A heparin-binding protein control and a method for its production

Abstract

The application discloses a heparin-binding protein control product and a preparation method thereof. The heparin-binding protein control product comprises HBP antigen and a control product diluent, the control product diluent comprises 10-100 mM buffer, 0.5-5 w / v% protective agent, 0.05-0.1 w / v% preservative and 0.2-1.0 w / v% ammonium sulfate; the control product is based on the buffer, the protective agent, the preservative and the ammonium sulfate are added into the buffer, and then the HBP antigen is added to obtain control product solutions with different levels. The control product provided by the application does not need to be freeze-dried, has the advantages of simple process, low cost, high accuracy, good uniformity, simple operation and the like, and the stability of the control product is not easily affected by environmental temperature; the control product can be stored for 90 days at 2-8 DEG C after being opened, and the use rate is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological products, in particular to a heparin-binding protein control and a preparation method thereof. BACKGROUND

[0002] Inflammation refers to a defensive response produced by the body when infected by bacteria or viruses and the like external stimuli, or a non-infectious inflammatory response caused by autoimmune deficiency and the like factors. An inflammation marker refers to a series of sensitive substances produced when the body is infected, wounded or subjected to other stimuli, and stably present in body fluid for a period of time after production, and is not affected by antibacterial drugs, inhibitors and the like, and thus has attracted widespread attention as an index for detecting inflammation.

[0003] Heparin-binding protein (HBP) is also known as azurocidin or cationic antimicrobial protein (CAP37), and is a multifunctional protein with bactericidal function and chemotactic properties, mainly existing in the secretory granules and azurophilic granules of neutrophils. Heparin-binding protein is the earliest marker to increase in inflammation caused by bacteria and the like, and when infection occurs, bacteria invade the blood vessels, stimulating neutrophils to release HBP, and the content of HBP in blood plasma increases. Traditional inflammation markers include procalcitonin (PCT), C-reactive protein (CRP), serum amyloid A (SAA) and the like, and each has certain limitations when used in a single clinical application, and in actual clinical detection, a plurality of indexes and a plurality of detection results are generally required to accurately determine the severity of inflammation. The infection change of heparin-binding protein occurs early, and has higher sensitivity and specificity, and has important clinical value for early warning, prediction and evaluation of inflammatory infectious diseases and observation of therapeutic effect.

[0004] At present, there are few types of quality control products on the market, and most of the reagent kits produced by manufacturers do not contain quality control products, and the preparation of quality control products by clinical laboratories is affected by many factors, so the quality control status in the clinic is not optimistic and needs to be improved. Some existing multi-item composite quality control products contain many detection items, have high cost, are easy to waste, and are mostly freeze-dried products, and the operation is relatively complex during detection. Therefore, there is an urgent need in the clinic for a commercial single-item inflammation marker quality control product with good stability and simple operation for indoor quality control and interlaboratory quality evaluation of laboratories. SUMMARY

[0005] Therefore, the present application aims to provide a heparin-binding protein (HBP) quality control product and a preparation method thereof, which has a simple preparation process, low cost, excellent stability, can be stored for a long time, has small inter-bottle differences, high accuracy, and can meet the clinical use requirements.

[0006] The technical scheme of the present application is as follows:

[0007] The first aspect of the present application provides a heparin binding protein control product, which comprises a HBP antigen and a control product diluent, wherein the control product diluent comprises 10-100 mM of a buffer, 0.5-5 w / v% of a protective agent, 0.05-0.1 w / v% of a preservative and 0.2%-1.0 w / v% of ammonium sulfate.

[0008] Ammonium sulfate is more used in protein purification in the prior art, and the present application finds that it can be used in the control product diluent of heparin binding protein to protect the activity of heparin binding protein, and the reason is that NH4 + It can be combined with the protein bend peptide chain through electrostatic action to make the protein structure more compact, so that the protein performance is more stable. However, too high salt ion concentration will compete for the water of the protein colloid, destroy the protein hydration layer, and cause the protein to aggregate and precipitate, so the appropriate concentration of ammonium sulfate is also one of the key factors to ensure the stability of the control product. In addition, ammonium sulfate is an inert substance and generally does not react with other active substances, so it can maximize the protective effect and thus ensure the monitoring effect of the control product.

[0009] It can be understood that "w / v%" in the present application means the volume-mass ratio of the components, for example, the control product diluent contains 0.5-5 w / v% of a protective agent, which means that 1 L of the control product diluent contains 5-50 g of the protective agent.

[0010] Further, in the above heparin binding protein control product, the buffer is a citrate buffer or a phosphate buffer; further, the concentration of the buffer is preferably 10-100 mM, and the pH is preferably 4.0-5.5; and the buffer is a pH 5.0, 50 mmol / L citric acid buffer.

[0011] Heparin binding protein belongs to the serine protease family, most of which are alkaline proteases, and generally has the best catalytic activity at pH 9-10, but is more stable in an acidic environment. In the present application, a citric acid buffer or a phosphate buffer is used to create an acidic environment with a pH of 4.0-5.5, which can maximize the stability of the heparin binding protein and thus prolong the shelf life of the product.

[0012] Further, in the above heparin binding protein control product, the protective agent is one or more of bovine serum albumin, casein, ovalbumin, gelatin, alanine and histidine.

[0013] Further, in the above heparin binding protein control product, the preservative is one or more of ProClin300, BND and gentamicin sulfate; and further, the preservative is preferably a combination of ProClin300 and BND.

[0014] Further, in the heparin-binding protein control article, high level control solution and low level control solution are included; in the high level control solution, the concentration of HBP antigen is 60-140 ng / mL; in the low level control solution, the concentration of HBP antigen is 12-28 ng / mL.

[0015] The second aspect of the present application provides the use of the above-mentioned heparin-binding protein control article in the preparation of a heparin-binding protein detection kit.

[0016] The third aspect of the present application provides a preparation method of the above-mentioned heparin-binding protein control article, specifically: taking a buffer solution as a base solution, adding a protective agent, a preservative and ammonium sulfate to the base solution in proportion, mixing uniformly, filtering to obtain a control article diluent; then adding HBP antigen to the control article diluent, quantifying and then storing after dispensing.

[0017] Further, in the above preparation method, the filtering can be performed using a 0.22 μm filter membrane.

[0018] Further, in the above preparation method, the content of HBP antigen in the control article solution can be detected using a full-automatic chemiluminescence immunoassay analyzer, and the dispensing is performed when the concentration detection value is within the given reference range.

[0019] Further, in the above preparation method, the storage temperature of the control article is preferably 2-8℃.

[0020] Compared with the prior art, the present application has at least the following advantages:

[0021] (1) The production process of the heparin-binding protein control article provided by the present application does not involve freeze-drying, and the process is simple and low in cost; the obtained control article is in liquid state, and does not need to be reconstituted when used, which is simple to operate.

[0022] (2) The heparin-binding protein control article provided by the present application uses a buffer agent base and adds an appropriate amount of protective agent, preservative and ammonium sulfate, the raw materials are easy to obtain and have good protection effect, and the heparin-binding protein has very high stability in this system.

[0023] (3) The heparin-binding protein control article provided by the present application has good clinical consistency and stability, and has no influence on the stability under the condition of 37℃ for 21 days, which ensures that the change of environmental temperature during transportation will not affect the stability, and the control article can be stored at 2-8℃ for 90 days after opening the bottle, which greatly ensures the use rate of the control article, reduces waste, has small inter-bottle variation, and enhances the accuracy of detection. DETAILED DESCRIPTION

[0024] For a better understanding of the present application, the following detailed description of the application is set forth in connection with specific examples. It is understood that the examples described herein are merely illustrative and are not intended to limit the application.

[0025] Unless otherwise specifically indicated, all experiments were performed in accordance with conventional protocols or as described in the manufacturer's instructions. The reagents and materials used were obtained from commercial sources unless otherwise specified.

[0026] Example 1

[0027] The present example provides a heparin binding protein (HBP) quality control product, which is composed of an HBP antigen and a quality control diluent, wherein the quality control diluent comprises 50 mM citric acid buffer (pH 5.0), 0.07 w / v% BND, 0.07 v / v% ProClin300, 1.0 w / v% bovine serum albumin, and 0.5 w / v% ammonium sulfate.

[0028] In the quality control product, high and low concentration levels of quality control solutions are included, and the final content of HBP antigen in the two concentration levels of quality control solutions is respectively preset as:

[0029] The content of HBP antigen in the low level of quality control solution is 20 ng / mL;

[0030] The content of HBP antigen in the high level of quality control solution is 100 ng / mL.

[0031] The present example also provides a preparation method of the above quality control product, and the specific process is as follows:

[0032] (1) Weigh the citric acid buffer and prepare 50 mM citric acid buffer, adjust the pH to 5.0 to obtain the basic buffer;

[0033] (2) Add 0.07 w / v% BND, 0.07 v / v% ProClin300, 1.0 w / v% bovine serum albumin, and 0.5 w / v% ammonium sulfate to the basic buffer, mix well, adjust the pH to 5.0, and filter with a filter membrane with a pore size of 0.22 μm to obtain the quality control matrix solution;

[0034] (3) Add HBP antigen to the quality control matrix solution to prepare high and low concentration levels of quality control solutions.

[0035] (4) Detect the high and low concentration levels of quality control solutions respectively, and if the detection results meet the following concentration standards, proceed to the next step of dispensing; if the detection results do not meet the following standards, the concentration can be adjusted by adding buffer dilution or continuing to add HBP antigen until the following concentration standards are met:

[0036] The content of HBP in the low level of quality control solution is 14-26 ng / mL; the content of HBP in the high level of quality control solution is 70-130 ng / mL;

[0037] The detection results of this example are shown in Table 1, which shows that the two levels of HBP quality control prepared in this example are within the given range.

[0038] (5) The high and low concentration levels of HBP quality control solution are divided into glass bottles to prepare liquid type quality control, and are sealed and stored at 2-8℃.

[0039] Table 1

[0040]

[0041] Example 2

[0042] This example provides a HBP quality control which is composed of HBP antigen and quality control diluent, wherein the quality control diluent comprises 10 mM citric acid buffer (pH 4.0), 0.05 w / v% BND, 0.05 v / v% ProClin300, 0.5 w / v% bovine serum albumin and 0.2 w / v% ammonium sulfate.

[0043] In the quality control, high and low concentration levels of quality control solution are included, and the final content of HBP antigen in the two concentration levels of quality control solution is respectively preset as:

[0044] The content of HBP antigen in the low level of quality control solution is 20 ng / mL;

[0045] The content of HBP antigen in the high level of quality control solution is 100 ng / mL.

[0046] This example also provides a preparation method of the above quality control, and the specific process is as follows:

[0047] (1) Weigh the citric acid buffer to prepare 10 mM citric acid buffer, adjust the pH to 4.0 to obtain the basic buffer;

[0048] (2) Add 0.05 w / v% BND, 0.05 v / v% ProClin300, 0.5 w / v% bovine serum albumin and 0.2 w / v% ammonium sulfate to the basic buffer, mix well, adjust the pH to 4.0, and filter with a filter membrane with a pore size of 0.22 μm to obtain the quality control matrix solution;

[0049] (3) Add HBP antigen to the quality control matrix solution to prepare high and low concentration levels of quality control solution, respectively.

[0050] (4) The high and low concentration level control solution is detected respectively, and the detection result meets the concentration requirement, and then the next step of sub-packaging is performed.

[0051] (5) The high and low concentration level heparin binding protein (HBP) control solution is sub-packaged in a glass bottle to prepare a liquid type control product, and is sealed and stored at 2-8°C.

[0052] Example 3

[0053] The example provides a heparin binding protein (HBP) control product composed of HBP antigen and control product diluent, wherein the control product diluent comprises 100 mM citric acid buffer (pH 5.5), 0.09 w / v% BND, 0.09 v / v% ProClin300, 5.0 w / v% bovine serum albumin and 1.0 w / v% ammonium sulfate.

[0054] In the control product, high and low concentration level control solutions are included, and the final content of HBP antigen in the two concentration level control solutions is designed as follows:

[0055] The content of HBP antigen in the low level control solution is 20 ng / mL;

[0056] The content of HBP antigen in the high level control solution is 100 ng / mL.

[0057] The example also provides a preparation method of the above control product, and the specific process is as follows:

[0058] (1) Citric acid buffer is weighed and prepared into 100 mM citric acid buffer, and the pH is adjusted to 5.5 to obtain a basic buffer;

[0059] (2) 0.09 w / v% BND, 0.09 v / v% ProClin300, 5.0 w / v% bovine serum albumin and 1.0 w / v% ammonium sulfate are added to the basic buffer, mixed uniformly, the pH is adjusted to 5.5, and the control product matrix solution is obtained by filtering with a filter membrane with a pore size of 0.22 μm;

[0060] (3) HBP antigen is added to the control product matrix solution to prepare high and low concentration level control solutions.

[0061] (4) The high and low concentration level control solution is detected respectively, and the detection result meets the concentration requirement, and then the next step of sub-packaging is performed.

[0062] (5) The high and low concentration levels of heparin binding protein (HBP) quality control solution were divided into glass bottles to prepare liquid type quality control products, and stored at 2-8°C under sealed conditions.

[0063] The quality control products prepared in Examples 1-3 were tested, which specifically included the following aspects:

[0064] 1. Uniformity test

[0065] Ten bottles of each level of quality control product were randomly numbered 1-10, and tested on a precision good automatic chemiluminescence detector. Each concentration level was repeated 3 times for each project. The 3 tests were performed in different orders, for example, 1, 3, 5, 7, 9, 2, 4, 6, 8, 10, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9. The test results were recorded, and when the test results had numerical values, F, S bb , S r and CV 瓶间 were calculated according to formulas (1)-(11).

[0066] Formula 1:

[0067] Formula 2:

[0068] Formula 3: SS 瓶内 = SS 总和 - SS 瓶间

[0069] Formula 4:

[0070] Formula 5:

[0071] Formula 6:

[0072] Formula 7: v1 = a-1;

[0073] Formula 8: v2 = N-a;

[0074] Formula 9:

[0075] Formula 10:

[0076]

[0077] In the formula, SS—variance; x i —designated parameter i-th measurement value or calculation result; —total average; n i —sample i repeated measurement times; x​ij - the jth result of sample i; MS - mean square; v - degrees of freedom; F - F test value; n0 - number of effective measurements; a - number of samples drawn; N - total number of tests; s bb - inter-bottle standard deviation; s r - intra-bottle standard deviation (repeatability standard deviation).

[0078] Acceptance criteria:

[0079] When F < 1, s r is replaced by s bb CV 瓶间 is calculated, and the result should be CV 瓶间 ≤ 10.0%;

[0080] When F < F 0.05(v1,v2) , the test result shows that there is no significant difference in inter-bottle uniformity, and the result should be CV 瓶间 ≤ 10.0%;

[0081] When s bb ≤ 0.3δ, it is considered that the inter-bottle uniformity is good, and the result should be CV 瓶间 ≤ 10.0%;

[0082] When s bb > 0.3δ, it is considered that the inter-bottle uniformity is poor, which does not meet the requirements.

[0083] The uniformity test results of the quality control sample of Example 1 are shown in Table 2.

[0084] Table 2

[0085]

[0086]

[0087] Table 2 shows that the inter-bottle variation of the heparin-binding protein quality control sample prepared in Example 1 at two levels is small, which can accurately reflect the true state of the detection system. The detection results of Example 2 and Example 3 are consistent with Example 1, which shows that the inter-bottle difference is small.

[0088] 2. Accelerated stability test.

[0089] Take each level of 6 bottles of quality control, of which 3 bottles are placed in the 2-8°C refrigerator to avoid light refrigeration, and the other three bottles are placed in the 37°C incubator. Then test one bottle of sample on the 7th, 14th, 21st day, and at the same time, take one bottle of refrigerated quality control for each node as a control sample for testing. When testing, 2 levels of samples are measured 3 times, and the average value is calculated. Calculate the relative deviation of the quality control determination mean value of each time node and the determination mean value of the refrigerated control quality control of each node, and the relative deviation should not exceed ±10%.

[0090] In which, the quality control accelerated stability results in example 1 are shown in table 3.

[0091] Table 3

[0092]

[0093]

[0094] From table 3, it can be seen that the stability deviation of the quality control prepared in example 1 is less than 10% for two levels of acceleration for 21 days. Similarly, the detection results of example 2 and example 3 are also less than 10%.

[0095] 3. Open bottle stability test.

[0096] Take each level of 6 bottles of quality control, of which 1 bottle is placed in the 2-8°C refrigerator to avoid light refrigeration, and the other 5 bottles are placed in the 2-8°C refrigerator to avoid light storage as the unopened control group. Take the opened sample for testing on the 5th, 15th, 30th, 60th, 90th day, and at the same time, take one bottle of unopened control sample for testing. When testing, 2 levels of samples are measured 3 times, and the average value is calculated. Calculate the relative deviation of the quality control determination mean value at the end of the opening period and the determination mean value of the unopened quality control, and the relative deviation should not exceed ±10%.

[0097] In which, the detection data of the quality control prepared in example 1 are shown in table 4.

[0098] Table 4

[0099]

[0100] From table 4, it can be seen that the stability deviation of the quality control prepared in example 1 is less than 10% for two levels of opening for 90 days, which has good opening stability, ensures that the quality control can be used to the maximum extent, and reduces waste. The opening stability detection results of example 2 and 3 are consistent with example 1, that is, the relative deviation is less than 10% within 90 days.

[0101] Example 4

[0102] In this example, three groups of heparin binding protein quality controls in different pH environments are prepared, and the specific process is as follows:

[0103] (1) Take the citric acid buffer, prepare 50mM citric acid buffer, adjust the pH to 5.0, and obtain the basic acidic buffer;

[0104] (2) Take the Tris-HCl buffer, prepare 50mM Tris buffer, adjust the pH to 7.0, and obtain the basic neutral buffer;

[0105] (3) Take the Tris-HCl buffer, prepare 50mM Tris buffer, adjust the pH to 9.0, and obtain the basic alkaline buffer;

[0106] (4) Add 0.07w / v% BND, 0.07v / v% ProClin300, 1.0w / v% bovine serum albumin, and 0.5w / v% ammonium sulfate to the three basic buffers prepared in (1), (2), and (3) respectively, mix well, adjust the pH to 5.0, 7.0, and 9.0 respectively, and filter with a filter membrane with a pore size of 0.22μm to obtain three quality control matrix solutions;

[0107] (5) Add HBP antigen to the above three quality control matrix solutions to prepare quality control solutions at high and low concentration levels respectively; the final content of HBP antigen in the two concentration levels of quality control solutions is respectively preset as:

[0108] The content of heparin-binding protein in the low-level quality control solution is 20ng / mL;

[0109] The content of heparin-binding protein in the high-level quality control solution is 100ng / mL;

[0110] (6) Detect the high and low concentration level quality control solutions respectively, and after meeting the concentration standard, package them in glass bottles to prepare liquid type quality controls, and store them at 2-8℃ in a sealed condition.

[0111] Take the heparin-binding protein quality controls prepared at different pH values in this example respectively, set 2 replicates for each quality control, store them at 4℃ for 1d, 2d, 3d, 4d, 5d, 6d, 7d, and continuously monitor the concentration value changes, and the specific monitoring data are shown in Table 5.

[0112] Table 5

[0113]

[0114]

[0115] As can be seen from Table 5, under the premise of containing the same protective agent, preservative, and ammonium sulfate, the appropriate pH environment is crucial to the stability of the quality control, and compared with the neutral and alkaline environment, the weak acidic environment can improve the stability of the quality control.

[0116] Example 5

[0117] Two different heparin-binding protein control products were prepared in this example, and the specific process was as follows:

[0118] (1) Citric acid buffer was weighed and prepared into 50 mM citric acid buffer, and the pH was adjusted to 5.0 to obtain a basic buffer;

[0119] (2) 0.07 w / v% BND, 0.07 v / v% ProClin300, 1.0 w / v% bovine serum albumin, and 0.5 w / v% ammonium sulfate were added to the basic buffer in the first group, and 0.07 w / v% BND, 0.07 v / v% ProClin300, 1.0 w / v% bovine serum albumin, and 0.5 w / v% sodium chloride were added to the basic buffer in the second group; After mixing well, the pH was adjusted to 5.0, respectively, and two kinds of control product matrix solutions were obtained by filtering with a filter membrane with a pore size of 0.22 μm;

[0120] (3) HBP antigen was added to the above two kinds of control product matrix solutions to prepare control product solutions with high and low concentration levels, respectively; The final content of HBP antigen in the control product solutions of the two concentration levels was pre-set as:

[0121] The content of heparin-binding protein in the low-level control product solution was 20 ng / mL;

[0122] The content of heparin-binding protein in the high-level control product solution was 100 ng / mL;

[0123] (4) The high and low concentration level control product solutions were detected respectively, and after meeting the concentration standard, they were divided into glass bottles to prepare liquid type control products, and stored at 2-8°C in a sealed condition.

[0124] Two kinds of heparin-binding protein control products prepared in this example were taken respectively, 2 replicates were set for each control product, and the concentration value change was continuously monitored after storage at 4°C for 1d, 2d, 3d, 4d, 5d, 6d, and 7d. The specific monitoring data is shown in Table 6.

[0125] Table 6

[0126]

[0127]

[0128] As can be seen from Table 6, compared with sodium chloride, ammonium sulfate has a better protective effect on heparin-binding protein, which significantly improves the stability of the control product.

[0129] In conclusion, the heparin binding protein control provided by the present application has superior stability, which has no influence on stability at 37℃ for 21 days, and can be stored for 90 days at 2-8℃ after opening, greatly ensuring the transportation and use of the quality control product, and being beneficial to commercial production.

[0130] The above-described embodiments are part, but not all, embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application, but merely to illustrate selected embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

Claims

1. A heparin-binding protein control product, characterized in that, It includes HBP antigen and quality control diluent, wherein the quality control diluent contains 10-100 mM buffer, 0.5-5 w / v % protectant, 0.05-0.1% w / v preservative and 0.2-1.0 w / v ammonium sulfate; The buffer solution is a citrate buffer or a phosphate buffer; The pH of the buffer solution is 4.0~5.

5.

2. The heparin-binding protein control product according to claim 1, characterized in that, The protective agent is one or more of bovine serum albumin, casein, ovalbumin, gelatin, alanine, and histidine.

3. The heparin-binding protein control product according to claim 1, characterized in that, The preservative is one or more of ProClin300, BND, and gentamicin sulfate.

4. The heparin-binding protein control product according to claim 1, characterized in that, The preservatives are ProClin300 and BND.

5. The heparin-binding protein control product according to claim 1, characterized in that, The quality control products include high-level and low-level quality control products. In the high-level quality control products, the concentration of HBP antigen is 60-140 ng / mL; in the low-level quality control products, the concentration of HBP antigen is 12-28 ng / mL.

6. The use of the heparin-binding protein control sample as described in any one of claims 1 to 5 in the preparation of a heparin-binding protein detection kit.

7. A method for preparing the heparin-binding protein control product according to any one of claims 1 to 5, characterized in that, Specifically, the process involves adding a protectant, preservative, and ammonium sulfate to the buffer solution, mixing thoroughly, filtering to obtain a quality control diluent, adding HBP antigen to the quality control diluent, quantifying, aliquoting, and storing.

8. The method according to claim 7, characterized in that, The storage temperature is 2~8℃.

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