A NSE quality control product and its preparation method and application

Abstract

The present invention provides a NSE quality control product and its preparation method and application. The NSE quality control product includes the following components: human serum as matrix liquid, containing NSE antigen 4.5~80 ng / mL, preservative 0.05~0.2v / v%, magnesium ion 10~40 mM, N-acetyl-L-cysteine ​​1~5 mM, bovine serum albumin 1~10 w / v% and sucrose 5~15 w / v%. The NSE quality control product of the present invention can be stored for at least 2 years at ≤-20°C, and is stable for 14 days at 2~8°C after dissolution, allowing one-time preparation and multiple uses, avoiding waste, greatly reducing frequent purchases and replacements due to storage or short-term failure, and further saving use costs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a NSE quality control product and a preparation method and application thereof. Background Art

[0002] Laboratory internal quality control (IQC) is an important part of the quality management system. It conducts quality control on the entire testing system, including sample aspiration, sample testing, and result reporting. The main purpose of quality control products is to determine that the measurement procedure is performed as expected to confirm that the results of patient specimens are suitable for providing medical services. In the design of quality control procedures, the 2022 version of ISO15189 should consider the use of third-party IQC materials as a substitute or supplement to the quality control materials provided by reagent or instrument manufacturers. At least two concentration levels (normal and abnormal levels) of quality control materials should be used.

[0003] The preparation of quality control products is generally divided into two ways: 1) Clinical laboratories collect high-concentration residual test specimens from daily testing as high-value samples, and normal-concentration residual test specimens as low-value samples, and use methods approved by the health licensing department to test at least four infectious diseases including hepatitis B surface antigen (HBsAg), hepatitis C antibody (Anti-HCV), AIDS antibody (HIV1 / 2 antibody), and syphilis spirochete-specific antibody (Anti-TP). After inactivation, they are packaged and frozen for storage; when it is difficult to reach the target concentration at high values, cryo-separation technology can be used to obtain high and low concentration levels of quality control samples, and prepare them into solutions or freeze-dried products according to the serum stability of the test items. 2) Instrument manufacturers or other quality control product manufacturers use natural or recombinant target antigen antibodies to commercial serum or stabilizer-added buffer systems, and adjust the input amount of additives to prepare fixed-value quality control products with target concentrations. However, the quality control products produced by medical institutions have the following disadvantages when collecting residual serum samples: the amount of each sample is small, generally less than 1 mL, which is difficult to meet the requirements of one-time mass production; it is difficult to collect high-value samples, and if it is a composite quality control product, it is difficult to achieve high or low values ​​for different analytes in the same product at the same time. Most commercial quality control products are imported brands from abroad, which are expensive and have a low degree of complexity. Some of them use a buffer system with added stabilizers as the matrix, which has a matrix effect with clinical samples and has poor stability, causing instability in clinical testing. Most freeze-dried quality control products need to be reconstituted with water when used by customers, which increases the time for manual operation, and the differences in reconstitution operations among different users may lead to large differences between bottles, which is not conducive to use.

[0004] Neuron specific enolase (NSE) is an isoenzyme of enolase. The biological half-life of NSE may be about 1.5 to 20 hours. The molecular weight of NSE is about 80kDa, and the isoelectric point is pH 4.7. It is an acidic protease that mainly participates in glycolysis and catalyzes 2-phosphoglycerate to enolphosphopyruvate. Enolase isoenzymes are composed of α, β, and γ subunits in the form of dimers, and there are five forms: αα, αβ, ββ, αγ, and γγ. Among them, the dimers αγ and γγ are called NSE. NSE plays an important regulatory role in the growth, differentiation, survival and death of neurons. Under normal circumstances, serum and cerebrospinal fluid contain trace amounts of NSE. Abnormally elevated NSE is often accompanied by brain damage or other nervous system diseases and tumor-related diseases, such as cerebral hemorrhage, cerebral ischemia, brain trauma, Alzheimer's disease, neuroblastoma, neuroendocrine diseases and small cell lung cancer. Clinically, NSE is mainly used for the diagnosis of small cell lung cancer (SCLC). It is the most sensitive and specific tumor marker for SCLC. The level of NSE in the serum of SCLC patients is significantly higher than that of patients with non-small cell lung cancer (lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, etc.), and can also be used for differential diagnosis. In addition, NSE is also used for the diagnosis of neuroblastoma, and its combined detection with S-100 protein is also used for the diagnosis of melanoma. Therefore, NSE in vitro diagnostic reagents play a very important role in the early diagnosis, disease monitoring, efficacy evaluation and recurrence prediction of the above diseases. Among the existing inventions and commercial products, most NSE quality control products are not 100% human serum matrix, and are expensive and have a short shelf life. They are generally stable for 1 to 3 days at 2 to 8 ° C after reconstitution or dissolution. If they are not used up within the shelf life after reconstitution, it is easy to cause reagent waste and increase costs. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes a NSE quality control product and a preparation method and application thereof.

[0006] The invention provides an NSE quality control product, which comprises the following components: 4.5-80 ng / mL of NSE antigen, 90-100 v / v of humanized serum, 0.05-0.2 v / v of preservative, 10-40 mM of magnesium ion, 1-5 mM of N-acetyl-L-cysteine, 1-10 w / v% of bovine serum albumin (BSA), and 5-15 w / v% of sucrose.

[0007] The human serum-containing matrix liquid provided by the present invention is used for the preparation of quality control products, which can significantly improve the stability of the chemical properties of NSE, maintain its spatial conformation, and improve its heat resistance. The present invention uses a specific stabilizer to compound with the human serum matrix to avoid the non-specific binding of components in the serum matrix (such as proteins, hormones, immune factors, etc.) with NSE, provide protection, and reduce protein denaturation or aggregation.

[0008] The present invention combines magnesium ions with N-acetyl-L-cysteine ​​to exert a synergistic effect, support the activity of antioxidant enzymes in cells, further enhance antioxidant capacity, and minimize the damage of oxidative stress to NSE. Through the support of magnesium ions on enzyme activity and stability and the antioxidant protection of NAC, the stability of biological molecules such as NSE can be improved at multiple levels.

[0009] The present invention adds a certain amount of BSA. As a protective protein, BSA can help NSE maintain its stable structure and reduce the contact with potential degradation factors in the environment, thereby improving its stability.

[0010] Sucrose is a common protective sugar with antifreeze, anti-drying and antioxidant properties. In protein solutions, sucrose can form a protective film to reduce the impact of water evaporation on NSE, and reduce the aggregation and denaturation of proteins that may occur during freezing or drying, thereby preventing their inactivation or degradation.

[0011] In some embodiments, the NSE quality control product uses human serum as the matrix liquid and includes the following components: 4.5~80 ng / mL of NSE antigen, 0.2v / v% of preservative, 20 mM of magnesium ion, 2.5 mM of N-acetyl-L-cysteine, 5 w / v% of bovine serum albumin BSA and 15 w / v% of sucrose.

[0012] In some embodiments, the NSE quality control product is a complete set of products, including low-concentration quality control products, medium-concentration quality control products, and high-concentration quality control products;

[0013] The low-concentration quality control product uses human serum as the matrix liquid, including NSE antigen 3.6~5.4 ng / mL, preservative 0.05~0.2v / v%, magnesium ion 10~40 mM, N-acetyl-L-cysteine ​​1~5 mM, bovine serum albumin BSA 1~10 w / v% and sucrose 5~15 w / v%;

[0014] The medium-concentration quality control product uses human serum as the matrix liquid, including NSE antigen 24~36 ng / mL, preservative 0.05~0.2v / v%, magnesium ion 10~40 mM, N-acetyl-L-cysteine ​​1~5 mM, bovine serum albumin BSA 1~10 w / v% and sucrose 5~15 w / v%;

[0015] The high-concentration quality control product uses human serum as the matrix liquid, including NSE antigen 64~96 ng / mL, preservative 0.05~0.2v / v%, magnesium ion 10~40 mM, N-acetyl-L-cysteine ​​1~5 mM, bovine serum albumin BSA 1~10 w / v% and sucrose 5~15 w / v%.

[0016] In some embodiments, the human serum is human serum that is negative in four tests: hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody, and syphilis treponema pallidum.

[0017] In some embodiments, the preservative is any one of proclin 300, sodium benzoate, sodium azide, potassium sorbate, and 2-methyl-4-isothiazoline-3-one.

[0018] The present invention also provides a method for preparing the NSE quality control product, comprising the following steps:

[0019] The NSE antigen, humanized serum, magnesium ions, N-acetyl-L-cysteine, bovine serum albumin BSA and sucrose are mixed to prepare a mixture, thereby obtaining the NSE quality control product.

[0020] The present invention also provides an NSE quality control product kit, comprising the NSE quality control product.

[0021] The present invention also provides the use of the NSE quality control product in preparing a kit for detecting small cell lung cancer.

[0022] The present invention also provides the use of the NSE quality control product in preparing a kit for neuroblastoma detection.

[0023] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0024] (1) The NSE quality control product of this application can be stored at ≤-20℃ for at least 2 years, which greatly reduces the frequent purchase and replacement due to storage or short-term failure. In addition, after dissolution, it can be prepared once and used multiple times at 2~8℃, and can be stably stored for 14 days, avoiding waste and thus saving usage costs. It is especially suitable for laboratory scenarios that require batch quality control. It eliminates the need for laboratory personnel to frequently prepare and repackage, reducing the workload and risk of errors, thereby improving detection efficiency.

[0025] (2) The NSE quality control product of this application optimizes the amount of stabilizer added in the formula design. Compared with the existing NSE quality control products, the stabilizer content is lower, which helps to reduce non-specific interference, thereby improving the accuracy and repeatability of detection, and is suitable for early screening and treatment monitoring of small cell lung cancer. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0027] The materials used in the present invention are all commercial products and can be purchased. The human serum matrix was purchased from TECHNOPATH.

[0028] Example 1 Exploring the optimal concentration of magnesium ions to stabilize NSE

[0029] This example verifies the effect of adding different concentrations of magnesium ions on the stability of NSE solution. The implementation steps are as follows:

[0030] (1) After commercial healthy human serum is thawed at 2-8°C, add proclin300 to 0.2% (v / v), mix well, and place at 2-8°C for later use;

[0031] (2) NSE antigen (purchased from Suzhou Jinan Protein Technology Co., Ltd.) was added to a final concentration of 4.5 ng / mL for level 1, 30 ng / mL for level 2, and 80 ng / mL for level 3. The quality control products for the three concentration levels were Rochecobas e411. The solutions containing NSE antigen at each concentration level were divided into 3 groups. Magnesium ions were added to the final concentrations of 10 mM, 20 mM, and 40 mM, respectively. No magnesium ions were added as a negative control. There were 4 groups with 12 samples in total.

[0032] (3) Each sample was divided into two parts, A and B. Sample A was stored at 2-8°C. Sample B was stored at -20°C after being packaged as a control group. The test was conducted continuously for 7 days. Each sample was tested in parallel 3 times at each time point. The average value was taken. The deviation between sample A and sample B was calculated. The change in the concentration value of the samples stored at 2-8°C was observed. The results are shown in Tables 1-4 below:

[0033] Table 1 Effect of 0 mM magnesium ion on NSE antigen concentration

[0034]

[0035] Table 2 Effect of adding 10 mM magnesium ions on the change of NSE antigen concentration

[0036]

[0037] Table 3 Effect of adding 20 mM magnesium ions on the change of NSE antigen concentration

[0038]

[0039] Table 4 Effect of adding 40 mM magnesium ions on the change of NSE antigen concentration

[0040]

[0041] According to the results in Tables 1 to 4, the addition of 20 mM magnesium ions can significantly improve the stability of NSE in the human serum matrix at 2 to 8 °C. With the deviation controlled at ±10%, it is considered stable for 3 days, which is higher than the stability level of current commercial quality control products, and the screening of stabilizers will continue.

[0042] Example 2

[0043] In conventional single-factor screening of stabilizers, the principle is to add as little stabilizer as possible to achieve the stability goal and reduce the matrix effect.

[0044] The stabilizer screening is as follows:

[0045] Reducing agents and antioxidants: dithiothreitol (DTT) 0.1~0.5g / L, disodium ethylenediaminetetraacetic acid 2~10mM, N-acetyl-L-cysteine ​​1~5mM;

[0046] Sugar protective agent: sucrose 5~15%, trehalose 5~15%;

[0047] Protein protective agent: casein 0.1~0.5%, bovine serum albumin BSA 1~10%;

[0048] Amino acid protective agent: arginine 1~5%, glycine 1~5%, lysine 0.1~0.5%;

[0049] Polyol: mannitol 1~5%;

[0050] The implementation steps are as follows:

[0051] (1) The following single-factor stabilizers were added to the human serum matrix of 0.2% proclin300, and the final concentrations were prepared as follows: dithiothreitol (DTT) 0.25 g / L, disodium ethylenediaminetetraacetic acid 5 mM, N-acetyl-L-cysteine ​​2.5 mM, sucrose 5%, sucrose 15%, trehalose 5%, trehalose 15%, casein 2.5%, bovine serum albumin BSA 5%, arginine 2.5%, glycine 2.5%, lysine 0.25%, mannitol 2.5%, and the input NSE antigen concentration was level 3 80 ng / mL. The negative control was human serum matrix with only 20 mM magnesium ions and 0.2% proclin300. A total of 14 samples.

[0052] (2) After preparation, each sample was stored at 2-8°C and tested at 0, 3 and 7 days. Each sample was tested three times in parallel at each time point and the average was taken. The deviation between the sample value at each time point and the sample value at time 0 was calculated, and the changes in the concentration values ​​of the samples stored at 2-8°C were observed.

[0053] The results are shown in Table 5 below:

[0054] Table 5 Effect of a single stabilizer on changes in NSE antigen concentration

[0055]

[0056] According to the above results, compared with the negative control, adding 1~5mM N-acetyl-L-cysteine ​​can improve the stability of NSE, among which 2.5mM concentration is the best, 1mM has a partial stabilizing effect, and when the concentration is increased to 5mM, it does not further improve the stability of NSE. Only adding 5% bovine serum albumin BSA or 15% sucrose can significantly slow down the degradation rate of NSE serum.

[0057] Example 3

[0058] This example explores the effect of the combination of single stabilizers screened in Example 2 on the stability of NSE serum. The matrix formula is obtained by mixing the final concentration of N-acetyl-L-cysteine ​​screened in Example 2, bovine serum albumin BSA and sucrose to make NSE serum stable at 2-8°C for 14 days.

[0059] The implementation steps are as follows:

[0060] (1) Preparation of composite matrix solution with stabilizer added:

[0061] Group A: After human serum was thawed at 2-8°C, proclin300 was added to 0.2% (v / v), magnesium ion was added to a final concentration of 20 mM, N-acetyl-L-cysteine ​​was added to a final concentration of 2.5 mM, bovine serum albumin BSA was added to 5% (w / v), and sucrose was added to 15% (w / v);

[0062] Group B: proclin300 was added to 0.2% (v / v), magnesium ion was added to a final concentration of 20 mM, N-acetyl-L-cysteine ​​was added to a final concentration of 1 mM, bovine serum albumin BSA was added to a final concentration of 1% (w / v), and sucrose was added to a final concentration of 5% (w / v);

[0063] Group C: proclin300 was added to 0.2% (v / v), magnesium ions were added to a final concentration of 20mM, N-acetyl-L-cysteine ​​was added to a final concentration of 5mM, bovine serum albumin BSA was added to a final concentration of 10% (w / v), and sucrose was added to a final concentration of 20% (w / v).

[0064] In addition, in this experiment, proclin300 was added to 0.2% (v / v), magnesium ions were added to a final concentration of 20mM, N-acetyl-L-cysteine ​​was added to a final concentration of 2.5mM, bovine serum albumin BSA was added to 5% (w / v), and sucrose was added to 15% (w / v). These three concentrations of single stabilizers were used as controls.

[0065] (2) Add NSE antigen to three target levels: Level 1: 4.5 ng / mL, Level 2: 30 ng / mL, Level 3: 80 ng / mL;

[0066] (3) Each sample was divided into two parts, A and B. Sample A was stored at 2-8°C, and sample B was packaged as a control group and stored at -20°C. The test was conducted for up to 14 days. Each sample was tested three times at each time point, and the average was taken. The deviation between sample A and sample B was calculated, and the changes in the concentration values ​​of samples stored at 2-8°C were observed.

[0067] The results are shown in Tables 6 to 8 below:

[0068] Table 6 Effect of compound stabilizer formula of group A on the stability of NSE serum

[0069]

[0070] Table 7 Effect of compound stabilizer formula of group B on the stability of NSE serum

[0071]

[0072] Table 8 Effect of compound stabilizer formula of group C on the stability of NSE serum

[0073]

[0074] According to the above results, adding 20mM magnesium ions to the serum matrix can significantly improve the stability of three concentrations of NSE at 2~8℃. Then, 13 single-factor stabilizers were screened. By calculating the deviation from 0 days at each time point of opening the bottle at 2~8℃, and testing for 7 days, it was found that adding N-acetyl-L-cysteine ​​at a final concentration of 2.5mM, bovine serum albumin BSA 5% (w / v), and sucrose 15% (w / v) can partially improve the stability of NSE in serum. These single-factor stabilizers were mixed and prepared, and stabilizers were added in different concentration gradients from low to high, a total of three combinations of A, B, and C. The stability of opening the bottle at 2~8℃ was tested for 28 days. At 14 days, the measured values ​​of the three concentration levels of A, B, and C were within ±10% of the calculated deviation of the samples under -20℃ storage conditions. It is believed that it can be stable for 14 days in this formula.

[0075] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A NSE quality control product, characterized in that: The NSE quality control product uses human serum as the matrix liquid and is composed of the following components: NSE antigen 4.5~80 ng / mL, preservative 0.2v / v%, magnesium ion 20 mM, N-acetyl-L-cysteine ​​2.5 mM, bovine serum albumin BSA 5 w / v% and sucrose 15 w / v%.

2. The NSE quality control product according to claim 1, characterized in that: The NSE quality control product is a complete set of products, consisting of low-concentration quality control products, medium-concentration quality control products and high-concentration quality control products; The low-concentration quality control product uses human serum as the matrix liquid and is composed of the following components: NSE antigen 3.6~5.4 ng / mL, preservative 0.05~0.2v / v%, magnesium ion 10~40 mM, N-acetyl-L-cysteine ​​1~5 mM, bovine serum albumin BSA 1~10w / v% and sucrose 5~15 w / v%; The medium-concentration quality control product uses human serum as the matrix liquid and is composed of the following components: NSE antigen 24~36 ng / mL, preservative 0.05~0.2v / v%, magnesium ion 10~40 mM, N-acetyl-L-cysteine ​​1~5 mM, bovine serum albumin BSA 1~10 w / v% and sucrose 5~15 w / v%; The high-concentration quality control product uses human serum as the matrix liquid and is composed of the following components: NSE antigen 64~96 ng / mL, preservative 0.05~0.2v / v%, magnesium ion 10~40 mM, N-acetyl-L-cysteine ​​1~5 mM, bovine serum albumin BSA 1~10 w / v% and sucrose 5~15 w / v%.

3. The NSE quality control product according to claim 1, characterized in that: The human serum is human serum that is negative for four tests: hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody and syphilis treponema.

4. The NSE quality control product according to claim 1, characterized in that: The preservative is any one of proclin 300, sodium benzoate, sodium azide, potassium sorbate, and 2-methyl-4-isothiazoline-3-one.

5. The method for preparing the NSE quality control product according to any one of claims 1 to 4, characterized in that: The steps include: The NSE antigen, humanized serum, magnesium ions, N-acetyl-L-cysteine, bovine serum albumin BSA and sucrose are mixed to prepare a mixture, thereby obtaining the NSE quality control product.

6. A NSE quality control kit, characterized in that: Including the NSE quality control product described in any one of claims 1 to 4.

7. Use of the NSE quality control product according to any one of claims 1 to 4 in the preparation of a kit for the detection of small cell lung cancer.

8. Use of the NSE quality control product according to any one of claims 1 to 4 in the preparation of a kit for detecting neuroblastoma.

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