Horseradish peroxidase marker storage solution and application thereof

Abstract

The present application relates to the technical field of in vitro diagnosis protein marker, in particular to a preservative for horseradish peroxidase marker and application thereof. The present application provides a preservative for horseradish peroxidase marker and application thereof. The preservative comprises lecithin 0.05-1.0 g / L, bovine serum albumin 2-10 g / L, sodium chloride 5-30 g / L, polyaspartic acid 2-10 g / L, and the rest is glycerol. The preservative can significantly improve the storage stability of enzyme-labeled product, and the activity of enzyme-labeled antibody can still maintain more than 70% after 21 days of storage at 37 DEG C. In the bottle opening stability test, the activity of enzyme-labeled antibody can maintain more than 87% after 45 days of storage at 4 DEG C.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic protein labeling technology, specifically to a preservation solution for horseradish peroxidase labeling and its application. Background Technology

[0002] Horseradish peroxidase (HRP) is a commonly used enzyme in clinical laboratory reagents. Widely distributed in the plant kingdom, HRP is abundant in horseradish. It is a glycoprotein composed of a colorless enzyme protein and a brown iron porphyrin, with a sugar content of approximately 18%. HRP-labeled proteins can be used for multiple biochemical assays and are widely used in immunoassay (ELISA) kits. The sodium periodate method, which couples HRP and glycoprotein, is a classic approach. This method utilizes sodium periodate to oxidize the sugar chains on glycoproteins (antibodies, glycosylated enzymes, etc.) into aldehyde groups. Under certain conditions, these aldehydes bind to antibodies to form Schiff bases. Sodium borohydride then reduces the Schiff bases to stable carbon-nitrogen single bonds and excess aldehyde groups in the glycoprotein molecule, thus yielding horseradish peroxidase. However, horseradish peroxidase labels suffer from low stability in solution; prolonged storage often degrades the performance of corresponding immunoassay kits, limiting their application.

[0003] Existing technologies attempt to improve the stability of enzyme-labeled products by modifying HRP labeling technology, such as by changing the dialysis solution, or by improving the storage stability by adding proteins, amino acids, salt ions, or chemicals with special properties. While these methods can improve the stability of enzyme-labeled products to some extent, the improvement is not significant, or there are issues such as the toxicity of the substances or excessive cost.

[0004] Therefore, there is an urgent need to find a low-cost, non-toxic technology that can effectively improve the stability of enzyme-labeled products. Summary of the Invention

[0005] In view of this, the present invention provides a preservation solution for horseradish peroxidase markers and their applications.

[0006] This invention provides a preservation solution for horseradish peroxidase (HPP) labeled products and its applications. The preservation solution comprises lecithin and polyamino acids, the addition of which significantly improves the preservation stability of the enzyme-labeled product. In particular, after 21 days of storage at 37°C, the activity of the enzyme-labeled antibody remains above 70%. In an open-bottle stability test, the enzyme-labeled antibody activity remained above 87% after 45 days of storage at 4°C using the preservation solution of this invention. The presence of lecithin and polyamino acids in this invention exhibits a synergistic effect, potentially altering the protein's state and stabilizing its structure at high temperatures, ultimately enhancing the stability of the enzyme-labeled product. Furthermore, all materials required in the preservation solution of this invention are non-toxic, low-cost, and easy to prepare, facilitating industrial production.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a preservation solution for horseradish peroxidase markers, comprising lecithin, bovine serum albumin, sodium chloride, polyaspartic acid, and glycerol.

[0009] In some specific embodiments of the present invention, the preservation solution, per 1L, comprises the following components:

[0010]

[0011] In some specific embodiments of the present invention, the preservation solution, per 1L, comprises the following components:

[0012]

[0013] In some specific embodiments of the present invention, the lecithin includes, but is not limited to, soybean lecithin.

[0014] In some specific embodiments of the present invention, the preservation solution further includes a preservative;

[0015] The preservative has a mass percentage content of 0.01% to 0.5%.

[0016] In some specific embodiments of the present invention, the preservatives include, but are not limited to, Proclin 300 and BND.

[0017] In some specific embodiments of the present invention, the average molecular weight of the polyaspartic acid includes 1000 to 5000.

[0018] In some specific embodiments of the present invention, the average molecular weight of the polyaspartic acid includes 3000 to 5000;

[0019] The average molecular weight of the polyaspartic acid is 3000-4000.

[0020] Preferably, the polyaspartic acid has an average molecular weight of 3500.

[0021] In some specific embodiments of the present invention, the pH of the preservation solution is 7.0 to 8.0.

[0022] In some specific embodiments of the present invention, the pH of the preservation solution is 7.0 to 7.5.

[0023] The present invention also provides a method for preparing the preservation solution, wherein lecithin, bovine serum albumin, sodium chloride, polyaspartic acid and glycerol are mixed in the prescribed amounts.

[0024] In some specific embodiments of the present invention, the method for preparing the preservation solution includes taking the prescribed amounts of lecithin, bovine serum albumin, sodium chloride, polyaspartic acid and glycerol, mixing them, and stirring to dissolve them;

[0025] The stirring speed is 100-300 rpm.

[0026] Based on the above research, the present invention also provides the application of the preservation solution or the preservation solution prepared by the above method in any of the following:

[0027] (1) Improve the stability of the standard product; and / or

[0028] (2) Preparation of horseradish peroxidase labeled solution.

[0029] The present invention also provides a horseradish peroxidase labeled solution, comprising an enzyme label and any of the following:

[0030] (1) the preservation solution; and / or

[0031] (2) The preservation solution prepared by the preparation method.

[0032] In some specific embodiments of the present invention, the enzyme label includes enzyme-labeled antibodies, enzyme-labeled antigens, and / or enzyme-labeled small molecule compounds.

[0033] In some specific embodiments of the present invention, the enzyme marker is an enzyme-labeled antibody;

[0034] The enzyme-labeled antibody is a cTnI antibody.

[0035] The present invention also provides a method for preparing the horseradish peroxidase labeled solution, wherein the enzyme-labeled antibody is mixed with the preservation solution or the preservation solution prepared by the preparation method to obtain the horseradish peroxidase labeled solution;

[0036] The volume percentage concentration of the preservation solution ranges from 40% to 60%.

[0037] In some specific embodiments of the present invention, the volume percentage concentration of the preservation solution includes 50-60%.

[0038] In some specific embodiments of the present invention, preferably, the volume percentage concentration of the preservation solution is 60%.

[0039] The present invention also provides a kit comprising any of the following:

[0040] (1) the preservation solution; and / or

[0041] (2) The preservation solution prepared by the method described above; and / or

[0042] (3) the horseradish peroxidase-labeled solution; and / or

[0043] (4) Horseradish peroxidase-labeled solution prepared by the above preparation method.

[0044] This invention provides a preservation solution for horseradish peroxidase-labeled products and its applications. The preservation solution comprises 0.05–1.0 g / L lecithin, 2–10 g / L bovine serum albumin, 5–30 g / L sodium chloride, 2–10 g / L polyaspartic acid, and the remainder being glycerol. The preservation solution of this invention significantly improves the preservation stability of enzyme-labeled products, especially after 21 days of storage at 37°C, where the enzyme-labeled antibody activity still remains above 70%. In the open-bottle stability test, under the preservation solution of this invention, the enzyme-labeled antibody activity remained above 87% after 45 days of storage at 4°C. Detailed Implementation

[0045] This invention discloses a preservation solution for horseradish peroxidase markers and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0046] To achieve the above objectives, the present invention provides a preservation solution for horseradish peroxidase labeling. By adding lipid substances and polyamino acids to the preservation system, the stability of the enzyme labeling solution is significantly improved. At the same time, the lipid substances and polyamino acids are non-toxic and low in cost, making them suitable for large-scale promotion and application.

[0047] Specifically, the present invention first provides a preservation solution for horseradish peroxidase-labeled compounds, wherein the preservation solution comprises 0.05-1.0 g / L lecithin, 2-10 g / L bovine serum albumin, 5-30 g / L sodium chloride, 2-10 g / L polyaspartic acid, and the remainder being glycerol.

[0048] In one embodiment of the present invention, the average molecular weight of the polyaspartic acid is 1000-5000, preferably 3000-5000, and most preferably 3500.

[0049] In one embodiment of the present invention, the preservation solution further includes a preservative.

[0050] In one embodiment of the present invention, the preservative is selected from one or more of Proclin 300 and BND.

[0051] In one embodiment of the present invention, the preservative has a mass percentage content of 0.01% to 0.5%.

[0052] In one embodiment of the present invention, the lecithin includes, but is not limited to, soybean lecithin.

[0053] In one embodiment of the present invention, the pH of the preservation solution is 7.0 to 8.0, preferably adjusted to 7.0-8.0 using 1M NaOH or 1M HCl.

[0054] This invention also provides a method for preparing the preservation solution of the above-mentioned horseradish peroxidase marker, which involves adding the weighed substances into glycerol and stirring at low speed to dissolve them.

[0055] In one embodiment of the present invention, the low speed is 100 to 300 rpm.

[0056] The present invention also provides a horseradish peroxidase labeled solution, comprising horseradish peroxidase labeled and the above-mentioned preservation solution.

[0057] In one embodiment of the present invention, the volume percentage of the horseradish peroxidase-labeled solution and the preservation solution is 40% to 60%.

[0058] In one embodiment of the present invention, the horseradish peroxidase label includes enzyme-labeled antibodies, enzyme-labeled antigens, enzyme-labeled small molecule compounds, etc.

[0059] The present invention also provides a method for preparing enzyme-labeled antibodies / antigens, wherein the method utilizes a modified sodium periodate method to label and obtain enzyme-labeled antibodies / antigens, and finally mixes the enzyme-labeled antibodies / antigens with the above-mentioned preservation solution at a ratio of 40-60%.

[0060] Preparation of preservation solution: Weigh out the required amounts of each substance in the formula, add them to glycerin, and mix thoroughly.

[0061] The preservation solution for horseradish peroxidase labeling provided by this invention and the raw materials and reagents used in its application are all commercially available.

[0062] The present invention will be further illustrated below with reference to the embodiments:

[0063] Example 1: Preparation of enzyme-labeled antibody

[0064] (1) Prepare an 8 mg / mL HRP solution and a 0.1 M sodium periodate solution using ultrapure water. Add 25 μL of sodium periodate solution per mg HRP to the HRP solution and react at 4°C in the dark for 2 h. Then, add ethylene glycol to stop the reaction and react at 4°C in the dark for 0.5 h. Dialyze the solution in 1 mM acetate buffer at pH 4.4 for 12 h, changing the buffer three times. At the same time, dialyze the cTnI antibody (2 mg / mL, 2 mL) in 0.01 mM carbonate buffer at pH 9.6 for 12 h, changing the buffer three times.

[0065] (2) Then, HRP and cTnI antibody were mixed at a molar ratio of 5:1, and 180 μL of 0.2 M carbonate solution with pH 9.6 was added to each mL of HRP solution to adjust the pH of the mixture to ≥ 9.5. The mixture was reacted at 4°C in the dark for 24 h.

[0066] (3) The reaction was terminated by adding 30 μL of 0.106 M sodium borohydride solution per mg HRP. The reaction was carried out at 4 °C in the dark for 2 h. Then, the solution was dialyzed in 0.067 M PBS buffer at pH 6.8 at 4 °C for 16 h, with the buffer changed 3 times during the process, to obtain enzyme-labeled cTnI antibody.

[0067] Example 2: Preservation of enzyme-labeled antibodies

[0068] An equal volume (v / v) of preservation solution was added to the enzyme-labeled antibody prepared in Example 1. The preservation solution contained 0.5 g / L lecithin, 6 g / L bovine serum albumin, 15 g / L sodium chloride, 4 g / L polyaspartic acid (average molecular weight 3500), 0.02% preservative Proclin 300, and the remainder was glycerol. The pH of the preservation solution was 7.0.

[0069] Example 3: Preservation of enzyme-labeled antibodies

[0070] An equal volume (v / v) of preservation solution was added to the enzyme-labeled antibody prepared in Example 1. The preservation solution contained 0.2 g / L lecithin, 9 g / L bovine serum albumin, 25 g / L sodium chloride, 7 g / L polyaspartic acid (average molecular weight 3500), 0.01% preservative Proclin 300, and the remainder was glycerol. The pH of the preservation solution was 7.0.

[0071] Example 4: Preservation of enzyme-labeled antibodies

[0072] The enzyme-labeled antibody prepared in Example 1 was added to a preservation solution (v / v) accounting for 60% of the total volume. The preservation solution contained 0.5 g / L lecithin, 6 g / L bovine serum albumin, 15 g / L sodium chloride, 4 g / L polyaspartic acid (average molecular weight of 3500), 0.02% preservative Proclin 300, and the remainder was glycerol. The pH of the preservation solution was 7.5.

[0073] Example 5: Preservation of enzyme-labeled antibodies

[0074] An equal volume (v / v) of preservation solution was added to the enzyme-labeled antibody prepared in Example 1. The preservation solution contained 0.9 g / L lecithin, 2 g / L bovine serum albumin, 7 g / L sodium chloride, 3 g / L polyaspartic acid (average molecular weight of 4000), and the remainder was glycerol. The pH of the preservation solution was 7.0.

[0075] Comparative Example 1: Preservation of Enzyme-Labeled Antibody

[0076] The enzyme-labeled antibody prepared in Example 1 was added to an equal volume of glycerol (v / v) for storage.

[0077] Comparative Example 2

[0078] An equal volume (v / v) of preservation solution was added to the enzyme-labeled antibody prepared in Example 1. The preservation solution contained 6 g / L bovine serum albumin, 15 g / L sodium chloride, 4 g / L polyaspartic acid (average molecular weight 3500), 0.02% preservative Proclin 300, and the remainder was glycerol. The pH of the preservation solution was 7.0.

[0079] Comparative Example 3

[0080] An equal volume (v / v) of preservation solution was added to the enzyme-labeled antibody prepared in Example 1. The preservation solution contained 0.5 g / L lecithin, 6 g / L bovine serum albumin, 15 g / L sodium chloride, 0.02% Proclin 300 preservative, and the remainder was glycerol. The pH of the preservation solution was 7.0.

[0081] Comparative Example 4

[0082] An equal volume (v / v) of preservation solution was added to the enzyme-labeled antibody prepared in Example 1. The preservation solution contained 0.5 g / L lecithin, 15 g / L sodium chloride, 4 g / L polyaspartic acid (average molecular weight 3500), 0.02% preservative Proclin 300, and the remainder was glycerol. The pH of the preservation solution was 7.0.

[0083] Comparative Example 5

[0084] An equal volume (v / v) of preservation solution was added to the enzyme-labeled antibody prepared in Example 1. The preservation solution contained 0.5 g / L lecithin, 6 g / L bovine serum albumin, 15 g / L sodium chloride, 4 g / L aspartic acid, 0.02% preservative Proclin 300, and the remainder was glycerol (with polyaspartic acid replaced by aspartic acid). The pH of the preservation solution was 7.0.

[0085] Example of effect 1

[0086] The preservation solution formulations for Examples 2-5 and Comparative Examples 1-5 are shown in Table 1 below.

[0087] Table 1. Preservative formulations for Examples 2-5 and Comparative Examples 1-5

[0088]

[0089]

[0090] Example 2: Storage Stability Test

[0091] The HRP-labeled cTnI antibody solutions obtained in Examples 2-5 and Comparative Examples 1-5 were subjected to accelerated assays in a 37°C incubator. After incubation for 7, 14, and 21 days, the enzyme-labeled reagent was removed and added to the wells of ELISA plates coated with cTnI antigen. cTnI antibody was added for competition, and the antigen-antibody reaction was carried out at 37°C for 1 hour. After washing the plates, luminol was added for color development, and the reaction was terminated by measuring the luminescence value. The retention rate of luminescence value is shown in Table 2 below.

[0092] Table 2. Accelerated test results of enzyme-labeled antibody preservation solutions in Examples 2-5 and Comparative Examples 1-5

[0093] Experiment number 37℃, 0 days 37℃, 7 days 37℃, 14 days 37℃, 21 days Example 2 100％ 98.2％ 95.4％ 78.6％ Example 3 100％ 97.9％ 92.3％ 73.2％ Example 4 100％ 98.1％ 94.8％ 76.9％ Example 5 100％ 97.7％ 92.4％ 76.3％ Comparative Example 1 100％ 61.8％ 34.7％ 11.1％ Comparative Example 2 100％ 92.5％ 80.6％ 56.0％ Comparative Example 3 100％ 91.3％ 77.1％ 55.2％ Comparative Example 4 100％ 96.7％ 85.2％ 63.7％ Comparative Example 5 100％ 91.7％ 75.9％ 54.3％

[0094] As shown in Table 1, the preservation solutions in Examples 2-5, when containing lecithin, polyaspartic acid, and bovine serum albumin, effectively improve the stability of enzyme-labeled antibodies. After storage at 37°C for 7 and 14 days, over 90% of the enzyme-labeled antibody activity is retained, and after 21 days, over 70% of the activity is retained, with a maximum retention of 78.6%. Furthermore, Example 5 demonstrates that even without the addition of preservatives, the composition of the preservation solution of this invention effectively maintains the stability of the enzyme-labeled antibody.

[0095] Compared with Example 2, when Comparative Example 1 used only glycerol as the preservation solution, its activity was only about 60% after 7 days of storage at 37°C, and only 34.7% of the activity was retained after 14 days of storage. It can be seen that using glycerol alone as the preservation solution will greatly affect the stability of enzyme-labeled antibodies.

[0096] Compared with Example 2, the preservation solutions of Comparative Examples 2-4 were missing lecithin, polyaspartic acid and bovine serum albumin, respectively. The results showed that the absence of any one of the three substances reduced the stability of the enzyme-labeled antibody, indicating that the three substances can synergistically improve the stability of the enzyme-labeled antibody.

[0097] Compared with Example 2, when polyaspartic acid was replaced with aspartic acid in Comparative Example 5, its effect on improving the stability of enzyme-labeled antibodies was not significant, indicating that polyaspartic acid is more beneficial to improving the stability of enzyme-labeled antibody activity than aspartic acid.

[0098] Example 3: Stability after opening

[0099] Three batches of reagent kits were opened and stored in a refrigerator at 4°C for 45 days. Samples were taken at 0, 15, 30 and 45 days after the date of storage. After each sampling, the cap was tightened and the kits were returned to 4°C for storage. The changes in absorbance were measured according to the above storage stability test. The results are shown in Table 3.

[0100] Table 3. Results of the open-bottle stability of the enzyme-labeled antibody preservation solutions of Examples 2-5 and Comparative Examples 1-5.

[0101] Experiment number 0d 15d 30d 45d Example 2 100％ 99.2％ 96.3％ 92.4％ Example 3 100％ 98.1％ 94.4％ 89.7％ Example 4 100％ 97.9％ 92.6％ 87.3％ Example 5 100％ 97.5％ 93.1％ 88.9％ Comparative Example 1 100％ 77.3％ 55.7％ 41.1％ Comparative Example 2 100％ 91.7％ 82.6％ 73.2％ Comparative Example 3 100％ 94.4％ 81.3％ 68.6％ Comparative Example 4 100％ 96.2％ 85.2％ 78.7％ Comparative Example 5 100％ 94.1％ 81.9％ 70.4％

[0102] As shown in Table 3, the diluted preservation solution of the present invention can ensure that the enzyme-labeled antibody retains more than 87% of its activity 45 days after opening, which is significantly better than comparative examples 1-5.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A preservation solution for horseradish peroxidase-labeled compounds, characterized in that, It consists of the following components per 1 liter: Lecithin 0.2~0.9g / L; Bovine serum albumin 2-9 g / L; Sodium chloride 7~25g / L; Polyaspartic acid 3~7 g / L; Glycerin balance.

2. A preservation solution for horseradish peroxidase-labeled compounds, characterized in that, It consists of the following components per 1 liter: Lecithin 0.2~0.9g / L; Bovine serum albumin 2-9 g / L; Sodium chloride 7~25g / L; Polyaspartic acid 3~7 g / L; Preservatives: 0.01%~0.5%; Glycerin balance.

3. The preservation solution according to any one of claims 1 to 2, characterized in that, The average molecular weight of the polyaspartic acid ranges from 1000 to 5000.

4. The preservation solution according to any one of claims 1 to 2, characterized in that, The pH of the preservation solution is 7.0~8.

0.

5. The method for preparing the preservation solution as described in claim 1, characterized in that, Mix the prescribed amounts of lecithin, bovine serum albumin, sodium chloride, polyaspartic acid, and glycerol.

6. The use of the preservation solution according to any one of claims 1 to 4 or the preservation solution prepared by the method according to claim 5 in any one of the following: (1) Improve the stability of enzyme-labeled products; (2) Preparation of horseradish peroxidase labeled solution.

7. A horseradish peroxidase-labeled solution, characterized in that, Including enzyme markers and any one of the following: (1) The preservation solution as described in any one of claims 1 to 4; (2) The preservation solution prepared by the method described in claim 5.

8. A reagent kit, characterized in that, Includes any one of the following: (1) The preservation solution as described in any one of claims 1 to 4; (2) The preservation solution prepared by the method described in claim 5; (3) The horseradish peroxidase labeled solution as described in claim 7.

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