A cyclic citrullinated peptide latex microsphere preservation solution and its application

Abstract

The present invention discloses a cyclic citrullinated peptide latex microsphere preservation solution, which contains either reduced glutathione or vitamin C, as well as a buffer, NaCl, an excipient, a stabilizer, and Tween 20. The preservation solution of the present invention can ensure the stability of the CCP latex microspheres, thereby reducing the probability of oxidation of the CCP latex microspheres during use, thereby improving the accuracy of detection. At the same time, it can increase the density of the preservation solution, thereby preventing the immune microspheres from firmly binding due to microsphere agglomeration or aggregation for too long.

Description

Technical Field

[0001] The present invention relates to immunoturbidimetry, and in particular to a cyclic citrullinated peptide latex microsphere preservation solution and application thereof. Background Art

[0002] In the anti-cyclic citrullinated peptide (CCP) antibody test (immunoturbidimetric assay), CCP coupled to latex microspheres can bind to anti-CCP antibodies in serum, causing the latex microspheres to form immune complexes and precipitate, enabling simple and rapid anti-CCP antibody detection. Synthetic CCPs contain a disulfide bond (-SS-) formed by two cysteines, which is thermally unstable and easily oxidized, causing the citrulline ring in the CCP peptide to transform from a cyclic structure to a linear structure. The coupling of CCP to the latex microspheres alters the groups on the latex microspheres, causing them to lose their original charge and aggregate and precipitate in a pure water storage system. Therefore, stable CCPs and dispersed, non-aggregating latex microspheres are crucial for detection.

[0003] Patent CN 110824160A discloses a test kit for measuring the concentration of anti-cyclic citrullinated peptide antibodies and its preparation method. The kit includes reagents R1 and R2. Reagent R1 comprises a first buffer, sodium chloride, a first active agent, a first preservative, and a first stabilizer. Reagent R2 comprises a second buffer, sodium chloride, a second active agent, a second preservative, a second stabilizer, and latex microspheres coupled with CCP antigen-BSA. The components and concentrations of reagent R2 are as follows: the second buffer is 25mM MES buffer; sodium chloride is at a concentration of 5%; the second active agent is Tween 20 at a concentration of 0.5%; the second preservative is Proclin at a concentration of 0.05%; the second stabilizer is bovine serum albumin at a concentration of 0.5%; and the latex microspheres coupled with CCP-BSA are at a concentration of 1%. Reagent R2 includes latex microspheres coupled with CCP-BSA and a preservation system thereof, but the preservation system fails to solve the problem that the artificially synthesized CCP contains a disulfide bond (-SS-) composed of two cysteines, has poor thermal stability, is easily oxidized, and has poor versatility.

[0004] Therefore, it is necessary to study a CCP latex microsphere preservation system that makes CCP have better thermal stability and improves the application range of the preservation system, which plays a key role in the application of anti-cyclic citrullinated peptide (CCP) antibody detection. Summary of the Invention

[0005] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide a cyclic citrullinated peptide latex microsphere preservation solution and its application.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a cyclic citrullinated peptide latex microsphere preservation solution, which contains any one of reduced glutathione or vitamin C, and also includes a buffer, NaCl, an excipient, a stabilizer, and Tween 20.

[0008] In some examples, the concentration of reduced glutathione added to the preservation solution is 0.25-0.5 mM.

[0009] In some examples, the concentration of vitamin C added to the preservation solution is 0.5-1 mM.

[0010] In some examples, the buffer is selected from any one of Tris buffer, PBS buffer, HEPES buffer, and sodium citrate-citric acid buffer, and the concentration of the buffer is 5-20 mM and the pH value is 6.8-7.8.

[0011] In some examples, the stabilizer is selected from any one of BSA and casein, and the mass concentration of the stabilizer is 0.7-1%.

[0012] In some examples, the concentration of NaCl in the preservation solution is 0.95-1.15 M, the mass concentration of the excipient is 3-5%, and the mass concentration of Tween 20 is 0.1-0.2%.

[0013] In some examples, the excipient is selected from at least one of sucrose, trehalose, mannitol, glucose, dextran, lactose, and maltose.

[0014] In some examples, the preservation solution further includes a preservative, and the preservative is selected from at least one of sodium sorbate, sodium benzoate, sodium azide, sodium nitrite, thimerosal, phenol, and PC300.

[0015] In another aspect, the present invention provides an anti-cyclic citrullinated peptide antibody detection kit, comprising cyclic citrullinated peptide latex microspheres and a preservation solution, wherein the preservation solution is the preservation solution described in the first aspect.

[0016] In some examples, the mass concentration of cyclic citrullinated peptide latex microspheres in the detection kit is 0.073% to 0.077%.

[0017] The beneficial effects of the present invention are:

[0018] The preservation solution prepared by the formula of this preservation system can ensure the stability of CCP latex microspheres, reduce the chance of CCP latex microspheres being oxidized during application, thereby maintaining the biological activity of the antibody to the greatest extent, extending the shelf life of the antibody molecules, and improving the accuracy of detection; and at the same time, it can increase the density of the preservation solution, allowing the microspheres to exist in a suspended form, reducing the probability of microsphere agglomeration and cross-linking, thereby achieving the effect of stable preservation of the microspheres, and avoiding the problem that the immune microspheres will be firmly bound due to microsphere agglomeration or agglomeration for too long, making it difficult to disperse them again through shaking. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a line graph of the stability decay rate of the storage solution of Examples 1-2 and Comparative Examples 1-2.

[0020] Figure 2 It is a line graph of the stability decay rate of the preservation solution of Examples 3 to 4 and Comparative Example 3.

[0021] Figure 3 It is a line graph of the stability decay rate of the preservation solution of Comparative Examples 4 to 7 and Comparative Example 1.

[0022] Figure 4 It is a line graph of the stability decay rate of the preservation solution of Comparative Examples 8 to 10 and Comparative Example 1.

[0023] Figure 5 It is a line graph of the stability decay rate of the preservation solution of Comparative Examples 11 to 12 and Comparative Example 1. DETAILED DESCRIPTION

[0024] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0025] The thermal stability test method of the preservation solution of each embodiment and comparative example is as follows:

[0026] 1) According to the formulation in Table 1, in each Example and Comparative Example, the buffer was selected from a PBS buffer system with a concentration of 10 mM and a pH of 7.4. The mass concentration of BSA, a stabilizer used to maintain the thermal stability of CCP latex microspheres, was 1%. 0.05% Proclin 300 was selected as a preservative. The compositions of the remaining components, such as sodium chloride, excipients trehalose, and Tween 20 (TW20), are shown in Table 1. Reduced glutathione (GSH) was added to Examples 1-2 and Comparative Examples 1-2, vitamin C was added to Examples 3-4 and Comparative Example 3, and citric acid was added to Comparative Examples 13-15 for comparison. Each Example and Comparative Example was then prepared.

[0027] 2) After stirring to fully dissolve, adjust the pH to 7.4;

[0028] 3) Filter the prepared solution through a 0.22 μm filter membrane to remove impurities;

[0029] 4) Using the prepared examples as diluents for CCP latex microspheres, the high-concentration CCP latex microspheres were diluted to a concentration of 0.075%, and the 10-day thermal stability of different formulations was studied (37°C);

[0030] 5) The test was performed using Mindray BS-400 biochemical analyzer. The test parameters of Mindray BS-400 biochemical analyzer are shown in Table 2. The test results of 100 U / ml sample are shown in Table 2. Figures 1 to 5 .

[0031] Table 1

[0032] Sodium chloride Trehalose TW20 GSH Vitamin C Citric acid Example 1 0.95 M 3% 0.20% 0.25 mM 0 0 Example 2 0.95 M 3% 0.20% 0.5 mM 0 0 Example 3 0.95 M 3% 0.20% 0 0.5 mM 0 Example 4 0.95 M 3% 0.20% 0 1 mM 0 Comparative Example 1 0.95 M 3% 0.20% 0 0 0 Comparative Example 2 0.95 M 3% 0.20% 1 mM 0 0 Comparative Example 3 0.95 M 3% 0.20% 0 2 mM 0 Comparative Example 4 0.15 M 3% 0.20% 0 0 0 Comparative Example 5 0.45 M 3% 0.20% 0 0 0 Comparative Example 6 0.75 M 3% 0.20% 0 0 0 Comparative Example 7 1.15 M 3% 0.20% 0 0 0 Comparative Example 8 0.95 M 3% 0 0 0 0 Comparative Example 9 0.95 M 3% 0.10% 0 0 0 Comparative Example 10 0.95 M 3% 0.05% 0 0 0 Comparative Example 11 0.95 M 0% 0.20% 0 0 0 Comparative Example 12 0.95 M 5% 0.20% 0 0 0 Comparative Example 13 0.95 M 3% 0.20% 0 0 1 mM Comparative Example 14 0.95 M 3% 0.20% 0 0 2.5 mM Comparative Example 15 0.95 M 3% 0.20% 0 0 5 mM

[0033] Table 2

[0034] instrument Mindray BS-400 Analytical methods 2 Point End Reading points 43-43；80-80 Main wavelength / sub-wavelength 546 Sample volume (μl) 5 R1 (μl) 150 R2 (μl) 50 Reaction direction rise

[0035] Experimental Summary

[0036] 1) Effects of GSH addition:

[0037] As shown in Tables 3 and 4, compared with Comparative Example 1, Examples 1-2 and Comparative Example 2 added GSH, and the stability decay was weaker in the stability test data from 7 to 10 days, indicating that the addition of GSH has a good stability effect. Among Examples 1-2 and Comparative Example 2, Example 1 has the best stability, with the test value changing by -1% at 10 days, which is still much less than 10%, achieving the effect of accelerated stability after 10 days. Figure 1 It shows that the change trend of Example 1 with the addition of 0.5 mM GSH is the smallest, indicating that Example 1 has the best stability.

[0038] Table 3 Stability values ​​of the storage solutions of Examples 1-2 and Comparative Examples 1-2

[0039] GSH addition amount / mM 0 0.25 0.5 1 0 d 1344.6 1413.8 1432.0 1395.0 1 d 1324.5 1427.4 1467.4 1400.2 3 d 1266.2 1486.7 1463.8 1361.8 5 d 1205.9 1503.5 1440.3 1320.4 7 d 1154.4 1521.8 1454.0 1327.3 10 d 1084.7 1604.0 1420.7 1208.9

[0040] Table 4 Stability decay rate of the storage solution of Examples 1-2 and Comparative Examples 1-2

[0041] GSH addition amount / mM 0 0.25 0.5 1 0 d 0% 0% 0% 0% 1 d -1% 1% 2% 0% 3 d -6% 5% 2% -2% 5 d -10% 6% 1% -5% 7 d -14% 8% 2% -5% 10 d -19% 13% -1% -13%

[0042] 2) Effect of adding vitamin C:

[0043] As shown in Tables 5 and 6, Example 3 added vitamin C relative to Comparative Example 1. The stability test data from 7 to 10 days showed that its stability decay degree was lower than that of Comparative Example 1, indicating that adding a certain amount of vitamin C has a good stability effect.

[0044] In addition, 1 mM and 2 mM were added to Example 4 and Comparative Example 3, respectively. The experimental data showed that the difference in the degree of change of the test value between Example 4 and Comparative Example 1 was not significant, indicating that the addition of 1 mM vitamin C in Example 4 did not promote the stability of CCP. The change in the test value between Comparative Example 3 and Comparative Example 1 was greater, and the stability was worse. The above experiments indicate that the amount of added vitamin C is particularly important for the stability of the CCP reagent, and does not have a positive effect.

[0045] The experimental results show that the addition of 0.5-1 mM vitamin C promotes the stability of the CCP reagent, especially the addition of 0.5 mM. The experimental results show that the change in the test value of Example 5 at 10 days is -9%, which is still much less than 10%, achieving the effect of accelerating stability for 10 days.

[0046] Figure 2 It is shown that the change trend of Example 3 in which 0.5 mM vitamin C is added is the smallest, indicating that Example 3 has the best stability.

[0047] Table 5 Stability values ​​of the storage solutions of Examples 3-4 and Comparative Example 3

[0048] Vitamin C added 0.5mM 1mM 2mM 0 d 1641.1 1381.1 1401.5 1 d 1667.2 1394.9 1407.0 3 d 1643.2 1436.8 1515.4 5 d 1653.8 1504.3 1578.1 7 d 1545.9 1576.4 1767.8 10 d 1499.3 1500.8 1787.8

[0049] Table 6 Stability decay rate of the storage solution of Examples 3-4 and Comparative Example 3

[0050] Vitamin C added 0.5mM 1mM 2mM 0 d 0% 0% 0% 1 d 2% 1% 0% 3 d 0% 4% 8% 5 d 1% 9% 13% 7 d -6% 14% 26% 10 d -9% 9% 28%

[0051] 3) Impact of adding sodium chloride:

[0052] In actual applications, aggregation and precipitation may occur in the existing storage system, which is also an important factor affecting the stability of CCP test values.

[0053] like Figure 3 As shown in Tables 7 and 8, in the comparative examples, the test value decreases less in the range of 0.4 to 1.15 M sodium chloride solution than in 0.15 M, and the most stable test value is in Comparative Example 1 at 0.95 M. At the same time, it can be seen from naked eye observation that the precipitate in Comparative Example 1 at 0.95 M sodium chloride solution is less than that in Examples 4 to 6.

[0054] Table 7 Stability values ​​of the storage solutions of Comparative Examples 4 to 7 and Comparative Example 1

[0055] Sodium chloride addition 0.15 M 0.45 M 0.75 M 0.95 M 1.15 M 0 d 1328.6 1364.9 1372.9 1242.4 1353.4 1 d 1235.7 1379.7 1453.0 1265.3 1348.3 3 d 1186.7 1335.7 1416.6 1290.0 1359.5 5 d 1090.4 1215.1 1272.0 1199.9 1285.1 7 d 1010.1 1099.9 1196.7 1127.0 1208.7 10 d 792.7 858.4 989.4 1031.2 1129.8

[0056] Table 8 Stability decay rate of the storage solution of Comparative Examples 4 to 7 and Comparative Example 1

[0057] Sodium chloride addition 0.15 M 0.45 M 0.75 M 0.95 M 1.15 M 0 d 0% 0% 0% 0% 0% 1 d -7% 1% 6% 2% 0% 3 d -11% -2% 3% 4% 0% 5 d -18% -11% -7% -3% -5% 7 d -24% -19% -13% -9% -11% 10 d -40% -37% -28% -17% -17%

[0058] 4) Impact of Tw20 addition:

[0059] like Figure 3 As shown in Tables 9 and 10, when the Tw20 of the comparative example is 0.2%, its test value is the most stable, which shows that 0.2% Tw20 is the optimal ratio.

[0060] Table 9 Stability values ​​of the storage solutions of Comparative Examples 8 to 10 and Comparative Example 1

[0061] Tw20 addition amount 0% 0.05% 0.10% 0.20% 0 d 1198.7 1226.3 1145.0 1242.4 1 d 1146.5 1242.3 1240.7 1265.3 3 d 1086.5 1146.5 1207.9 1290.0 5 d 1007.3 1075.7 1130.4 1199.9 7 d 979.7 949.4 1031.8 1127.0 10 d 841.4 819.7 913.6 1031.2

[0062] Table 10 Stability decay rate of the storage solution of Comparative Examples 8 to 10 and Comparative Example 1

[0063] Tw20 addition amount 0% 0.05% 0.10% 0.20% 0 d 0% 0% 0% 0% 1 d -4% 1% 8% 2% 3 d -9% -7% 5% 4% 5 d -16% -12% -1% -3% 7 d -18% -23% -10% -9% 10 d -30% -33% -20% -17%

[0064] 5) Effect of adding trehalose:

[0065] like Figure 5 As shown in Tables 11 and 12, the test value of the comparative example is the most stable when the trehalose content is 3%. Therefore, it can be seen that the 3% trehalose example is the optimal ratio.

[0066] Table 11 Stability values ​​of the storage solutions of Comparative Examples 11-12 and Comparative Example 1

[0067] Trehalose added amount 0% 3% 5% 0 d 1142.3 1300.3 1379.0 1 d 1202.7 1342.0 1274.3 3 d 1011.0 1218.7 1266.7 5 d 1022.7 1215.0 1258.0 7 d 986.3 1196.5 1215.0 10 d 903.8 1098.3 1165.8

[0068] Table 12 Stability decay rate of the storage solution of Comparative Examples 11-12 and Comparative Example 1

[0069] Trehalose added amount 0% 3% 5% 0 d 0% 0% 0% 1 d 5% 3% -8% 3 d -11% -6% -8% 5 d -10% -7% -9% 7 d -14% -8% -12% 10 d -21% -16% -15%

[0070] 6) Effect of adding citric acid:

[0071] It can be seen from Table 4, Table 6, and Tables 13 to 14 that Examples 1 to 4 have better stability than Comparative Examples 13 to 15 in which citric acid was added, indicating that the addition of GSH or vitamin C is more effective than citric acid.

[0072] Table 13 Stability values ​​of the storage solutions of Comparative Examples 13 to 15

[0073] Citric acid addition 1mM 2.5mM 5mM 0 d 1447.7 1433.1 1415.4 1 d 1469.3 1396.3 1327.8 3 d 1464.5 1356.5 1348.7 5 d 1320.2 1232.8 1205.7 7 d 1077.8 999.7 1018.4 10 d 824.4 804.3 810.6

[0074] Table 14 Stability decay rate of the storage solution of Comparative Examples 13 to 15

[0075] Citric acid addition 1mM 2.5mM 5mM 0 d 0% 0% 0% 1 d 1% -3% -6% 3 d 1% -5% -5% 5 d -9% -14% -15% 7 d -26% -30% -28% 10 d -43% -44% -43%

[0076] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. The use of reduced glutathione or vitamin C in the preparation of a preservation solution for cyclic citrullinated peptide latex microspheres, characterized in that: The preservation solution is composed of: any one of reduced glutathione or vitamin C, a buffer, NaCl, an excipient, a stabilizer, and Tween 20. The concentration of reduced glutathione in the preservation solution is 0.25-0.5 mM, the concentration of vitamin C in the preservation solution is 0.5-1 mM, the concentration of NaCl in the preservation solution is 0.95-1.15 M, the mass concentration of the excipient is 3-5%, and the mass concentration of Tween 20 is 0.1-0.2%.

2. The use according to claim 1, characterized in that The buffer is selected from any one of Tris buffer, PBS buffer, HEPES buffer, and sodium citrate-citric acid buffer. The concentration of the buffer is 5-20 mM and the pH value is 6.8-7.

8.

3. The use according to claim 1 or 2, characterized in that The stabilizer is selected from any one of BSA and casein, and the mass concentration of the stabilizer is 0.7-1%.

4. The use according to claim 1, characterized in that The excipient is selected from at least one of sucrose, trehalose, mannitol, glucose, dextran, lactose and maltose.

5. The use according to claim 1 or 2, characterized in that: The preservation solution also includes a preservative, which is selected from at least one of sodium sorbate, sodium benzoate, sodium azide, sodium nitrite, thimerosal, phenol, and PC300.

6. An anti-cyclic citrullinated peptide antibody detection kit, comprising cyclic citrullinated peptide latex microspheres and a preservation solution, characterized in that: The storage solution is the storage solution according to any one of claims 1 to 5.

7. The detection kit according to claim 6, characterized in that The mass concentration of the cyclic citrullinated peptide latex microspheres in the detection kit is 0.073% to 0.077%.

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