Stable composite special protein control product and preparation method thereof

Abstract

The invention relates to the technical field of medical examination, in particular to a stable compound type special protein control product and a preparation method thereof.The preparation method comprises the steps that purified water is taken, a buffer solution, a protective agent, a first stabilizer, a chelating agent, C-reactive protein, antistreptolysin O, rheumatoid factors and retinol binding protein are sequentially added, the mixture is fully stirred till being completely dissolved, and the stable compound type special protein control product is obtained; putting into a constant-temperature box for aging to obtain a dissolving solution; taking out the dissolved solution, standing to room temperature, and filtering by using a peristaltic pump and a bag type filter to obtain a filtrate; and sequentially adding a second stabilizer, cystatin C protein, a third stabilizer, an ion balance agent, lipoprotein (a) and a preservative into the filtrate, and adjusting the pH value to obtain the composite special protein control product. The method can be used for quality control of biochemical item detection processes of retinol binding protein, cystatin C protein, C-reactive protein, antistreptolysin 'O', rheumatoid factors and lipoprotein (a) at the same time.

Description

Technical Field

[0001] The present invention relates to the field of medical testing technology, and in particular to a stable composite special protein control product and a preparation method thereof. Background Art

[0002] With the improvement of living standards, the level of medical technology in my country has been continuously improving. However, the development of in vitro diagnostic reagents still lags behind that of foreign countries. Whether it is instruments, reagents, antibodies or test quality control materials, they are more dependent on imports. At present, most of the composite quality control products for blood lipids, renal function and rheumatism-related specific proteins rely on Randox in the UK and other imported brands. There are not many independently developed products in my country, and there are no relevant and stable composite quality control products that cover the three biochemical detection directions of blood lipids, renal function and rheumatism. This invention aims to fill the gap in this product in my country and improve the convenience of customer use.

[0003] At present, most of the quality control products related to special proteins used in China are single items or single fields, such as three items in the rheumatology category. There are few domestic public patents for special protein composite quality control products. Among them, the Chinese invention patent with patent number "CN201811621004.6" and patent name "A composite quality control product of blood lipids and special proteins" can detect a total of 12 substances including complement C3, complement C4, apolipoprotein A1, apolipoprotein B, high-density lipoprotein, low-density lipoprotein, C-reactive protein, IgA, IgM, IgG, total cholesterol and glycerol, but does not involve lipoprotein (a) and antistreptolysin "O", rheumatoid factor (routine clinical rheumatology test items), and cannot realize the detection and application of renal function and rheumatism at the same time, which to a certain extent restricts the scope of application of the product. Another Chinese invention patent with the patent number "CN201610674109.2" and the patent name "A special protein liquid composite quality control product with strong stability for biochemistry" discloses a quality control product that can detect 12 substances, including anti-streptococcal O, C-reactive protein, complement C3, complement C4, IgA, IgM, IgG, prealbumin, rheumatoid factor, transferrin, total protein, and albumin, but does not involve lipoprotein (a) and cystatin C protein, and cannot be used for detection of blood lipids and renal function at the same time. In addition, the validity period is only 12 months, which also restricts the scope of application of the product to a certain extent. Summary of the Invention

[0004] The purpose of the present invention is to provide a stable composite special protein control product and a preparation method, aiming to solve the problem that existing quality control products do not involve lipoprotein (a) and cystatin C protein and cannot be used for simultaneous detection of blood lipids and renal function.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing a stable composite special protein control product, comprising the following steps:

[0006] Take purified water at room temperature;

[0007] Adding a buffer, a protective agent, a first stabilizer, a chelating agent, C-reactive protein, antistreptolysin "O", rheumatoid factor, and retinol binding protein to the purified water in sequence, stirring thoroughly until completely dissolved, and placing in a constant temperature box for aging to obtain a dissolved solution;

[0008] The dissolved solution was taken out and allowed to stand at room temperature, and then filtered using a capsule filter with a peristaltic pump to obtain a filtrate;

[0009] A second stabilizer, cystatin C protein, a third stabilizer, an ion balance agent, lipoprotein (a) and a preservative are sequentially added to the filtrate, and the pH value is adjusted to obtain a composite special protein control product.

[0010] Wherein, the purified water is 80% of the prepared volume;

[0011] The temperature of the incubator is 37°C;

[0012] The capsule filter is a 0.45 μm capsule filter;

[0013] The aging time is 24h;

[0014] The pH value is 6.80±0.05.

[0015] The buffer is one of 50 mmol / L HEFES buffer, MES buffer, PBS buffer, and Tris-HCl buffer at 25° C. and pH 6.8±0.5;

[0016] The first stabilizer is one or more combinations of PEG4000, PEG6000, and PEG8000;

[0017] The second stabilizer is one or more combinations of trehalose, glucose, fructose, and sucrose;

[0018] The third stabilizer is one or more combinations of lysine and mannitol;

[0019] The protective agent is one or more combinations of bovine serum albumin and polyvinyl pyrrolidone;

[0020] The chelating agent is one or more combinations of EDTA, EGTA, DTPA, and CDTA;

[0021] The preservative is one or more combinations of proclin 300, sodium azide, gentamicin sulfate, Germa 115, and sodium benzoate;

[0022] The ion balance agent is one or more combinations of potassium chloride, sodium chloride, sodium acetate, and magnesium acetate.

[0023] The method further comprises:

[0024] The composite special protein control product is freeze-dried to obtain a freeze-dried product.

[0025] In the second aspect, the present invention provides a stable composite special protein control product, including 30-100 mmol / L of buffer; 5-50 g / L of a first stabilizer; 5-50 g / L of a second stabilizer; 10-100 g / L of a third stabilizer; 5-100 g / L of a protective agent; 0.1-5.0 g / L of a chelating agent; 0.5-10 g / L of a preservative; 0.5-10 g / L of an ion balancer; 20-80 mg / L of retinol binding protein; 0.7-2.5 mg / L of cystatin C protein; 10-80 mg / L of reactive protein; 80-240 IU / mL of antistreptolysin "O"; 15-80 IU / mL of rheumatoid factor; and 120-360 mg / L of lipoprotein (a).

[0026] The buffer is one of 50 mmol / L HEFES buffer, MES buffer, PBS buffer, and Tris-HCl buffer at 25° C. and pH 6.8±0.5;

[0027] The first stabilizer is one or more combinations of PEG4000, PEG6000, and PEG8000;

[0028] The second stabilizer is one or more combinations of trehalose, glucose, fructose, and sucrose;

[0029] The third stabilizer is one or more combinations of lysine and mannitol;

[0030] The protective agent is one or more combinations of bovine serum albumin and polyvinyl pyrrolidone;

[0031] The chelating agent is one or more combinations of EDTA, EGTA, DTPA, and CDTA;

[0032] The preservative is one or more combinations of proclin 300, sodium azide, gentamicin sulfate, Germa 115, and sodium benzoate;

[0033] The ion balance agent is one or more combinations of potassium chloride, sodium chloride, sodium acetate, and magnesium acetate.

[0034] The present invention provides a method for preparing a stable composite special protein control product, comprising the steps of: obtaining purified water at room temperature; sequentially adding a buffer, a protective agent, a first stabilizer, a chelating agent, C-reactive protein, antistreptolysin "O", rheumatoid factor and retinol binding protein to the purified water, stirring the mixture until completely dissolved, and placing the mixture in a constant temperature box for aging to obtain a dissolved solution; taking out the dissolved solution, allowing the solution to stand at room temperature, and then filtering the solution using a capsule filter with a peristaltic pump to obtain a filtrate; sequentially adding a second stabilizer, cystatin C protein, a third stabilizer, an ion balance agent, lipoprotein (a) and a preservative to the filtrate, and adjusting the pH value to obtain a composite special protein control product. The above steps are simple to operate, have good stability and low production cost, and the prepared composite special protein control product can be simultaneously used for quality control in the detection process of biochemical items of retinol binding protein, cystatin C protein, C-reactive protein, antistreptolysin "O", rheumatoid factor and lipoprotein (a). The invention solves the problem that the existing quality control products do not involve lipoprotein (a) and cystatin C protein and cannot be used for the detection of blood lipids and renal function at the same time.

[0035] Beneficial effects:

[0036] 1) The present invention comprises retinol binding protein, cystatin C protein, C-reactive protein, antistreptolysin "O", rheumatoid factor and lipoprotein (a)

[0037] 2) The buffer used in the present invention can effectively control the pH value without any influence on the components.

[0038] 3) The stabilizers and protective agents used in the present invention can ensure that there is no cross-reaction between the components, which can greatly improve the stability of the product. The chelating agent can prevent the influence of heavy metal substances on the protein, and the ion balance agent can effectively activate the protein, which are all very helpful for stability.

[0039] 4) Aging in a 37°C thermostat can make the reaction system more balanced and allow some impurities to precipitate.

[0040] 5) The present invention uses freeze-drying technology, and the loss of some volatile components in the substance is very small. Moreover, since the drying is carried out in a vacuum with very little oxygen, some easily oxidizable substances are protected. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 The present invention provides a flow chart of a method for preparing a stable composite special protein control product. DETAILED DESCRIPTION

[0043] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0044] See also Figure 1 In a first aspect, the present invention provides a method for preparing a stable composite special protein control product, comprising the following steps:

[0045] S1 takes purified water at room temperature;

[0046] Specifically, the purified water accounts for 80% of the prepared volume.

[0047] S2: adding a buffer, a protective agent, a first stabilizer, a chelating agent, C-reactive protein, antistreptolysin "O", rheumatoid factor, and retinol binding protein to the purified water in sequence, stirring thoroughly until completely dissolved, and placing in a constant temperature box for aging to obtain a dissolved solution;

[0048] Specifically, the temperature of the constant temperature box is 37°C; the aging time is 24 hours; the buffer is one of 50 mmol / L HEFES buffer, MES buffer, PBS buffer, and Tris-HCl buffer at 25°C and a pH of 6.8±0.5; the first stabilizer is one or more of PEG4000, PEG6000, and PEG8000; the protective agent is one or more of bovine serum albumin and polyvinyl pyrrolidone; and the chelating agent is one or more of EDTA, EGTA, DTPA, and CDTA.

[0049] S3: taking out the dissolved solution and letting it stand at room temperature, and then filtering it with a capsule filter using a peristaltic pump to obtain a filtrate;

[0050] Specifically, the capsule filter is a 0.45 μm capsule filter.

[0051] S4: adding a second stabilizer, cystatin C protein, a third stabilizer, an ion balance agent, lipoprotein (a) and a preservative to the filtrate in sequence, and adjusting the pH value to obtain a composite special protein control product.

[0052] Specifically, the pH value is 6.80±0.05. The second stabilizer is one or more combinations of trehalose, glucose, fructose, and sucrose; the third stabilizer is one or more combinations of lysine and mannitol; the preservative is one or more combinations of proclin 300, sodium azide, gentamicin sulfate, Germa 115, and sodium benzoate; and the ion balance agent is one or more combinations of potassium chloride, sodium chloride, sodium acetate, and magnesium acetate.

[0053] S5 freeze-drying the composite special protein control product to obtain a freeze-dried product.

[0054] Beneficial effects

[0055] 1) The present invention comprises retinol binding protein, cystatin C protein, C-reactive protein, antistreptolysin "O", rheumatoid factor and lipoprotein (a)

[0056] 2) The buffer used in the present invention can effectively control the pH value without any influence on the components.

[0057] 3) The stabilizers and protective agents used in the present invention can ensure that there is no cross-reaction between the components, which can greatly improve the stability of the product. The chelating agent can prevent the influence of heavy metal substances on the protein, and the ion balance agent can effectively activate the protein, which are all very helpful for stability.

[0058] 4) Aging in a 37°C thermostat can make the reaction system more balanced and allow some impurities to precipitate.

[0059] 5) The present invention uses freeze-drying technology, and the loss of some volatile components in the substance is very small. Moreover, since the drying is carried out in a vacuum with very little oxygen, some easily oxidizable substances are protected.

[0060] In the second aspect, the present invention provides a stable composite special protein control product, including 30-100 mmol / L of buffer; 5-50 g / L of a first stabilizer; 5-50 g / L of a second stabilizer; 10-100 g / L of a third stabilizer; 5-100 g / L of a protective agent; 0.1-5.0 g / L of a chelating agent; 0.5-10 g / L of a preservative; 0.5-10 g / L of an ion balancer; 20-80 mg / L of retinol binding protein; 0.7-2.5 mg / L of cystatin C protein; 10-80 mg / L of reactive protein; 80-240 IU / mL of antistreptolysin "O"; 15-80 IU / mL of rheumatoid factor; and 120-360 mg / L of lipoprotein (a).

[0061] Specifically, the buffer is one of 50 mmol / L HEFES buffer, MES buffer, PBS buffer, and Tris-HCl buffer at 25° C. and pH 6.8±0.5;

[0062] The first stabilizer is one or more combinations of PEG4000, PEG6000, and PEG8000;

[0063] The second stabilizer is one or more combinations of trehalose, glucose, fructose, and sucrose;

[0064] The third stabilizer is one or more combinations of lysine and mannitol;

[0065] The protective agent is one or more combinations of bovine serum albumin and polyvinyl pyrrolidone;

[0066] The chelating agent is one or more combinations of EDTA, EGTA, DTPA, and CDTA;

[0067] The preservative is one or more combinations of proclin 300, sodium azide, gentamicin sulfate, Germa 115, and sodium benzoate;

[0068] The ion balance agent is one or more combinations of potassium chloride, sodium chloride, sodium acetate, and magnesium acetate.

[0069] The present invention is described below in conjunction with specific implementations. A stable composite special protein control product of the present invention includes two levels: Level 1 and Level 2.

[0070] Example 1

[0071] In this embodiment, the specialty protein composite quality control product level 1 includes:

[0072]

[0073]

[0074] In this embodiment, the specialty protein composite quality control product level 2 includes:

[0075]

[0076] The prepared freeze-dried products were tested using a Guilin Urit-8280 biochemical analyzer and corresponding registered reagent kits. The experimental results are shown in Tables 1, 2, 3, and 4.

[0077] Table 1 Thermal stability and full-term stability data of quality control materials at level 1

[0078]

[0079] Table 2 Freezing stability data of quality control material level 1 after reconstitution

[0080]

[0081]

[0082]

[0083] Table 3 Thermal stability and full-term stability data of quality control materials at level 2

[0084]

[0085] Table 4 Freezing stability data of quality control material level 2 after reconstitution

[0086]

[0087]

[0088] As can be seen from Tables 1 and 2, the two levels of quality control materials prepared in this example can simultaneously control the quality of retinol binding protein, C-reactive protein, antistreptolysin "O", rheumatoid factor, cystatin C protein, and lipoprotein (a).

[0089] Example 2

[0090] In this embodiment, the specialty protein composite quality control product level 1 includes:

[0091]

[0092] In this embodiment, the specialty protein composite quality control product level 2 includes:

[0093] Example 3

[0094] In this embodiment, the specialty protein composite quality control product level 1 includes:

[0095]

[0096] In this embodiment, the specialty protein composite quality control product level 2 includes:

[0097]

[0098]

[0099] In summary, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here, and any obvious variations or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

[0100] The above disclosure is only a preferred embodiment of a stable composite special protein control product and preparation method of the present invention. Of course, this cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiments and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for preparing a stable composite special protein control product, characterized in that: The following steps are involved: Take purified water at room temperature; Adding a buffer, a protective agent, a first stabilizer, a chelating agent, C-reactive protein, antistreptolysin "O", rheumatoid factor, and retinol binding protein to the purified water in sequence, stirring thoroughly until completely dissolved, and placing in a constant temperature box for aging to obtain a dissolved solution; The dissolved solution was taken out and allowed to stand at room temperature, and then filtered using a capsule filter with a peristaltic pump to obtain a filtrate; A second stabilizer, cystatin C protein, a third stabilizer, an ion balance agent, lipoprotein (a) and a preservative are sequentially added to the filtrate, and the pH value is adjusted to obtain a composite special protein control product.

2. The method for preparing a stable composite special protein control product according to claim 1, wherein: The purified water is 80% of the prepared volume; The temperature of the incubator is 37°C; The capsule filter is a 0.45 μm capsule filter; The aging time is 24h; The pH value is 6.80±0.

05.

3. The method for preparing a stable composite special protein control product according to claim 2, wherein: The buffer is one of 50 mmol / L HEFES buffer, MES buffer, PBS buffer, and Tris-HCl buffer at 25° C. and pH 6.8±0.5; The first stabilizer is one or more combinations of PEG4000, PEG6000, and PEG8000; The second stabilizer is one or more combinations of trehalose, glucose, fructose, and sucrose; The third stabilizer is one or more combinations of lysine and mannitol; The protective agent is one or more combinations of bovine serum albumin and polyvinyl pyrrolidone; The chelating agent is one or more combinations of EDTA, EGTA, DTPA, and CDTA; The preservative is one or more combinations of proclin 300, sodium azide, gentamicin sulfate, Germa 115, and sodium benzoate; The ion balance agent is one or more combinations of potassium chloride, sodium chloride, sodium acetate, and magnesium acetate.

4. The method for preparing a stable composite special protein control product according to claim 3, wherein: The method further comprises: The composite special protein control product is freeze-dried to obtain a freeze-dried product.

5. A stable composite special protein control product, prepared by the method for preparing a stable composite special protein control product according to claim 4, characterized in that: The invention comprises a buffer solution of 30 to 100 mmol / L; a first stabilizer of 5 to 50 g / L; a second stabilizer of 5 to 50 g / L; a third stabilizer of 10 to 100 g / L; a protective agent of 5 to 100 g / L; a chelating agent of 0.1 to 5.0 g / L; a preservative of 0.5 to 10 g / L; an ion balance agent of 0.5 to 10 g / L; a retinol-binding protein of 20 to 80 mg / L; a cystatin C protein of 0.7 to 2.5 mg / L; a reactive protein of 10 to 80 mg / L; an antistreptolysin "O" of 80 to 240 IU / mL; a rheumatoid factor of 15 to 80 IU / mL; and a lipoprotein (a) of 120 to 360 mg / L.

6. The method for preparing a stable composite special protein control product according to claim 5, characterized in that: The buffer is one of 50 mmol / L HEFES buffer, MES buffer, PBS buffer, and Tris-HCl buffer at 25° C. and pH 6.8±0.5; The first stabilizer is one or more combinations of PEG4000, PEG6000, and PEG8000; The second stabilizer is one or more combinations of trehalose, glucose, fructose, and sucrose; The third stabilizer is one or more combinations of lysine and mannitol; The protective agent is one or more combinations of bovine serum albumin and polyvinyl pyrrolidone; The chelating agent is one or more combinations of EDTA, EGTA, DTPA, and CDTA; The preservative is one or more combinations of proclin 300, sodium azide, gentamicin sulfate, Germa 115, and sodium benzoate; The ion balance agent is one or more combinations of potassium chloride, sodium chloride, sodium acetate, and magnesium acetate.

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