Preparation method of stable rheumatism kidney function composite calibration quality control raw material

Abstract

The present application relates to a kind of preparation methods of stable rheumatoid nephrology composite calibration quality control raw materials, comprising the following steps;Step 1, preparation composite quality control matrix liquid;Step 2, preparation quality control raw material additive;Step 3, the prepared quality control raw material additive is mixed into composite quality control matrix liquid;Step 4, the mixed solution is filtered and can be used.The present application uses rheumatoid factor (RF), C-reactive protein (CRP), anti-streptolysin (ASO), alpha 1 microglobulin (alpha 1-mg), beta 2 microglobulin (beta 2-mg) high-purity antigen as raw material, through composite debugging and assignment, and liquid protective agent (Tween 80, trehalose, anhydrous calcium chloride, ETDA, glycerol) is added, to obtain a kind of stable rheumatoid nephrology composite calibration quality control raw material.The rheumatoid nephrology composite calibration quality control raw material prepared has good stability, simple preparation process, low cost, high process repeatability, can be industrialized production, has potential and extensive application value in clinical diagnosis, in vitro diagnosis.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for preparing a stable rheumatic kidney function composite calibration quality control raw material. Background Technology

[0002] Rheumatic diseases and renal dysfunction often occur together, and clinical testing requires simultaneous monitoring of indicators such as rheumatoid factor (RF), C-reactive protein (CRP), anti-streptolysin (ASO), α1-microglobulin (α1-mg), and β2-microglobulin (β2-mg).

[0003] Currently, medical institutions typically use biochemical analysis platforms and specific protein analysis platforms for related testing. However, the calibrators and quality control products for these two platforms are often used independently. Furthermore, the use of single calibrators and quality control products lacks multi-indicator composite analysis, resulting in large systematic errors. Existing quality control products are prone to protein denaturation, degradation, or loss of enzyme activity during long-term storage or transportation, affecting the reliability of testing. Traditional preparation methods often employ simple mixing or freeze-drying processes, lacking systematic optimization of protein stability, matrix effects, and interoperability, leading to problems such as large batch-to-batch variations, poor traceability, and high costs.

[0004] Therefore, there is a need to provide reagent manufacturers with a composite calibration quality control raw material that covers key indicators of rheumatism and renal function, is highly stable, and suitable for multiple platforms, for use in conjunction with reagent kits. The method for preparing a stable, reproducible, and batch-to-batch variability composite calibration quality control raw material for rheumatism and renal function provided by this invention is of great significance. Summary of the Invention

[0005] In summary, to overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a method for preparing a stable rheumatic kidney function composite calibration quality control raw material, which has stable production, repeatable preparation process, and small batch-to-batch variation.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a method for preparing a stable rheumatic kidney function composite calibration quality control raw material, comprising the following steps;

[0007] Step 1: Prepare the composite quality control matrix solution;

[0008] Step 2: Prepare the quality control raw material additives;

[0009] Step 3: Mix the prepared quality control raw material additives into the composite quality control matrix solution obtained in Step 1 to obtain a mixture;

[0010] Step 4: Filter the mixture obtained in Step 3 to obtain the raw material for the rheumatoid renal function composite calibration quality control.

[0011] Based on the above technical solution, the present invention can be further improved as follows:

[0012] Further, step 1 specifically involves weighing 10-15g bovine serum albumin, 5-8g citric acid, 4-6g disodium hydrogen phosphate dodecahydrate, 0.1-0.5% Tween 80, 4-8g trehalose, 0.5-1.5g anhydrous calcium chloride, 0.03% sodium azide, 0.8-1.2 mmol / L ETDA, and 15-30% glycerol. Then, add 50ml of sterile purified water sequentially and adjust the pH to 7.0 using sodium hydroxide to obtain the composite quality control matrix solution, which is then stored for later use.

[0013] Furthermore, the concentration of the sodium hydroxide is 0.2 mmol / L.

[0014] Furthermore, the obtained composite quality control matrix solution was stored at 2-8℃ for later use.

[0015] Further, step 2 specifically involves weighing 70-90 IU / mL of rheumatoid factor, 55-70 mg / L of C-reactive protein, 450-500 IU / mL of anti-streptolysin, 90-110 mg / L of α1-microglobulin, and 7-10 mg / L of β2-microglobulin.

[0016] Further, step 3 specifically involves adding the rheumatoid factor, C-reactive protein, anti-streptolysin, α1-microglobulin, and β2-microglobulin weighed in step 2 to the composite quality control matrix solution in sequence, and mixing and shaking well.

[0017] Further, add it to 50 ml of composite quality control matrix solution.

[0018] Furthermore, in step 4, the material is filtered through a 0.22μm filter membrane.

[0019] The beneficial effects of this invention are as follows: Using high-purity antigens of rheumatoid factor (RF), C-reactive protein (CRP), anti-streptolysin (ASO), α1-microglobulin (α1-mg), and β2-microglobulin (β2-mg) as raw materials, a stable composite calibration quality control raw material for rheumatoid and renal function is obtained through compounding, adjustment, and value assignment, with the addition of liquid protective agents (Tween 80, trehalose, anhydrous calcium chloride, ETDA, and glycerol). The prepared composite calibration quality control raw material for rheumatoid and renal function exhibits good stability, simple preparation process, low cost, high process repeatability, and industrial production capability, and has potential and wide-ranging application value in clinical diagnosis and in vitro diagnostics. Detailed Implementation

[0020] The principles and features of the present invention are described below with reference to specific examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0021] A method for preparing a stable quality control raw material for rheumatic renal function composite calibration includes the following steps;

[0022] Step 1: Prepare composite quality control matrix solution

[0023] Weigh 10-15g bovine serum albumin, 5-8g citric acid, 4-6g disodium hydrogen phosphate dodecahydrate, 0.1-0.5% Tween 80, 4-8g trehalose, 0.5-1.5g anhydrous calcium chloride, 0.03% sodium azide, 0.8-1.2 mmol / L ETDA, and 15-30% glycerol. Add 50ml of sterile purified water sequentially. Adjust the pH to 7.0 using 0.2 mmol / L sodium hydroxide to obtain the composite quality control matrix solution. Store at 2-8℃ for later use.

[0024] The matrix solution serves as a "preservation carrier" for target antigens (RF, CRP, etc.). Its core function is to maintain a stable pH (matching the human physiological environment and preventing protein denaturation), inhibit microbial contamination, and reduce antigen degradation through the synergistic effect of a buffer system (citric acid, disodium hydrogen phosphate dodecahydrate), protein protectants (Tween 80, trehalose, anhydrous calcium chloride, ETDA, glycerol), and preservatives (sodium azide). This provides a stable thermodynamic environment for the subsequent addition of high-purity antigens and reduces matrix effects (avoiding matrix components from interfering with detection results).

[0025] Key Component Synergistic Mechanism: Bovine serum albumin (BSA) acts as a carrier protein, encapsulating the target antigen and reducing oxidative degradation; citrate-disodium hydrogen phosphate dodecahydrate forms a buffer pair, precisely regulating pH; Tween 80 (a nonionic surfactant) reduces interfacial tension and prevents protein aggregation; trehalose / glycerol acts as a lyophilization protectant (enhancing the stability of liquid formulations even without lyophilization), binding water molecules through hydrogen bonds and reducing protein dehydration and denaturation; sodium azide acts as a preservative, inhibiting bacterial growth; EDTA / anhydrous calcium chloride regulates ionic strength and maintains the protein's spatial conformation.

[0026] Step 2, prepare the quality control raw material additives.

[0027] Weigh out 70-90 IU / mL of rheumatoid factor (RF), 55-70 mg / L of C-reactive protein (CRP), 450-500 IU / mL of anti-streptolysin (ASO), 90-110 mg / L of α1-microglobulin (α1-mg), and 7-10 mg / L of β2-microglobulin (β2-mg).

[0028] Step 3: Mix the prepared quality control raw material additives into the composite quality control matrix solution obtained in Step 1 to obtain a mixture.

[0029] Add the rheumatoid factor, C-reactive protein, anti-streptolysin, α1-microglobulin, and β2-microglobulin weighed in step 2 to 50 ml of composite quality control matrix solution and mix well.

[0030] Step 4: Filter the mixture obtained in Step 3 through a 0.22μm filter membrane to obtain the raw material for the rheumatoid renal function composite calibration quality control.

[0031] High-purity antigens (RF, CRP, ASO, α1-mg, β2-mg) for key indicators of rheumatism and kidney function are precisely added to the composite matrix solution. Through uniform mixing and filtration purification, stable composite of multiple indicator antigens is achieved, while ensuring the homogeneity of raw materials (reducing batch-to-batch variation) and purity (removing impurities that interfere with detection). The core logic is "precise matching of antigen concentration + optimization of matrix and antigen compatibility," ensuring that the antigens of each indicator do not interfere with each other and maintain their activity in the same system.

[0032] The beneficial effects of the present invention will be illustrated below through examples.

[0033] Example 1

[0034] 1. Preparation of composite quality control matrix solution

[0035] Weigh out 10g bovine serum albumin, 5g citric acid, 4g disodium hydrogen phosphate dodecahydrate, 0.1% Tween 80, 4g trehalose, 0.5g anhydrous calcium chloride, 0.03% sodium azide, 0.8mmol / L ETDA, and 15% glycerol. Add 50ml of sterile purified water and adjust the pH to 7.0 using 0.2mmol / L sodium hydroxide to obtain the composite quality control matrix solution. Store at 2-8℃ for later use.

[0036] 2. Preparation of quality control raw material additives

[0037] Weigh out 8000 IU of rheumatoid factor (RF), 6 mg of C-reactive protein (CRP), 50000 IU of anti-streptolysin (ASO), 10 mg of α1-microglobulin (α1-mg), and 1 mg of β2-microglobulin (β2-mg) high-purity antigen, and add them sequentially to 50 ml of composite quality control matrix solution. Mix well and then filter through a 0.22 μm filter membrane to obtain the rheumatoid and renal function composite calibration quality control raw material. The obtained rheumatoid and renal function composite calibration quality control raw material is aliquoted into 3 ml brown lyophilized bottles, with 1 ml per bottle. One bottle is tested using a fully automated biochemical analyzer. Each item is tested three times, and the average value is taken as the 0-day test result of the rheumatoid and renal function composite calibration quality control raw material.

[0038] 3. Accelerated stability verification: The packaged samples were sealed and stored in a 37°C constant temperature oven. They were taken out on the 11th day for testing and compared with the test results on the 0th day. The test deviation is shown in Table 1.

[0039] Table 1 Accelerated stability test results

[0040]

[0041] Conclusion: Through accelerated simulation stability testing, this product has a shelf life of at least 12 months when stored at 2-8℃.

[0042] 4.2-8℃ Long-term stability verification: The aliquoted samples were sealed and stored in an environment of 2-8℃, and the same batch of reagents were stored at 2-8℃. The samples were taken out at 6 months, 12 months, and 15 months for testing, and the results were compared with the results from day 0. The test deviations are shown in Table 2 below.

[0043] Table 2 Long-term stability monitoring data

[0044]

[0045] Conclusion: Using the modified formulation process, the product can be stored at 2-8℃ for 12 months with a target protein concentration decrease of less than 10%, maintaining relative stability. However, at the 15th month, the RF concentration decreased by more than 10%.

[0046] Example 2

[0047] 1. Preparation of composite quality control matrix solution

[0048] Weigh out 15g bovine serum albumin, 8g citric acid, 6g disodium hydrogen phosphate dodecahydrate, 0.5% Tween 80, 8g trehalose, 1.5g anhydrous calcium chloride, 0.03% sodium azide, 1.2 mmol / L ETDA, and 30% glycerol. Add these to 50ml of sterile purified water, stir to dissolve, adjust the pH to 7.2, and bring the volume to 100ml. Store at 2-8℃ for later use.

[0049] 2. Quality control of raw material additives preparation

[0050] Weigh 8000 IU of rheumatoid factor (RF), 6 mg of C-reactive protein (CRP), 50000 IU of anti-streptolysin (ASO), 10 mg of α1-microglobulin (α1-mg), and 1 mg of β2-microglobulin (β2-mg) high-purity antigen, and add them sequentially to 50 ml of composite quality control matrix solution. Mix well, then filter through a 0.22 μm filter membrane, and aliquot into 3 ml brown lyophilized bottles for storage. Divide each bottle into 1 ml portions and test one bottle using a fully automated biochemical analyzer. Test each item three times and take the average value as the target value of the rheumatoid renal function composite calibration quality control raw material.

[0051] 3. Accelerated stability verification: The packaged samples were sealed and stored in a 37°C constant temperature oven. They were taken out on the 11th day for testing and compared with the test results on the 0th day. The test deviation is shown in Table 3.

[0052] Table 3 Accelerated stability test results

[0053]

[0054] Conclusion: Through accelerated simulation stability testing, this product has a shelf life of at least 12 months when stored at 2-8℃.

[0055] 4.2-8℃ Long-term stability verification: The aliquoted samples were sealed and stored in an environment of 2-8℃, and the same batch of reagents were stored in the same environment of 2-8℃. The samples were taken out at 6 months, 12 months and 15 months and tested respectively. The results were compared with the test results at 0 days. The test deviations are shown in Table 4.

[0056] Table 4 Long-term stability test results

[0057] In summary, the optimization of the matrix solution composition resulted in a better improvement in the long-term stability of RF.

[0058] Example 3

[0059] 1. Preparation of composite quality control matrix solution

[0060] Weigh out 12.5g bovine serum albumin, 7g citric acid, 5g disodium hydrogen phosphate dodecahydrate, 0.3% Tween 80, 6g trehalose, 1g anhydrous calcium chloride, 0.03% sodium azide, 1mmol / L ETDA, and 25% glycerol. Add these to 50ml of sterile purified water, stir to dissolve, adjust the pH to 7.2, and bring the volume to 100ml. Store at 2-8℃ for later use.

[0061] 2. Quality control of raw material additives preparation

[0062] Weigh 8000 IU of rheumatoid factor (RF), 6 mg of C-reactive protein (CRP), 50000 IU of anti-streptolysin (ASO), 10 mg of α1-microglobulin (α1-mg), and 1 mg of β2-microglobulin (β2-mg) high-purity antigen, and add them sequentially to 50 ml of composite quality control matrix solution. Mix well, then filter through a 0.22 μm filter membrane, and aliquot into 3 ml brown lyophilized bottles for storage. Divide each bottle into 1 ml portions and test one bottle using a fully automated biochemical analyzer. Test each item three times and take the average value as the target value of the rheumatoid renal function composite calibration quality control raw material.

[0063] 3. Accelerated stability verification: The aliquoted samples were sealed and stored in a 37℃ incubator. They were taken out on day 11 for testing, and the results were compared with those from day 0. The test deviations are shown in Table 5 below.

[0064] Table 5 Accelerated stability test results

[0065]

[0066] Conclusion: Through accelerated simulation stability testing, this product has a shelf life of at least 12 months when stored at 2-8℃.

[0067] 4.2-8℃ Long-term stability verification: The aliquoted samples were sealed and stored in an environment of 2-8℃, and the same batch of reagents were stored in the same environment of 2-8℃. The samples were taken out at 6 months, 12 months and 15 months and tested respectively. The results were compared with the test results at 0 days. The test deviations are shown in Table 6.

[0068] Table 6 Long-term stability test results

[0069]

[0070] In summary, by optimizing the composition of the matrix solution as described above, Case 2 demonstrates a greater effectiveness in improving the overall stability of the project.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a stable composite calibration and quality control raw material for rheumatic and renal function, characterized in that, The steps include the following; Step 1: Prepare the composite quality control matrix solution; Step 2: Prepare the quality control raw material additives; Step 3: Mix the prepared quality control raw material additives into the composite quality control matrix solution obtained in Step 1 to obtain a mixture; Step 4: Filter the mixture obtained in Step 3 to obtain the raw material for the rheumatoid renal function composite calibration quality control.

2. The preparation method of the stable rheumatic renal function composite calibration quality control raw material according to claim 1, characterized in that, Step 1 is as follows: Weigh 10-15g bovine serum albumin, 5-8g citric acid, 4-6g disodium hydrogen phosphate dodecahydrate, 0.1-0.5% Tween 80, 4-8g trehalose, 0.5-1.5g anhydrous calcium chloride, 0.03% sodium azide, 0.8-1.2mmol / L ETDA, and 15-30% glycerol. Add 50ml of sterile purified water in sequence, and adjust the pH to 7.0 using sodium hydroxide to obtain the composite quality control matrix solution for later use.

3. The method for preparing stable rheumatic renal function composite calibration quality control raw materials according to claim 2, characterized in that, The concentration of sodium hydroxide is 0.2 mmol / L.

4. The preparation method of the stable rheumatic renal function composite calibration quality control raw material according to claim 2, characterized in that, The obtained composite quality control matrix solution should be stored at 2-8℃ for later use.

5. The method for preparing stable rheumatic renal function composite calibration quality control raw materials according to claim 1, characterized in that, Step 2 specifically involves weighing 70-90 IU / mL of rheumatoid factor, 55-70 mg / L of C-reactive protein, 450-500 IU / mL of anti-streptolysin, 90-110 mg / L of α1-microglobulin, and 7-10 mg / L of β2-microglobulin.

6. The method for preparing stable rheumatic renal function composite calibration quality control raw materials according to claim 5, characterized in that, Step 3 is as follows: The rheumatoid factor, C-reactive protein, anti-streptolysin, α1-microglobulin, and β2-microglobulin weighed in step 2 are added to the composite quality control matrix solution in sequence and mixed well.

7. The preparation method of the stable rheumatic renal function composite calibration quality control raw material according to claim 6, characterized in that, Add to 50 ml of composite quality control matrix solution.

8. The method for preparing stable rheumatic renal function composite calibration quality control raw materials according to any one of claims 1 to 7, characterized in that, In step 4, the material is filtered through a 0.22μm filter membrane.

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