal disease diagnostic reagents / kits through the synergistic effect of multiple stabilizer components, so as to ensure the accuracy and repeatability of the ELISA detection result.

Description

Technical Field

[0001] This invention belongs to the technical field of protective agents and stabilizers for animal disease immunology diagnostic reagents / kits, and relates to an antibody stabilizer composition for maintaining the stability of ELISA detection performance and its application. Background Technology

[0002] Immunodiagnostic reagents are a class of in vitro diagnostic preparations based on the principle of antigen-antibody specific binding reactions. They provide a scientific basis for disease diagnosis by quantitatively or qualitatively detecting target analytes in biological samples, such as proteins, pathogen markers, and tag proteins. These reagents are generally used independently or in combination as reagents, kits, calibrators, and quality control products. The stability of the physical, chemical, and biological activities of each component in an immunodiagnostic reagent is directly and closely related to the quality and efficacy of the diagnostic reagent. It is the core foundation for ensuring the overall stability of the product, and its performance stability plays a decisive role in maintaining the reliability, repeatability, and consistency of the reagent's detection reactions.

[0003] With the development and maturation of various detection technologies, commercial diagnostic reagents / kits in my country are showing a diversified development trend. Their core advantages are mainly reflected in: standardized production ensuring repeatability and consistency of results; simplified operation adapting to high-throughput detection; and pre-packaged design reducing the risk of exposure to toxic reagents (such as concentrated sulfuric acid stop solution), improving safety, and reducing cross-contamination. Studies have found that the stability and accuracy of reagent kits are easily affected by various factors such as abnormal antigenic determinants, blocking effects, or interference from non-specific components of the coating antigen, leading to false negative or false positive results. Meanwhile, uneven antigen coating, unstable antigen-antibody binding, incomplete substrate color development, and manufacturing processes also significantly affect the stability of reagent kits. Furthermore, research indicates that diagnostic reagents / kits require strict temperature control during transportation; temperature changes or abnormalities can reduce the stability and sensitivity of diagnostic reagents and shorten the shelf life of kits. For example, excessively high temperatures can lead to enzyme denaturation and decomposition of chromogenic agents, while excessively low temperatures can cause freezing and thus destroy enzyme activity. Therefore, the stability of diagnostic reagents is an extremely important indicator in the comprehensive evaluation of product quality and a crucial basis for determining storage conditions and shelf life.

[0004] In recent years, foreign countries have conducted research on stabilizer component screening and formulation combinations. However, after systematically reviewing the progress of stabilizer research in China, we found that my country's research and exploration in this field are relatively limited, started late, and lack systematicity, resulting in slow progress. This problem is particularly evident in the field of animal disease diagnosis and testing. Therefore, there is still a significant gap in the research and development of stabilizers for diagnostic reagents in my country. Early research focused on optimizing single performance aspects, such as short-term stability and heat tolerance, but lacked systematic research and evaluation on long-term comprehensive stability, optimization of different formulation combinations, establishment of instability models, component action mechanisms, and clinical applicability. Furthermore, my country's technical methods for diagnostic reagent stability research are unclear, its technical system is immature, its technical platform is incomplete, and related commercial products are scarce. In practice, these factors often lead to dependence on foreign companies, resulting in the so-called "bottleneck" problem. Stabilizers and protective agents, key excipients for diagnostic reagents, have both significant scientific research value and broad application prospects in improving formulation stability and bioactivity. However, as mentioned above, my country's research in this field is relatively lagging. Therefore, improving the stability of animal disease diagnostic reagents in my country, so as to independently develop sensitive, specific, and reproducible animal disease diagnostic reagents, has become an important scientific problem and practical need that urgently needs to be solved. Thus, this invention can promote technological innovation and industrial upgrading of veterinary diagnostic product enterprises in my country, significantly improve their economic benefits and international competitiveness, effectively improve the key technological level of animal disease diagnostic products in my country, alleviate the pressure of animal disease prevention and control, and enhance the level of animal disease prevention and control technology in my country. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody stabilizer composition for maintaining the stability of ELISA detection performance and its application.

[0006] This invention, through the synergistic effect of multiple stabilizer components, breaks through the common problem of poor stability in domestically produced animal disease diagnostic reagents / kits, thereby ensuring the accuracy and repeatability of ELISA test results.

[0007] This invention provides an antibody stabilizer composition for maintaining the stability of ELISA detection performance, comprising the following components: The final concentration of the buffer is 10~100 mM, the mass percentage of the sugar and / or polyol is 1~15%, and the final concentration of the amino acid is 1~300 mM.

[0008] The antibody stabilizer composition described above further includes at least one of a polymeric stabilizer, an inorganic salt, a surfactant, and a preservative.

[0009] In the above-mentioned antibody stabilizer composition, the mass percentage concentration of the polymeric stabilizer can be 0-2%; The final concentration of the inorganic salt can be 50~150 mM; The mass percentage concentration of the surfactant can be 0~0.2%; The mass percentage concentration of the preservative can be 0~0.1%.

[0010] In this invention, the antibody stabilizer composition specifically comprises at least one of the following: a buffer with a final concentration of 10-100 mM, a sugar and / or polyol with a mass percentage concentration of 1-15%, an amino acid with a final concentration of 1-300 mM, a polymeric stabilizer with a mass percentage concentration of 0-2%, an inorganic salt with a mass percentage concentration of 50-150 mM, a surfactant with a mass percentage concentration of 0-0.2%, and a preservative with a mass percentage concentration of 0-0.1%.

[0011] In the above-mentioned antibody stabilizer composition, the buffer is selected from at least one of phosphate buffer solution, histidine buffer system and Tris buffer solution; The phosphate buffer solution includes a dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer pair.

[0012] In the above-mentioned antibody stabilizer composition, the sugar and / or polyol is selected from at least one of trehalose, sorbitol and glycerol.

[0013] In the above-mentioned antibody stabilizer composition, the amino acid is selected from at least one of L-methionine, arginine, glycine, proline, and glutamic acid.

[0014] In the above-mentioned antibody stabilizer composition, the polymeric stabilizer is selected from at least one of polyvinylpyrrolidone, gelatin, and collagen peptides; The inorganic salt is selected from sodium chloride and / or ammonium sulfate; The surfactant is selected from Tween-20 and / or Tween-80. The preservatives include Proclin-type preservatives.

[0015] In the above-mentioned antibody stabilizer composition, the pH value of the antibody stabilizer composition can be 6.0~8.0.

[0016] In this invention, the antibody stabilizer composition is preferably one of the compositions listed in 1) to 4) below, based on the final concentration (moles / volume) or final mass percentage (%) of each component in the composition: 1) Potassium dihydrogen phosphate and potassium dihydrogen phosphate buffer (50mM) + trehalose (10%) + PVP (polyvinylpyrrolidone) (1%) + L-methionine (10mM); 2) Potassium dihydrogen phosphate buffer (50mM) + Trehalose (10%) + PVP (polyvinylpyrrolidone) (1%) + Sorbitol (1%) + L-methionine (10mM) + Tween 80 (0.1%) + Proclin 300 (0.09%) + Proline (10mM); 3) Histidine (50mM) + Glycine (50mM) + Trehalose (5%) + NaCl (150mM) + Fish collagen peptide powder (0.5%, 0.5g) + Tween-20 (0.05%, 50μL) + Proclin 300 (0.05%). 4) Histidine (20mM) + Ammonium sulfate (50mM) + Arginine (50mM) + Trehalose (5%) + Gelatin (1%) + Tween-20 (0.05%) + Proclin 300 (0.05%).

[0017] The present invention also provides the use of the above-described antibody stabilizer composition in the preparation of antibody stabilizers for the preservation, dilution or use of antibodies in ELISA assays.

[0018] In the above applications, the antibody stabilizer is used to inhibit the aggregation or decrease in activity of antibodies used in ELISA during storage and use, thereby improving the repeatability of test results.

[0019] The present invention further provides a method for storing, diluting or using the antibody stabilizer composition described above for ELISA detection, comprising the following steps: dissolving or diluting the antibody for ELISA detection in the antibody stabilizer composition described above for use in ELISA detection.

[0020] The present invention has the following beneficial effects: 1. The antibody stabilizer composition of the present invention is used for the preservation, dilution or use of antibodies in ELISA detection.

[0021] 2. The antibody stabilizer composition of the present invention is used to inhibit the aggregation or activity reduction of antibodies used in ELISA during storage and use, thereby improving the repeatability of test results.

[0022] 3. This invention utilizes the ELISA method to detect OD 450 Changes in value and potency are used to accurately determine the effect of the stabilizer composition of the present invention. Attached Figure Description

[0023] Figure 1 To measure the OD after placing the sample at different temperatures for 5 minutes in Example 2 of this invention. 450 value.

[0024] Figure 2 To measure the OD after placing at different temperatures for 1 hour in Example 2 of this invention 450 value.

[0025] Figure 3 The original dilution OD in Example 3 of this invention 450 Bar chart of average values ​​(mean ± SD).

[0026] Figure 4 The 1:2 dilution OD in Example 3 of this invention 450 Bar chart of average values ​​(mean ± SD).

[0027] Figure 5 The OD at a 1:4 dilution in Example 3 of this invention 450 Bar chart of average values ​​(mean ± SD).

[0028] Figure 6 The OD at a 1:8 dilution in Example 3 of this invention 450 Bar chart of average values ​​(mean ± SD).

[0029] Figure 7 The OD at a 1:16 dilution in Example 3 of this invention 450 Bar chart of average values ​​(mean ± SD). Detailed Implementation

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Example 1: Preparation of Antibody Stabilizer Composition The stabilizer composition is specifically the following composition, expressed as a final concentration or final mass percentage of each component in the composition: Composition 1: Dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer (50mM) + trehalose (10%) + PVP (polyvinylpyrrolidone) (1%) + L-methionine (10mM); Composition 2: Dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer (50mM) + Trehalose (10%) + PVP (polyvinylpyrrolidone) (1%) + L-methionine (10mM) + Proclin 300 (0.09%). Composition 3: Dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer (50mM) + Trehalose (10%) + PVP (polyvinylpyrrolidone) (1%) + Sorbitol (1%) + L-methionine (10mM) + Proclin 300 (0.09%). Composition 4: Dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer (50mM) + Trehalose (10%) + PVP (polyvinylpyrrolidone) (1%) + Sorbitol (1%) + L-methionine (10mM) + Tween 80 (0.1%) + Proclin 300 (0.09%). Composition 5: Dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer (50mM) + Trehalose (10%) + PVP (polyvinylpyrrolidone) (1%) + Sorbitol (1%) + L-methionine (10mM) + Tween 80 (0.1%) + Proclin 300 (0.09%) + Proline (10mM); Composition 2.1: Histidine (50mM) + Glycine (50mM) + Trehalose (5%) + NaCl (150mM) + Tween-20 (0.05%, 50μL) + Proclin 300 (0.05%); Composition 2.2: Histidine (50mM) + Glycine (50mM) + Trehalose (5%) + NaCl (150mM) + Fish collagen peptide powder (0.5%, 0.5g) + Tween-20 (0.05%, 50μL) + Proclin 300 (0.05%). Composition 2.3: Histidine (20mM) + Ammonium sulfate (50mM) + Arginine (50mM) + Trehalose (5%) + Gelatin (1%) + Tween-20 (0.05%) + Proclin 300 (0.05%); Composition 2.4: Tris (50mM) + L-arginine (50mM) + L-glutamic acid (50mM) + proline (0.3M) + NaCl (150mM) + ammonium sulfate (100mM) + trehalose (5%) + glycerol (1%) + Tween 20 (0.05%) + Proclin300 (0.05%).

[0033] The above nine stabilizer compositions (i.e., referred to as Formulas 1 to 5 and Formulas 2.1 to 2.4 respectively) are all prepared with a total volume of 100 mL, and the components of the compositions are prepared in the order and proportion described.

[0034] The preparation of compositions 1 to 5 above: the final pH value of the solution should be between 7.2 and 7.4; after adding histidine to compositions 2.1, 2.2, and 2.3, the pH value needs to be adjusted to 6.2 with HCl before adding other components; after adding trehalose to composition 2.3, the pH value needs to be adjusted to 6.2 again; when preparing Tris-HCl buffer for composition 2.4, the pH value needs to be adjusted to 7.2. Specifically, when preparing composition 2.3, since gelatin is a solid, it needs to be pretreated before adding it: take 1g of gelatin powder and put it into 20mL of water, heat it at 50-60℃ to melt it, let it cool to room temperature until it becomes a paste, and immediately stir it evenly. This is used as the raw material for preparing the composition.

[0035] Example 2: Establishment of an "antibody-temperature" instability model To establish a method for evaluating the effects of the nine stabilizer compositions in Example 1 of this invention, an "instability model" was constructed. The antibody was transformed into a variable through a stress experiment, and its OD was measured after being placed at different temperatures for different times. 450 The values ​​and antibody titers were determined, and the time and temperature range at which antibody activity decreased by at least half (titer significantly reduced) were established. An instability model was then used to evaluate the effect of the stabilizer.

[0036] African swine fever virus antibody-positive serum was placed at 37℃, 56℃, and 75℃ for 5 min and 1 h, respectively, before testing. Untreated samples were used as a control group and tested simultaneously. This serum was a "qualitative standard sample of African swine fever virus antibody-positive serum" jointly developed by the China Animal Disease Prevention and Control Center and Inner Mongolia Jinmaishi Biotechnology Co., Ltd. It was developed based on a national standard sample development project officially issued by the Standardization Administration of China (SAC), as detailed in the "Notice of the Standardization Administration of China on Issuing the First Batch of National Standard Sample Development and Replication Plan Projects in 2021" (SAC

[2021] No. 4), project number: S2021067. Each sample underwent a 2× serial dilution, and the model was determined by the decrease in indirect ELISA titer. Results are as follows... Figure 1 and Figure 2 As shown. Among them, Figure 1 In the middle, negative control OD 450 The average value was 0.119, and the positive control OD... 450 The average value is 1.895; Figure 2 In the middle, negative control OD 450 The average value was 0.129, and the positive control OD... 450 The average value is 2.039.

[0037] The S / P values ​​of indirect ELISA detection under various temperature conditions after 5 minutes of incubation are shown in Table 1; the S / P values ​​of indirect ELISA detection under various temperature conditions after 1 hour of incubation are shown in Table 2.

[0038] Table 1. Indirect ELISA detection results (S / P values) after samples were placed for 5 minutes under different temperature conditions.

[0039] Table 2. Indirect ELISA detection results (S / P values) after samples were placed for 1 hour under different temperature conditions.

[0040] To further explore and optimize experimental conditions, the storage time was extended at 37℃ and 56℃: African swine fever virus serum was placed at 37℃ and 56℃ for 6 hours and 24 hours, respectively, before testing. Untreated samples were used as a control group and tested simultaneously. Each sample underwent a 2× serial dilution, and the model was successfully established by observing changes in titer. The measured OD... 450 The results are shown in Tables 3 and 5; the S / P values ​​at various temperatures after 6 h and 24 h are shown in Tables 4 and 6. In Table 3, the OD values ​​of the negative control are shown. 450 The average value was 0.1315, and the positive control OD... 450 The average value is 2.01.

[0041] Table 3. Indirect ELISA detection results (OD) after samples were placed for 6 hours under different temperature conditions. 450 value)

[0042] Table 4. ELISA detection results (S / P values) of samples after 6 hours of storage under different temperature conditions.

[0043] Table 5. ELISA detection results (OD) of samples after 24 hours under different temperature conditions. 450 value)

[0044] Table 6. ELISA detection results (S / P values) of samples after 24 hours of storage under different temperature conditions.

[0045] The OD was measured after being placed at 37℃ and 56℃ for 48 hours respectively. 450 Value and potency determination, and OD obtained at 56℃ 450 Independent samples were compared between the values ​​and the control group data. tThe results showed that the samples treated at 56℃ for 48 hours were significantly different from the control group, indicating that the model was valid. The results are shown in Tables 7 and 8.

[0046] Table 7. ELISA detection results (OD) of samples after 48 hours under two different temperature conditions. 450 value)

[0047] Table 8. ELISA detection results (S / P values) of samples after 48 hours under two different temperature conditions.

[0048] Table 1-8 and Figure 1-2 The results showed that ELISA tests were performed on samples placed at 37℃, 56℃, and 75℃ for 5 minutes and 1 hour, respectively. The titers at 37℃ and 56℃ were both 1:4, while the titers at 75℃ were all negative, similar to the control group. Tests were also performed on samples placed at 37℃ for 6 hours, 24 hours, and 48 hours, with no significant changes. However, when samples were placed at 56℃ for 6 hours, the OD... 450 The S / P value decreased to about one-third, and with the extension of the storage time, the S / P value gradually decreased, reaching about 50% after 48 hours. These results indicate that the S / P value of the sample decreased with increasing temperature and storage time at different temperatures and for different durations. The sample was clearly unstable after 48 hours at 56℃.

[0049] Example 3: Preliminary screening of stabilizer compositions According to Example 2 of the present invention, the method for detecting the effects of screening nine stabilizer formulations comprises the following steps: The nine compositions from Example 1 of the present invention, sterile deionized water (as a negative control), and the aforementioned African swine fever serum are mixed evenly at a ratio of 1:1 (v / v), with a control group not subjected to temperature treatment. The African swine fever indirect ELISA kit (batch number: ZE014G250901) produced by Beijing Nabai Biotechnology Co., Ltd. is used for detection according to the kit's instruction manual, and three repeatability tests are performed at different time points.

[0050] Experimental group: Compositions 1-5, 2.1-2.4 and sterile deionized water in Example 1 of this invention were mixed with African swine fever serum at a ratio of 1:1 and placed in an incubator at 56°C for 48 hours.

[0051] Control group: Compositions 1-5, 2.1-2.4 and sterile deionized water in Example 1 of this invention were mixed with African swine fever serum at a ratio of 1:1, without temperature treatment.

[0052] The detection procedure was performed in accordance with the national standard "Diagnostic Techniques for African Swine Fever" (GB / T 18648-2020) and the instructions for the African Swine Fever Virus ELISA Antibody Detection Kit. The steps are as follows: Preparation of 1× Wash Buffer (Note: This wash buffer is the 25× wash buffer provided in the African Swine Fever Virus Indirect ELISA Antibody Detection Kit manufactured by Beijing Nabai Biotechnology Co., Ltd. The ELISA test steps are as described in the kit's instruction manual): Dilute the 25× wash buffer provided in the kit by 25× using sterile ultrapure water (after dilution, it can be stably stored at 2–8℃ for 1 week). For example: to prepare 125mL of 1× wash buffer, add 120mL of sterile ultrapure water to 5mL of 25× wash buffer. Caution: If crystals are found in the 25× wash buffer, be sure to dissolve them completely in a water bath at room temperature or 37℃ before use.

[0053] (1) All samples were serially diluted 2× using the sample diluent provided in the kit: original, 1:2, 1:4, 1:8, 1:16, for a total of 5 dilutions. Each dilution was then diluted 50×. Negative and positive control sera were not diluted.

[0054] (2) Add 100 μL of negative control serum to wells A1 and B1 respectively; add 100 μL of positive control serum to wells C1 and D1 respectively; add the above 9 diluted samples to the other wells of the coated plate, 100 μL / well, and repeat the process.

[0055] (3) Cover the reaction plate and incubate at room temperature (25℃) for 30 minutes (±1 minute).

[0056] (4) Discard the solution in the wells, add 300 μL of 1× washing solution to each well, and discard the washing solution after washing. Repeat the above washing 3 times, and gently pat dry on absorbent paper. It is strictly forbidden for the well plate to dry between each step.

[0057] (5) Add 100 μL of the HRP-labeled anti-pig IgG antibody provided in the kit to each well. Cover the reaction plate and incubate at room temperature for 30 minutes (±1 minute).

[0058] (6) Repeat step (4) above.

[0059] (7) Add 100 μL of TMB substrate solution to each well. Cover the reaction plate and incubate at room temperature for 15 minutes (±1 minute).

[0060] (8) Add 50 μL of stop solution to each well to terminate the enzymatic reaction.

[0061] (9) Read the OD of each well on the microplate reader. 450 value.

[0062] (10) Calculate the S / P value and analyze the data.

[0063] The data obtained above were preprocessed using the Q test (only applicable to small samples, typically 3 ≤ n ≤ 10, and with only one suspicious value) and the Grubbs test (G test) (which can handle multiple outliers), before data analysis was performed to obtain the original dilution OD. 450 Average value, results as follows Figure 3 As shown.

[0064] Original dilution: One-way ANOVA and homogeneity of variance tests were performed on the data from the control group and the nine compositions (discarding questionable values); as shown in the normality test of the above data. P <0.05, does not satisfy normal distribution, and has a homogeneity of variance test with temperature treatment. P If the variance is less than 0.05, the homogeneity of variance is not satisfied, and the KW nonparametric test should be applied.

[0065] Data analysis revealed no significant differences among the formulations without temperature treatment, indicating that the formulation components had no impact on the test results. After treatment at 56℃ for 48 hours, significant differences were observed between the formulation groups. Multiple comparison analysis showed significant differences between the control group and formulations 1, 2, 3, 4, 5, 2.1, 2.2, 2.3, and 2.4.

[0066] For dilutions of 1:2, 1:4, 1:8, and 1:16, OD 450 For detailed statistical results of the average values, please refer to [link / reference]. Figures 4-7 The results, along with SPSS software analysis of experimental data for each formulation under untreated and 56°C-treated conditions for 48 hours, showed a significant difference between the untreated control group and the experimental group treated at 56°C for 48 hours, confirming the validity of the "antibody-temperature instability model." Although slight differences existed between the two experimental groups for each composition, these differences were not significant and were less pronounced than the differences between the control group and the control group. The above analysis indicates that the nine compositions of this invention have stabilizing effects.

[0067] Analysis based solely on original dilution data: Significant comparison of the original volume of each formulation between untreated and 56°C treated (independent samples) t In the test, the results of the control group, compositions 1-5, and compositions 2.1-2.4 are shown in Table 9-18: Table 9 Independent Samples Test for the Control Group

[0068] The experimental data analysis results in Table 9 show that the control group data follows a normal distribution, and the variance test results are... P=0.08, significance >0.05, then look at the significance (two-tailed) of the assumption of equal variance (first row). P A value >0.05 indicates that the null hypothesis is rejected, meaning there is no significant difference; in this case, it is 0.000. P <0.001 indicates extremely significant (***), proving that the established model holds true.

[0069] Table 10 Independent Sample Testing of Composition 1

[0070] The results of the experimental data analysis in Table 10 show that, P =0.015 < 0.05, indicating a significant difference (*). Compared to the highly significant difference in the control group, this significance is relatively small, proving that composition 1 of the present invention has some effect, but the effect is weak. (Note: The smaller the significance, the better the effect of the composition.) Table 11 Independent Sample Testing of Composition 2

[0071] The data analysis results in Table 11 show that, P =0.005<0.05 indicates a significant difference (**). Compared with the highly significant control, the significance is relatively low, indicating that the composition has an effect but it is very weak and can be ignored.

[0072] Table 12 Independent Sample Testing of Composition 3

[0073] The results of the experimental data analysis in Table 12 show that, P <0.001 indicates a highly significant difference (***), proving that composition 3 is significantly similar to the control group, and that the composition has no effect.

[0074] Table 13 Independent Sample Testing of Composition 4

[0075] Analysis of the experimental data in Table 13 shows that, P <0.001 indicates a highly significant difference (***), proving that composition 4 is significantly similar to the control group, and that the composition has no effect.

[0076] Table 14 Independent Sample Testing of Composition 5

[0077] The results of the experimental data analysis in Table 14 show that, P=0.03<0.05 indicates a significant difference (*), which is highly significant compared to the control, indicating that the significance is not high. This composition is effective and stronger than composition 1.

[0078] If any of the data in composition 2.1 does not conform to a normal distribution, a nonparametric test is used.

[0079] Table 15 Statistical analysis of composition 2.1

[0080] The results of the experimental data analysis in Table 15 show that the significance is... P =0.01<0.05, indicating a significant difference (*). Compared to the control, the difference is extremely significant, indicating that the significance is relatively low, proving that composition 2.1 has some effect, but the effect is relatively weak.

[0081] Table 16 Independent Sample Testing of Composition 2.2

[0082] The results of the experimental data analysis in Table 16 show that, P =0.174>0.05, which proves that there is no significant difference, and composition 2.2 has the best effect.

[0083] Table 17 Statistical Analysis of Composition 2.3

[0084] The results of the experimental data analysis in Table 17 show that... P =0.025<0.05, which indicates a significant difference (*), suggesting that composition 2.3 is effective, but the effect is weak.

[0085] Table 18 Independent Sample Testing of Composition 2.4

[0086] The results of the experimental data analysis in Table 18 show that, P <0.001 indicates extremely significant (***), meaning that composition 2.4 has no effect.

[0087] Based on the above method, the OD values ​​obtained under two conditions (i.e., the experimental group and the control group) were analyzed. 450 Values ​​for independent sampling tAnalysis of the results showed that the test results of the samples treated at 56℃ for 48 hours were not significantly different from those of the control group. Four compositions were screened and found to have protective effects. Analysis of the above results and data from SPSS software on each formulation after treatment at 56℃ for 48 hours without temperature treatment, as shown in Tables 9-18, revealed a significant difference between the control group (without temperature treatment) and the experimental group treated at 56℃ for 48 hours, thus confirming the validity of the "antibody-temperature instability model." Although there were slight differences between the two experimental groups for each formulation, the differences were less pronounced than those in the control group, demonstrating that all nine formulations had a stabilizing effect (Note:). P The higher the value, the better the formula's effect, because P The larger the value, the less significant the difference between the untreated and treated data, and the better the formulation effect.

[0088] Based on independent samples of each composition at its original dilution t The test analysis results show that: Composition 2.2 ( P =0.174) has the best protective effect, followed by composition 5 ( P =0.03), composition 2.3 ( P =0.025), Composition 1 ( P =0.015), composition 2.1 ( P =0.01).

[0089] Example 4: Determination of stabilizer ratio According to the method in Example 3 of the present invention, African swine fever serum antibodies were uniformly mixed with Composition 1, Composition 5, Composition 2.2, and Composition 2.3 in seven ratios: 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, and 1:8, respectively. A control group was set up, and three repeatability tests were performed at different times. Statistical analysis of variance was performed on the test results. Experimental group: Composition 1, Composition 5, Composition 2.2, Composition 2.3, deionized water and African swine fever serum antibody were mixed evenly in 7 proportions and placed in an incubator at 56℃ for 48 hours.

[0090] Control group: Composition 1, Composition 5, Composition 2.2, Composition 2.3, deionized water and African swine fever serum were mixed in 7 proportions and no temperature treatment was performed.

[0091] The OD obtained above 450 The values ​​were tested for normality and independent samples. One group (56℃) did not conform to a normal distribution, so a nonparametric test was used. The results are shown in Tables 19-23, demonstrating that: (1) Deionized water as the control group: significance test results P<0.05 indicates a significant difference, which proves that all proportions of the experiment are statistically significant and all proportions satisfy the unstable model. (2) The ratio of composition 2.2 to African swine fever serum antibody at 4:1 was ineffective or had no effect; the ratio of composition 2.2 to African swine fever serum at 1:2 had no effect. (3) The ratio of composition 2.3 to African swine fever serum antibody was 8:1, 1:2, 1:4, and 1:8. The results showed no effect. The ratio of composition 2.3 to African swine fever serum antibody was also ineffective. (4) Except for the above-mentioned compositions and corresponding proportions which have no effect, the other proportions of compositions 1, 5, 2.2 and 2.3 have all been shown to have an effect; (5) Data analysis of the results shows that the optimal mixing ratio (v / v) of African swine fever serum antibody and stabilizer is as follows: the optimal ratio of composition 1 is 2:1, the optimal ratio of composition 5 is 1:1, the optimal ratio of composition 2.2 is 1:1, and the optimal ratio of composition 2.3 is 2:1.

[0092] Table 19 Activity retention rate of deionized food

[0093] Table 20 Activity retention rate of Composition 1

[0094] As shown in Table 20 above, considering only the average activity rate of composition 1, the 2:1 ratio exhibits the highest activity rate. Furthermore, the activity rate of composition 1 with a 2:1 ratio is significantly higher than that of water (control) with a 2:1 ratio. This aligns with the previously calculated significance analysis results. p If the optimal ratios are 2:1 and 1:8 (maximum value), then the optimal ratio is determined to be 2:1.

[0095] Table 21 Activity retention rate of composition 5

[0096] As shown in Table 21 above, considering only the average activity rate of composition 5, the 1:1 ratio exhibits the highest activity rate. Furthermore, the activity rate of the 1:1 ratio in composition 5 is significantly higher than that of water (control) at a 1:1 ratio. This aligns with the previously calculated significance analysis results. p If the value is the largest, then the optimal ratio is determined to be 1:1.

[0097] Table 22 Activity retention rate of composition 2.2

[0098] As shown in Table 22 above, considering only the average activity rate of compositions 2.2, the 1:4 ratio exhibits the highest activity rate, followed by 1:8 and 1:1. Furthermore, the activity rates of compositions 5 at the 1:8, 1:4, and 1:1 ratios are significantly higher than those corresponding to water (control). Considering the previously calculated significance analysis results, the variance of the 1:4 ratio is unequal, suggesting that its optimal ratio is either 1:1 or 1:8. However, the 1:8 ratio has insufficient antibody content, making it difficult to ensure OD (Obstacle Course). 450 Regarding the accuracy of the data, we reserve our opinion for now and determine the optimal ratio as 1:1.

[0099] Table 23 Activity retention rate of composition 2.3

[0100] As shown in Table 23 above, considering only the average activity rate of formulation 2.3, the 2:1 ratio exhibits the highest activity rate. Furthermore, the activity rate of the 2:1 ratio in formulation 2.3 is significantly higher than that of water (control) at a 2:1 ratio. This is consistent with the significance analysis results calculated from the preceding data. p If the value is the largest, then the optimal ratio is determined to be 2:1.

[0101] Example 5: Forced Oxidation Degradation Experiment The composition with the best protective effect prepared in Example 4 of this invention was mixed with the above-mentioned African swine fever serum antibody in the optimal ratio, placed in an open glass container, and treated under an ozone atmosphere of 0.3~0.5 ppm for 10~15 min at a treatment temperature of 22~25 °C. After treatment, the sample was sealed and allowed to stand at 4 °C for 1 h.

[0102] The detection was performed according to the method in Embodiment 2 of the present invention, and the OD was calculated according to the instruction manual. 450 The activity retention rate was calculated using the S / P value or the control group as a reference.

[0103] Experimental group: Composition 2.2, deionized water and African swine fever serum antibody were mixed evenly in the corresponding proportions and placed in an ozone generator for oxidation treatment.

[0104] Control group: Composition 2.2, deionized water and African swine fever serum antibody were mixed evenly in the same proportion without oxidation treatment.

[0105] The results of the above experiments are shown in Table 24 below.

[0106] Table 24 Activity rate of composition 2.2 oxidation test

[0107] Analysis of the above data shows that the stabilizer composition of the present invention can significantly improve the antibody ELISA activity rate under ozone oxidation conditions, and the activity rate of the preferred composition can reach 60-80%.

[0108] Example 6: Mechanical Oscillation Stability Experiment According to the method in Examples 2-3 of this invention, the four prepared compositions were mixed with the above-mentioned African swine fever serum antibody in the optimal ratio, and the samples were treated under shaking conditions (300 rpm, 24 h). The antibody activity was then detected by ELISA. The results were analyzed.

[0109] Experimental group: Composition 2.2, deionized water and African swine fever serum antibody were mixed evenly in the corresponding proportions and processed in a shaker.

[0110] Control group: Composition 2.2, deionized water and African swine fever serum antibody were mixed evenly in the corresponding proportions without shaking.

[0111] Table 25 Activity rate of composition 2.2 shaking test

[0112] The results showed that the activity loss in the experimental group was significantly lower than that in the control group, indicating that the composition of the present invention can effectively inhibit antibody inactivation caused by transportation or shaking.

[0113] Example 7: Repeated freeze-thaw stability test According to the method in Examples 2-3 of the present invention, the prepared composition with the best protective effect was mixed with the above-mentioned African swine fever serum antibody in the optimal ratio, dispensed into sealed containers, frozen at -80 °C and thawed at 25 °C, and this process was repeated 0 and 10 times. After the freeze-thaw cycle was completed, the antibody immunomodulatory activity was detected by ELISA. Table 26 Activity rate of composition 2.2 after 10 freeze-thaw cycles

[0114] After multiple freeze-thaw cycles, the antibody activity retention rate of the stabilizer composition samples of the present invention can still be maintained at 75%~95%, while the activity of the control group usually drops to about 70%, indicating that the composition of the present invention has good freeze-thaw stability protection ability.

[0115] Example 8: Broad-spectrum verification experiment Antibodies from various sources and targeting different targets were selected, including African swine fever antibodies, porcine reproductive and respiratory syndrome (PRRS) antibodies, porcine circovirus disease (PCV) antibodies, and classical swine fever antibodies (all antibodies were prepared by the Veterinary Diagnostic Laboratory of the China Animal Disease Prevention and Control Center and the National / WOAH Reference Laboratory for Porcine Reproductive and Respiratory Syndrome). These antibodies were mixed with the stabilizer composition of this invention, and stability tests were conducted under the established instability model conditions. Their activity was detected using an ELISA method.

[0116] Control group: African swine fever antibody, porcine reproductive and respiratory syndrome antibody, porcine circovirus antibody, classical swine fever antibody and composition 2.2 were mixed uniformly in a 1:1 ratio, and then subjected to ELISA detection without temperature treatment.

[0117] Experimental group: African swine fever antibody, porcine reproductive and respiratory syndrome antibody, porcine circovirus antibody, classical swine fever antibody and composition 2.2 were mixed uniformly in a 1:1 ratio, treated at 56℃ for 48h and then detected by ELISA.

[0118] Table 27 Activity rates of four antibodies

[0119] The stabilizer can significantly improve the stability of antibodies from different sources and targeting antibodies, with the activity rate generally maintained between 70% and 90%, proving that the stabilizer has good broad-spectrum applicability.

[0120] Example 9: Short-term storage stability test According to the method in Examples 2-3 of this invention, the four prepared compositions were mixed with antibodies in corresponding proportions and stored at 4°C for 3, 7, 15, and 30 days; at 25°C for 3, 5, 7, and 15 days; and at 37°C for 3, 5, and 7 days. The antibodies were then simultaneously tested with samples stored for 0 days using the ELISA method to detect their immunomodulatory activity. The OD was calculated according to the instructions. 450 The activity retention rate was calculated using the S / P value or S / P value, with day 0 as the control group and as a reference.

[0121] Table 28 Short-term stability activity rate

[0122] During the storage period, the stabilizer composition of the present invention can still maintain antibody immune activity of about 70% to 90%, proving that the stabilizer of the present invention can be stable for at least 30 days at 4°C, at least 15 days at 25°C, and at least 7 days at 37°C.

[0123] Example 10: Long-term storage stability test According to the method in Examples 2-3 of this invention, the four prepared compositions were mixed with antibodies in corresponding proportions and stored at 4°C for 1, 3, 5, 7, 12, and 24 months before testing. The antibody immunomodulatory activity was detected using the ELISA method. OD was calculated according to the instructions. 450 The activity retention rate was calculated by setting a control group as a reference.

[0124] Table 29 Long-term stability activity rate

[0125] The results are shown in Table 29. During the storage periods of 0 days, 1 month, 3 months, 5 months, 7 months, 1 year, and 2 years, the stabilizer composition of the present invention can still maintain antibody immune activity above 80%, which is greater than that of the control group, proving that the stabilizer of the present invention has good long-term stability.

Claims

1. An antibody stabilizer composition for maintaining the stability of ELISA detection performance, characterized in that, The components include the following concentrations: The final concentration of the buffer is 10~100 mM, the mass percentage of the sugar and / or polyol is 1~15%, and the final concentration of the amino acid is 1~300 mM.

2. The antibody stabilizer composition according to claim 1, characterized in that, The antibody stabilizer composition further includes at least one of a polymeric stabilizer, an inorganic salt, a surfactant, and a preservative.

3. The antibody stabilizer composition according to claim 2, characterized in that, In the antibody stabilizer composition, the mass percentage concentration of the polymeric stabilizer is 0-2%; The final concentration of the inorganic salt is 50~150mM; The surfactant has a mass percentage concentration of 0-0.2%; The mass percentage concentration of the preservative is 0~0.1%.

4. The antibody stabilizer composition according to any one of claims 1-3, characterized in that, The buffer is selected from at least one of phosphate buffer solution, histidine buffer system and Tris buffer solution; The phosphate buffer solution includes a dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer pair.

5. The antibody stabilizer composition according to any one of claims 1-4, characterized in that, The sugars and / or polyols are selected from at least one of trehalose, sorbitol and glycerol.

6. The antibody stabilizer composition according to any one of claims 1-5, characterized in that, The amino acid is selected from at least one of L-methionine, arginine, glycine, proline, and glutamic acid.

7. The antibody stabilizer composition according to any one of claims 2-6, characterized in that, The polymeric stabilizer is selected from at least one of polyvinylpyrrolidone, gelatin, and collagen peptides; The inorganic salt is selected from sodium chloride and / or ammonium sulfate; The surfactant is selected from Tween-20 and / or Tween-80; The preservatives include Proclin-type preservatives.

8. The antibody stabilizer composition according to any one of claims 1-7, characterized in that, The pH value of the antibody stabilizer composition is 6.0~8.

0.

9. Use of the antibody stabilizer composition according to claims 1-8 in the preparation of antibody stabilizers for the preservation, dilution or use of antibodies in ELISA assays.

10. The application according to claim 9, characterized in that, The antibody stabilizer is used to inhibit the aggregation or decrease in activity of antibodies used in ELISA during storage and use.

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