Biochemical multi-phase liquid quality control product containing glycocholic acid and preparation method of biochemical multi-phase liquid quality control product

Abstract

The invention belongs to the technical field of biochemical multi-item liquid quality control products containing glycocholic acid, and particularly relates to a biochemical multi-item liquid quality control product containing glycocholic acid and a preparation method of the biochemical multi-item liquid quality control product containing glycocholic acid. Comprising a buffer solution, a mixed anti-interference agent of acetylcysteine and cyclodextrin, a stabilizer, a surfactant and a preservative, and the concentration of the mixed anti-interference agent in the quality control product is 0.1%-2% (w / v); the analytes comprise glycocholic acid, total bile acid and other biochemical markers. In order to overcome the defects in the prior art, human serum is taken as a matrix, and a mixed anti-interference agent of acetylcysteine and cyclodextrin is added, so that the problems that glycocholic acid is easy to oxidize and degrade, cross-reacts with total bile acid and the stability of a liquid quality control product is insufficient are solved; the method can realize long-term stable and accurate detection of the liquid quality control product containing CG and multiple biochemical markers.

Description

Technical Field

[0001] The present invention mainly relates to the technical field of biochemical multiple liquid quality control products containing glycocholic acid, and specifically relates to a biochemical multiple liquid quality control product containing glycocholic acid and a preparation method thereof. Background Art

[0002] Glycocholic acid (CG) is a conjugated bile acid formed by the combination of cholic acid and glycine through an amide bond. After hepatocyte metabolism, about 95% of CG is reabsorbed through the enterohepatic circulation. Under normal circumstances, its peripheral blood concentration is very low, about 1.3 ± 0.8 mg / L. CG is regarded as an important biomarker for liver and gallbladder diseases: the serum CG level in patients with primary liver cancer can exceed 20 times the normal value, and its sensitivity is better than that of alpha-fetoprotein; in patients with liver cirrhosis, the increase in CG is closely related to pathological progression; in hepatitis, CG can reflect hepatocyte damage earlier, and its sensitivity and specificity are also better than traditional indicators such as ALT / AST. For patients with intrahepatic cholestasis of pregnancy (ICP), the serum CG level may increase by 10 to 100 times and is directly related to risks such as fetal distress and premature birth. Therefore, it has been clinically used as the core indicator for ICP screening. In addition, CG also has significant value in the early warning of alcoholic liver injury and the diagnosis of biliary obstruction (such as cholelithiasis and jaundice). The CG level in patients with obstructive liver diseases can increase by 10 to 20 times.

[0003] The stability of CG is affected by its molecular structure characteristics: the three hydroxyl groups on the cholic acid mother nucleus are easily oxidized into keto or carboxylic acid groups; the amide bond is easily hydrolyzed in an acidic or alkaline environment or under high humidity; the combination of carboxylic acid groups and metal ions will catalyze degradation. Secondly, the amphiphilicity of CG will cause micelle aggregation, and heterologous matrices (such as bovine serum) will accelerate its degradation. Repeated freezing and thawing or storage under high humidity may damage its structure, and photooxidation and hydrolysis will also occur continuously during long-term storage.

[0004] Current technologies have improved stability to a certain extent through composite stabilizers (such as sugars or chelating agents) and low-temperature light protection, however, batch differences and multi-component compatibility issues still need to be further optimized. In addition, when CG is added as a test item to a composite biochemical quality control product containing bile acids, due to the structural similarity between the two (such as the common cholic acid core), cross-reaction may occur, resulting in a false increase in the CG test value.

[0005] The above stability challenges and the interference of total bile acids further exacerbate the difficulty of developing composite biochemical quality control products containing CG. At the same time, quality control products containing CG on the current market still face multiple technical shortcomings: First, structural analogues such as total bile acids interfere with the detection of CG, resulting in the detection value of CG being too high and exceeding the established target value range, making it difficult to meet clinical needs; Second, the special amphiphilic structure of CG has relatively strict requirements for the matrix of the quality control product. Heterologous matrices not only cause the accelerated degradation of CG, but also other substances introduced are prone to non-specific binding or matrix effects such as enzyme activity inhibition, with a large deviation from the detection of clinical samples, affecting the reliability of laboratory quality control.

[0006] Therefore, there is an urgent need to develop a liquid composite biochemical quality control product with low interference in CG detection, using a standardized human serum matrix and having good batch-to-batch stability, which will fill the deficiencies of existing products in terms of project comprehensiveness, matrix authenticity, and mass production stability, provide key technical support for improving the accuracy of liver disease diagnosis and laboratory quality control level, and have significant clinical application value and market prospects. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, one of the purposes of the present invention is to provide a biochemical multi-item liquid quality control product containing glycocholic acid and its preparation method. Using human serum as the matrix, by adding a mixed anti-interference agent of acetylcysteine and cyclodextrin, the problems of easy oxidation and degradation of glycocholic acid (CG), cross-reaction with total bile acid (TBA), and insufficient stability of the liquid quality control product are solved, and a method for long-term stable and accurate detection of the liquid quality control product containing CG and multiple biochemical markers is realized.

[0008] The present invention for the first time provides the following ideas for the matrix effect interference problem of biochemical multi-item liquid quality control products containing glycocholic acid and total bile acid, especially the problems related to the stability of glycocholic acid and the interference of structural analogues in detection: stabilizing the structure of glycine, strengthening the exposure of its specific epitopes, and masking its common steroid ring structure. The core of the present invention is to provide a specific anti-interference agent in this specific quality control product composition. The anti-interference ability of the biochemical multi-item liquid quality control product containing glycocholic acid and total bile acid is significantly improved by the mixed anti-interference agent of acetylcysteine and cyclodextrin, and the homogeneity effect is significantly enhanced. Especially when bile acid and glycocholic acid are detected in combination, the structure of CG can be kept stable and can be efficiently detected, avoiding false positive or false negative results caused by cross-reaction during detection due to the same steroid ring structure of bile acid and glycocholic acid.

[0009] In traditional biochemical quality control products, the stability protection of protein or enzyme analytes usually relies on sugars (such as sucrose, trehalose) or amino acids (such as glycine) as stabilizers. However, for the oxidation degradation and cross-reaction problems of small molecule substances such as bile acids like CG, traditional stabilizers have obvious limitations: Those skilled in the art usually prefer EDTA as a metal ion chelator or vitamin C / E as a free radical scavenger. However, EDTA can only inhibit metal-catalyzed oxidation and cannot solve the conformational isomerization problem of CG; vitamin C / E is prone to oxidation and inactivation in a liquid environment and may even chemically react with CG.

[0010] Although NAC is widely used as an expectorant or antidote in the medical field and its antioxidant effect of the mercapto group (-SH) is well-known, its application in biochemical quality control products has the following technical biases: 1. High-concentration NAC (>5% w / v) may damage the structures of other proteinaceous analytes (such as enzymes, antibodies) in the quality control product through thiol-disulfide exchange reactions, and those skilled in the art usually avoid using it in multi-index quality control products; 2. The amphiphilic structure of NAC (acetyl + mercaptopropionic acid) may change the surface tension of human serum matrix, resulting in the aggregation or precipitation of lipoprotein analytes (such as sdLDL), affecting the uniformity of detection results.

[0011] 3. Regarding the structural similarity problem between CG and TBA, the prior art mainly solves it through antibody modification (such as screening highly specific monoclonal antibodies) or chemical blockers (such as adding excessive structural analogs to compete for binding sites), but these methods are costly and may introduce new interfering substances.

[0012] Therefore, those skilled in the art have not realized that by stabilizing the CG molecular conformation rather than directly blocking antibody binding, the cross-reaction rate can be indirectly reduced. To solve the above technical problems, the core of the present invention lies in first introducing a mixed anti-interference agent of acetylcysteine and cyclodextrin into a liquid quality control product containing glycocholic acid. On the one hand, acetylcysteine scavenges reactive oxygen species and chelates metal ions through the mercapto group, and inhibits the oxidative degradation, metal-catalyzed damage and conformational isomerization of CG through the hydrophobic interaction between the acetyl group and the steroid nucleus of CG and the formation of hydrogen bonds between the carboxylic acid group and the hydroxyl group of CG, stabilizing its molecular structure; on the other hand, cyclodextrin specifically includes the steroid nucleus structure of CG through a hydrophobic cavity, covering the Si-Lea epitope shared with total bile acids, and at the same time exposing the specific epitope of the glycine side chain of CG, enhancing the specific binding with antibodies during detection, thereby reducing cross-reactions. The two work together to keep CG structurally stable in the liquid quality control product and achieve accurate detection, synergistically solving the problems of CG stability and detection interference.

[0013] Specifically, the technical solutions adopted by the present invention are as follows: In the first aspect, the present invention provides a biochemical multi-index liquid quality control product containing glycocholic acid, which uses human serum as a matrix and contains the following components: Non - analytes, including buffer, mixed anti - interference agent, stabilizer, surfactant and preservative, the mixed anti - interference agent is a mixture of acetylcysteine and cyclodextrin, and the concentration of the mixed anti - interference agent in the quality control product is 0.1% - 2% (w / v); Analytes, including glycocholic acid, total bile acid and other biochemical markers.

[0014] In the present invention, human serum as a matrix can simulate the protein composition (such as albumin, globulin) and ionic environment (pH, salt concentration) of clinical samples, reducing non - specific binding or enzyme activity inhibition caused by heterologous matrix (such as bovine serum).

[0015] In the present invention, adding acetylcysteine to the quality control product containing glycocholic acid and total bile acid can significantly improve its stability, which is mainly achieved through the following multi - dimensional action mechanisms: (1) Scavenging reactive oxygen species: The sulfhydryl group (-SH) of acetylcysteine can directly neutralize oxidizing substances such as hydroxyl radicals and hydrogen peroxide, blocking the oxidative cleavage of the glycocholic acid steroid nucleus (such as 7α - hydroxyl, C - 24 side chain).

[0016] (2) Inhibiting the Fenton reaction: Acetylcysteine forms stable chelates with transition metal ions such as Fe 2+ , Cu 2+ etc., blocking the hydrogen bonds generated by metal catalysis and reducing the oxidation rate of iron - mediated glycocholic acid.

[0017] (3) Hydrophobic interaction and hydrogen bond formation: The acetyl group of acetylcysteine binds to the glycocholic acid steroid nucleus through van der Waals forces, reducing conformational isomerization caused by molecular thermal motion. At the same time, the carboxylic acid group forms hydrogen bonds with the C - 12 hydroxyl group, enhancing the rigidity of the steroid skeleton.

[0018] In summary, through the synergistic effects of antioxidant, molecular stabilization, environmental regulation, etc., acetylcysteine effectively overcomes the key stability problems such as easy oxidation degradation and conformational changes of glycocholic acid in the quality control product, providing technical guarantee for the accurate quality control of bile acid - related biomarkers.

[0019] On the basis of adding acetylcysteine, the present invention also adds cyclodextrin to form a "stability - anti - interference complex". Acetylcysteine is responsible for maintaining the structural stability of glycocholic acid, and cyclodextrin is responsible for coating the steroid nucleus of glycocholic acid and exposing the glycine side chain of glycocholic acid, enhancing its specific binding with the antibody in the kit during detection and reducing the cross - reaction of bile acids.

[0020] In some realizable ways, the mass ratio of acetylcysteine to cyclodextrin is 0.1:1 - 1:1.

[0021] In some realizable ways, the cyclodextrin is selected from one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. Preferably, the cyclodextrin is β-cyclodextrin, which encapsulates the CG steroid nucleus through its hydrophobic cavity, masks the Si-Lea epitope, and exposes the LSTa epitope of the glycine side chain in a directed manner, reducing the cross-reaction with TBA.

[0022] In some realizable ways, the stabilizer includes: A saccharide stabilizer selected from at least one of sucrose, trehalose, and mannitol, preferably trehalose, which inhibits water loss and ice crystal damage through a hydrogen bond network of hydroxyl groups; its concentration in the quality control product is 0.1% - 4% (w / v).

[0023] A protein stabilizer selected from at least one of casein, ovalbumin, and antifreeze glycoprotein, preferably antifreeze glycoprotein, and its concentration in the quality control product is 0.1% - 2% (w / v).

[0024] In some realizable ways, the buffer is selected from at least one of HEPES buffer, MES buffer, PIPES buffer, and Tris-HCl buffer, preferably HEPES buffer, and its concentration is 20 - 100 mM, and the pH is 6.0 - 8.0.

[0025] And / or, the surfactant is selected from at least one of Tween 20 and Triton 100, preferably Tween 20.

[0026] And / or, the preservative is selected from at least one of sodium azide, Proclin 300, Proclin 950, and gentamicin, preferably Proclin 300.

[0027] And / or, the pH value range of the liquid quality control product is 6.0 - 8.0.

[0028] In some realizable ways, the concentration of HEPES buffer is 50 mM, and its pH is 7.5. Maintaining neutrality can not only effectively inhibit the hydrolysis of the glycocholic acid amide bond but also stabilize the active conformation of enzyme analytes, ensuring the compatibility of multi-item detection.

[0029] In some realizable ways, other biochemical markers include at least one of homocysteine, β-hydroxybutyric acid, glutamate dehydrogenase, β2-microglobulin, C-reactive protein, aspartate aminotransferase, glutathione reductase, 5'-nucleotidase, glycated albumin, glycated serum protein, small and dense low-density lipoprotein cholesterol, lipoprotein (a), apolipoprotein E, retinol-binding protein, cystatin C, creatine kinase isoenzyme, α-fucosidase, and adenosine deaminase, meeting the requirements of multi-item combined quality control.

[0030] When co - detecting glycocholic acid and total bile acid, acetylcysteine stabilizes the conformation of glycocholic acid, and cyclodextrin is responsible for coating the steroid nucleus of glycocholic acid and exposing the glycine side chain of glycocholic acid, enhancing its specific binding with the antibody in the kit during detection and reducing the cross - reaction of bile acids.

[0031] In a second aspect, the present invention also provides a method for preparing a multi - biochemical liquid quality control product containing glycocholic acid for preparing the above - mentioned liquid quality control product. The preparation method includes the following steps: (1) Matrix preparation: Take negative human serum for infectious diseases, and sequentially add buffer, a mixed anti - interference agent of acetylcysteine and cyclodextrin, a stabilizer and a preservative, and stir to dissolve. First add acetylcysteine to the serum matrix to ensure its prior binding with glycocholic acid molecules to form a stable complex.

[0032] (2) pH adjustment: Adjust the pH of the system to 6.0 - 8.0 with acid - base reagents; (3) Analyte addition: Add glycocholic acid, total bile acid and other biochemical markers at a set concentration, and stir until completely dissolved; (4) Filtration and sub - packaging: Sterilize through a 0.2 - 0.45μm filter membrane, and store frozen after sub - packaging.

[0033] In some realizable ways, the shelf life of the prepared liquid quality control product is ≥3 years, and the concentration fluctuation of CG is ≤±5% after 38 months of storage; the coefficient of variation (CV) of each analyte in the liquid quality control product is <3% within the bottle and <3% between bottles, meeting the long - term clinical quality control requirements. The present invention adds a mixed anti - interference agent (acetylcysteine + cyclodextrin). The two items of glycocholic acid and bile acid in the quality control product can be stably stored for 38 months, and can still be effectively detected with less interference, and the measured value fluctuation is within the allowable range.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Significantly improved stability: The mercapto group of acetylcysteine scavenges reactive oxygen species, chelates metal ions, and forms hydrophobic interactions and hydrogen bonds with CG to inhibit the oxidation, hydrolysis and conformational isomerization of CG. At the same time, cyclodextrin inclusion of the CG steroid nucleus further enhances the structural rigidity. After storing the quality control product at - 20°C for 38 months, the concentration fluctuation of CG is ≤±5%, the CV of each analyte is ≤3% within the bottle and ≤3% between bottles, and the shelf life is ≥3 years, breaking through the stability bottleneck of traditional liquid quality control products.

[0035] (2) Effective inhibition of cross - reaction: NAC stabilizes the conformation of CG, reducing the exposure of the common steroid ring structure with total bile acid. Cyclodextrin selectively includes the CG steroid nucleus and exposes the specific epitope LSTa of the glycine side chain, blocking the non - specific binding of the antibody to the common Si - Lea epitope, reducing the cross - reaction rate of CG and TBA from 15% - 20% of the traditional method to less than 5%, significantly improving the detection accuracy.

[0036] (3) Excellent matrix compatibility and homogeneity: The human serum matrix is used to simulate the protein composition and ionic environment of clinical samples, reducing non-specific binding caused by heterologous matrices. At the same time, there is no significant interaction between the mixed anti-interference agents (NAC and cyclodextrin mixed anti-interference agents) and other biochemical markers (such as HCY, sdLDL, etc.) in the quality control product, resulting in the within-bottle coefficient of variation (CV within-bottle) of each analyte ≤ 3% and the between-bottle coefficient of variation (CV between-bottles) ≤ 3%. The homogeneity is significantly better than that of traditional heterologous matrix quality control products.

[0037] (4) Improved operational convenience: The quality control product is in the form of a frozen liquid, eliminating the need for reconstitution steps, avoiding human errors introduced by reconstituting freeze-dried quality control products, significantly improving the detection efficiency, reducing laboratory labor and time costs, and being more suitable for clinical rapid detection requirements.

[0038] (5) Good multi-component synergistic stability: Through precise concentration optimization of the mixed anti-interference agents, while stabilizing CG, it does not affect the activity and structure of other analytes (such as enzymes, lipoproteins, etc.), achieving the simultaneous long-term stability of glycocholic acid, total bile acid, and more than 20 other biochemical markers, meeting the clinical multi-item joint quality control requirements and solving the problem of poor multi-component compatibility of traditional composite quality control products.

[0039] The present invention will be explained and described in detail below in conjunction with the accompanying drawings and specific embodiments. Brief Description of the Drawings

[0040] Figure 1 It is a result diagram of the stability experiment of analytes TBA and CG in the liquid quality control product at level 3 of the horizontal concentration in Example 1 of the present invention. The abscissa is the time span from 0 to 38 months, and the ordinate is the corresponding concentration of analytes TBA and CG. Detailed Embodiments

[0041] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant accompanying drawings. Several embodiments of the present invention are given in the drawings, but the present invention can be implemented in different forms and is not limited to the embodiments described in the text. On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0043] Example 1: The present invention provides a method for preparing a multi-biochemical liquid quality control product containing glycocholic acid, which includes the following steps: (1) Matrix preparation: Take negative human serum for infectious diseases, and successively add 50 mM HEPES buffer, 2% w / v of a mixed anti-interference agent, 4% w / v of trehalose, 2% w / v of antifreeze glycoprotein, and 1 mL / L of ProClin 300, and stir to dissolve. The mass ratio of acetylcysteine to cyclodextrin in the mixed anti-interference agent is 1:1.

[0044] (2) pH adjustment: Adjust the pH of the system to 7.5 with acid-base reagents; (3) Analyte addition: Add glycocholic acid, total bile acid, and other biochemical markers at different concentration levels set in Table 1, and stir until completely dissolved; (4) Filtration and sub-packaging: Sterilize through a 0.2 μm filter membrane, and freeze and store after sub-packaging.

[0045] Table 1 shows the analyte concentrations at level 3 Project Name Concentration Unit Level 3 TBA μmol / L 7.6 B2MG mg / L 8.23 CRP mg / L 120.3 mAST U / L 4 GR U / L 224.1 5'NT U / L 31.8 GA μmol / L 508 GSP mmol / L 3.74 D3H mmol / L 1.252 sdLDL mmol / L 0.84 LPa mg / L 434 APOE mg / dL 10.43 RBP mg / L 86.6 CysC mg / L 2.973 CK-MB U / L 62 HCY μmol / L 36.8 AFU U / L 17.2 CG μg / mL 2.26 ADA U / L 11.9 GLDH U / L 29.9 Perform an analytical performance evaluation experiment on the liquid quality control product at level concentration 3: Take 10 bottles of the liquid quality control product at level concentration 3, measure each bottle 3 times on a suitable immunoassay analyzer, and calculate the within-bottle coefficient of variation (CV within-bottle) and the between-bottle coefficient of variation (CV between-bottles).

[0046] Table 2 shows the analytical performance test results of quality control products at different concentration levels after adding 2% of the mixed anti-interference agent Level 3 TBA B2MG CRP mAST GR CV within vial 0.6% 1.4% 1.5% 1.5% 0.7% CV between vials 0.3% 0.9% 0.5% 0.8% 1.1% Project Name 5'NT GA GSP D3H sdLDL CV within vial 1.0% 0.9% 1.2% 1.0% 0.8% CV between vials 0.4% 0.9% 0.5% 1.1% 0.4% Project Name LPa APOE RBP CysC CK-MB CV within vial 1.1% 0.9% 1.5% 0.7% 0.8% CV between vials 0.9% 0.4% 0.8% 1.1% 1.2% Project Name HCY AFU CG ADA GLDH CV within vial 1.3% 0.7% 0.7% 0.8% 0.9% CV between vials 1.2% 0.8% 0.5% 1.4% 1.2%

[0047] It can be obtained from Tables 1 and 2 above that the CVs of the analytes in the quality control product at concentration level 3 in this example do not exceed 1.5%, indicating good homogeneity.

[0048] Store the multi-biochemical liquid quality control product with the analyte concentration at level 3 at -20 °C and below. Take out 2 bottles for testing at 0, 6, 12, 18, 24, 30, 36, and 38 months respectively, take the average value by measuring each bottle 3 times, and perform a significance test for differences using the t-test method, requiring that the differences in the test results are not significant.

[0049] Table 3 shows the stability evaluation results of the multi-biochemical liquid quality control product Level 3 Total Bile Acid - TBA β2-Microglobulin - B2MG C-Reactive Protein - CRP Mitochondrial Isoenzyme of Aspartate Aminotransferase - mAST Glutathione Reductase - GR Unit μmol / L mg / L mg / L U / L U / L Month 0 7.83 8.15 122.22 3.75 227.17 Month 6 7.85 8.13 122.02 3.73 226.08 Month 12 7.87 8.14 121.42 3.73 226.22 Month 18 7.8 8.13 122.03 3.75 225.58 Month 24 7.8 8.15 121.42 3.73 225.98 Month 30 7.83 8.14 121.87 3.75 225.88 Month 36 7.85 8.12 121.87 3.72 226.25 Month 38 7.88 8.1 122.23 3.72 225.68 Syx 0.0312 0.0135 0.3438 0.0123 0.4215 t0.05,n-2 2.4469 2.4469 2.4469 2.4469 2.4469 s(b1) 0.0008 0.0004 0.0093 0.0003 0.0114 Sum of Squared Deviations 1358 1358 1358 1358 1358 Square Root of Sum of Squared Deviations 36.85 36.85 36.85 36.85 36.85 b1 0.0004 0.0006 0.0008 0.0006 0.0212 t0.05,n-2 × s(b1) 0.0021 0.0009 0.0228 0.0008 0.028 Result Judgment |b1| < t0.05,n-2 × s(b1), the trend is not significant |b1| < t0.05,n-2 × s(b1), the trend is not significant |b1| < t0.05,n-2 × s(b1), the trend is not significant |b1| < t0.05,n-2 × s(b1), the trend is not significant |b1| < t0.05,n-2 × s(b1), the trend is not significant Level 3 5'-Nucleotidase - 5'NT Glycated Albumin - GA Glycated Serum Protein (Fructosamine) - GSP D-3-Hydroxybutyric Acid (β-Hydroxybutyric Acid) - D3H Small Dense Low Density Lipoprotein Cholesterol - sdLDL Unit U / L μmol / L mmol / L mmol / L mmol / L Month 0 32.68 510.17 3.7 1.3 0.86 Month 6 32.43 506.83 3.66 1.29 0.85 Month 12 32.43 505.17 3.67 1.29 0.85 Month 18 32.45 504.67 3.67 1.3 0.85 Month 24 32.55 503.17 3.66 1.28 0.85 Month 30 32.42 507.5 3.67 1.29 0.85 Month 36 32.38 505.67 3.66 1.29 0.85 Month 38 32.43 506.83 3.65 1.29 0.85 Syx 0.0844 2.1272 0.0121 0.0041 0.0027 t0.05,n-2 2.4469 2.4469 2.4469 2.4469 2.4469 s(b1) 0.0023 0.0577 0.0003 0.0001 0.0001 Sum of Squared Deviations 1358 1358 1358 1358 1358 Square Root of Sum of Squared Deviations 36.85 36.85 36.85 36.85 36.85 b1 0.0042 0.054 0.0006 0.0002 0 t0.05,n-2 × s(b1) 0.0056 0.1412 0.0008 0.0003 0.0002 Result Judgment |b1| < t0.05,n-2 × s(b1), the trend is not significant |b1| < t0.05,n-2 × s(b1), the trend is not significant |b1| < t0.05,n-2 × s(b1), the trend is not significant |b1| < t0.05,n-2 × s(b1), the trend is not significant |b1| < t0.05,n-2 × s(b1), the trend is not significant Level 3 Lipoprotein (a) - LPa Apolipoprotein E - APOE Retinol Binding Protein - RBP Cystatin C - CysC Creatine Kinase Isoenzyme - CK-MB Unit mg / L mg / dL mg / L mg / L U / L Month 0 457.83 10.71 87.3 3.11 66.22 The 6th month 452.17 10.57 86.23 3.05 65 The 12th month 451.83 10.59 85.6 3.05 64.8 The 18th month 452 10.54 85.85 3.06 65.15 The 24th month 451 10.53 86.28 3.06 65.25 The 30th month 452.33 10.56 85.8 3.07 64.77 The 36th month 452.33 10.59 86 3.04 65.25 The 38th month 453.17 10.58 85.6 3.04 65.13 Syx 2.0174 0.0497 0.464 0.0191 0.4424 t0.05,n-2 2.4469 2.4469 2.4469 2.4469 2.4469 s(b1) 0.0547 0.0013 0.0126 0.0005 0.012 Sum of squared deviations from the mean 1358 1358 1358 1358 1358 Square root of the sum of squared deviations from the mean 36.85 36.85 36.85 36.85 36.85 b1 0.0702 0.0019 0.0252 0.001 0.0138 t0.05,n-2×s(b1) 0.134 0.0033 0.0308 0.0013 0.0294 Result determination |b1|<t0.05,n-2×s(b1), the trend is not significant |b1|<t0.05,n-2×s(b1), the trend is not significant |b1|<t0.05,n-2×s(b1), the trend is not significant |b1|<t0.05,n-2×s(b1), the trend is not significant |b1|<t0.05,n-2×s(b1), the trend is not significant Level 3 Homocysteine - HCY α-L-Fucosidase - AFU Glycocholic acid - CG Adenosine deaminase - ADA Glutamate dehydrogenase - GLDH Unit μmol / L U / L μg / mL U / L U / L The 0th month 37.05 16.78 2.19 12.35 30.05 The 6th month 36.28 16.52 2.14 12.15 29.58 The 12th month 36.27 16.43 2.15 12.13 29.53 The 18th month 36.47 16.4 2.14 12.17 29.58 The 24th month 36.35 16.52 2.15 12.1 29.57 The 30th month 36.42 16.45 2.14 12.15 29.57 The 36th month 36.42 16.43 2.15 12.12 29.53 The 38th month 36.37 16.43 2.16 12.2 29.63 Syx 0.2421 0.101 0.0151 0.0736 0.1551 t0.05,n-2 2.4469 2.4469 2.4469 2.4469 2.4469 s(b1) 0.0066 0.0027 0.0004 0.002 0.0042 Sum of squared deviations from the mean 1358 1358 1358 1358 1358 Square root of the sum of squared deviations from the mean 36.85 36.85 36.85 36.85 36.85 b1 0.0081 0.0058 0.0005 0.0028 0.0068 t0.05,n-2×s(b1) 0.0161 0.0067 0.001 0.0049 0.0103 Result determination |b1|<t0.05,n-2×s(b1), the trend is not significant |b1|<t0.05,n-2×s(b1), the trend is not significant |b1|<t0.05,n-2×s(b1), the trend is not significant |b1|<t0.05,n-2×s(b1), the trend is not significant |b1|<t0.05,n-2×s(b1), the trend is not significant

[0050] As can be seen from Table 3 above, the CG concentration decreased from 2.19 μg / mL to 2.16 μg / mL, with a fluctuation ≤ 0.03 μg / mL. The difference was not significant by t-test. The concentration fluctuations of other markers (such as TBA, GR, etc.) at each time point were all < 3%. Trend analysis showed no statistical significance. Therefore, the mixed anti-interference agent in the embodiments of the present invention enables the quality control product to maintain high stability during long-term storage, and the shelf life can be up to three years, meeting the long-term clinical quality control requirements.

[0051] Example 2: The embodiments of the present invention provide a method for preparing a biochemical multi-item liquid quality control product containing glycocholic acid. The basic steps are the same as those in Example 1, except that in this example, the concentration of the mixed anti-interference agent is 1% (w / v), and the mass ratio of acetylcysteine to cyclodextrin in the mixed anti-interference agent is 0.1:1.

[0052] Perform an analytical performance evaluation experiment on the liquid quality control product of the analyte at level concentration 3 in Table 1: Take 10 bottles of the liquid quality control product at level concentration 3, measure each bottle 3 times on a suitable immunoanalyzer, and calculate the within-bottle coefficient of variation (CV within-bottle) and the between-bottle coefficient of variation (CV between-bottle).

[0053] Table 4 shows the analytical performance test results of quality control products at different concentration levels after adding 1% of the mixed anti-interference agent Level 3 TBA B2MG CRP mAST GR CV within the vial 0.6% 1.0% 0.4% 1.1% 0.5% CV between vials 1.2% 0.5% 0.5% 1.1% 1.2% Item name 5'NT GA GSP D3H sdLDL CV within the vial 1.1% 0.2% 0.5% 0.5% 0.3% CV between vials 0.8% 0.6% 1.0% 0.8% 0.1% Item name LPa APOE RBP CysC CK-MB CV within the vial 0.5% 1.1% 0.9% 0.8% 0.9% CV between vials 0.4% 0.3% 0.6% 0.2% 0.6% Item name HCY AFU CG ADA GLDH CV within the vial 0.2% 0.7% 1.1% 1.1% 0.9% CV between vials 0.2% 0.2% 0.3% 0.3% 0.6%

[0054] As can be obtained from Table 4 above, the CVs of the analytes in the quality control products at different concentration levels in this example do not exceed 1.5%, indicating good homogeneity.

[0055] Example 3: The embodiments of the present invention provide a method for preparing a biochemical multi-item liquid quality control product containing glycocholic acid. The basic steps are the same as those in Example 1, except that in this example, the concentration of the mixed anti-interference agent is 0.1% (w / v), and the mass ratio of acetylcysteine to cyclodextrin in the mixed anti-interference agent is 0.5:1.

[0056] Perform an analytical performance evaluation experiment on the liquid quality control product of the analyte at level concentration 3 in Table 1: Take 10 bottles of the liquid quality control product at level concentration 3, measure each bottle 3 times on a suitable immunoanalyzer, and calculate the within-bottle coefficient of variation (CV within-bottle) and the between-bottle coefficient of variation (CV between-bottle).

[0057] Table 5 shows the analytical performance test results of quality control products at different concentration levels after adding 0.1% of the mixed anti-interference agent Level 3 TBA B2MG CRP mAST GR CV within the vial 0.8% 0.2% 1.1% 0.8% 0.9% CV between vials 0.3% 0.1% 1.1% 0.2% 0.6% Item name 5'NT GA GSP D3H sdLDL CV within the vial 0.9% 1.0% 0.5% 0.1% 0.5% CV between vials 0.1% 0.4% 0.7% 0.9% 0.1% Item name LPa APOE RBP CysC CK-MB CV within the vial 0.3% 0.6% 0.5% 0.2% 1.0% CV between vials 0.8% 0.4% 1.1% 0.4% 1.1% Item name HCY AFU CG ADA GLDH CV within the vial 1.0% 0.2% 0.9% 0.9% 0.4% CV between vials 1.1% 0.2% 0.4% 0.6% 0.9% As can be seen from Table 5 above, the CVs of the QC analyte at different concentration levels in this example do not exceed 1.5%, indicating good homogeneity. Among them, the within-bottle CV of CG is 0.9% and the between-bottle CV is 0.4%, both meeting the homogeneity requirement (≤1.5%), but slightly higher than the CV value in Example 1; for other markers such as the within-bottle CV of TBA is 0.8% and the between-bottle CV of HCY is 1.1%, the homogeneity is close to the optimal concentration group, but the dispersion of some indicators is slightly higher.

[0058] Comparative Example 1: In this comparative example, no mixed anti-interference agent was added. The following substances were added to human serum, and stirred and dissolved repeatedly: Non-analyte: 50 mM HEPES buffer, 4% trehalose, 2% antifreeze glycoprotein, 0.5% Tween 20 and 1 mL / L ProClin 300, and the pH was adjusted to 7.5. Analyte: See specific level 3 in Table 1 Table 6 shows the effect of the absence of acetylcysteine on the homogeneity of the CG biochemical multi-item liquid quality control product Level 3 TBA B2MG CRP mAST GR CV within the vial 7.9% 1.7% 1.0% 1.2% 2.8% CV between vials 6.4% 2.6% 0.6% 1.0% 1.7% Item name 5'NT GA GSP D3H sdLDL CV within the vial 1.2% 2.6% 1.6% 2.6% 1.7% CV between vials 1.9% 1.9% 2.3% 1.9% 1.3% Item name LPa APOE RBP CysC CK-MB CV within the vial 2.4% 1.6% 2.1% 2.1% 2.1% CV between vials 2.3% 0.6% 0.7% 0.7% 1.7% Item name HCY AFU CG ADA GLDH CV within the vial 2.6% 2.2% 8.7% 1.8% 1.2% CV between vials 2.0% 3.0% 7.0% 2.5% 1.4% From the data in Table 6 above, it can be seen that the within-bottle CV of CG is 8.7% and the between-bottle CV is 7.0%, significantly higher than that in Example 1; for other markers such as the within-bottle CV of TBA is 7.9% and the between-bottle CV is 6.4%, the homogeneity is seriously insufficient. It shows that the absence of the mixed anti-interference agent leads to a significant decrease in the stability of CG and multi-components, verifying the necessity of NAC and cyclodextrin for inhibiting degradation.

[0059] Comparative Example 2: In this comparative example, 0.01% acetylcysteine was added. The following substances were added to human serum, and stirred and dissolved repeatedly: Non-analyte: 50 mM HEPES buffer, 4% trehalose, 4% antifreeze glycoprotein, 0.5% Tween 20 and 1 mL / L ProClin 300 and 0.01% acetylcysteine, and the pH was adjusted to 7.5. Analyte: See specific level 3 in Table 1.

[0060] Table 7 shows the effect of adding 0.01% acetylcysteine on the homogeneity of the CG biochemical multi-item liquid quality control product Level 3 TBA B2MG CRP mAST GR CV within the vial 4.8% 1.5% 1.3% 1.4% 1.6% CV between vials 3.5% 1.1% 1.5% 1.8% 2.0% Item name 5'NT GA GSP D3H sdLDL CV within the vial 1.8% 1.7% 1.6% 1.7% 1.5% CV between vials 1.1% 1.3% 1.8% 1.4% 1.6% Item name LPa APOE RBP CysC CK-MB CV within the vial 1.4% 1.1% 1.1% 1.0% 1.8% CV between vials 1.6% 1.6% 1.7% 1.6% 1.5% Item name HCY AFU CG ADA GLDH CV within the vial 1.2% 1.1% 5.2% 1.4% 1.8% CV between vials 1.2% 1.5% 4.3% 1.4% 1.4% As can be seen from Table 7 above, adding 0.01% acetylcysteine has a certain improvement effect on the homogeneity of the QC product containing CG, but the improvement effect on the homogeneity of the CG item in the QC product is very limited. The within-bottle CV of CG is 5.2% and the between-bottle CV is 4.3%. Although it is improved compared with Comparative Example 1, it does not meet the usage requirements. The antioxidant ability of low-concentration NAC is insufficient to effectively inhibit the multiple degradation paths of CG.

[0061] Comparative Example 3: In this comparative example, 10% of a mixed anti-interference agent was added. The mass ratio of acetylcysteine to cyclodextrin in the mixed anti-interference agent was 1:1. The following substances were added to human serum, and the mixture was stirred and dissolved repeatedly: Non-analyte: 50 mM HEPES buffer, 4% w / v trehalose, 4% w / v antifreeze glycoprotein, 0.5% w / v Tween 20, 1 mL / L ProClin 300, and 10% w / v mixed anti-interference agent. The pH was adjusted to 7.5. Analyte: See specifically Level 3 in Table 1.

[0062] Table 8 shows the effect of adding 10% of the mixed anti-interference agent on the homogeneity of the multi-item liquid quality control product containing CG biochemicals. Level 3 TBA B2MG CRP mAST GR CV within the vial 8.6% 0.8% 0.6% 0.8% 0.9% CV between vials 7.4% 0.9% 0.6% 1.1% 0.7% Item name 5'NT GA GSP D3H sdLDL Within the CV vial 1.1% 0.6% 0.9% 0.8% 1.1% Between CV vials 1.0% 1.1% 0.6% 0.8% 0.9% Project name LPa APOE RBP CysC CK-MB Within the CV vial 1.1% 0.5% 0.7% 0.9% 0.7% Between CV vials 0.9% 0.8% 1.1% 0.8% 0.8% Project name HCY AFU CG ADA GLDH Within the CV vial 0.5% 0.7% 0.6% 0.8% 0.9% Between CV vials 1.1% 0.9% 1.0% 0.5% 0.8% As can be seen from the above table, after the excessive addition of the mixed anti-interference agent, the cyclodextrin has too strong an inclusion effect on the steroid ring nucleus of bile acids, resulting in the inability of bile acids to be effectively recognized by the antibodies in the kit. The deviation rate of the CG test value > 10%. Due to the excessive embedding of the steroid nucleus by cyclodextrin, the antibody cannot recognize the LSTa epitope; lipoprotein analytes (such as sdLDL) aggregate, and CV between bottles = 1.1%, which is higher than the normal level. Therefore, the exposure of the CG conformation is damaged at too high a concentration, proving that the mixed anti-interference agent requires precise concentration optimization.

[0063] Comparative Example 4: In this comparative example, 2% EDTA was added. The following substances were added to human serum, and the mixture was stirred and dissolved repeatedly: Non-analyte: 50 mM HEPES buffer, 10% w / v trehalose, 5% w / v antifreeze glycoprotein, 0.5% w / v Tween 20, 1 mL / L ProClin 300, and 2% w / v EDTA. The pH was adjusted to 7.5. Analyte: See specifically Level 3 in Table 1.

[0064] Table 9 shows the effect of adding 2% EDTA on the homogeneity of the multi-item liquid quality control product containing CG biochemicals. Level 3 TBA B2MG CRP mAST GR Within the CV vial 2.5% 1.9% 2.1% 2.2% 2.6% Between CV vials 2.1% 2.5% 1.8% 1.6% 2.7% Project name 5'NT GA GSP D3H sdLDL Within the CV vial 1.7% 2.4% 1.7% 2.3% 2.3% Between CV vials 2.2% 2.2% 1.7% 1.7% 1.8% Project name LPa APOE RBP CysC CK-MB Within the CV vial 1.8% 1.8% 2.0% 2.3% 2.6% Between CV vials 2.3% 2.2% 2.3% 2.3% 2.1% Project name HCY AFU CG ADA GLDH Within the CV vial 2.4% 1.7% 2.2% 1.7% 2.0% Between CV vials 2.5% 2.4% 2.0% 1.8% 2.5% As can be seen from the above table, the effect of adding 2% EDTA on the homogeneity of the quality control product containing CG is worse than that of Example 1. The CV within the bottle of CG = 2.2%, and the CV between bottles = 2.0%, both of which are significantly higher than those in Example 1; the cross-reactivity is large. Since EDTA only chelates metal ions and cannot solve the problems of conformational isomerization and epitope exposure. It shows that a single-mechanism stabilizer (such as EDTA) cannot break through the bottleneck of the existing technology, and the multi-dimensional effect of the mixed anti-interference agent is irreplaceable.

[0065] Any numerical value recited herein includes all values from the lower value to the upper value incremented by one unit therebetween, provided that there is a separation of at least two units between any lower value and any higher value. For example, if a value for the number of components or a process variable (such as temperature, pressure, time, etc.) is stated to be from 1 to 90, preferably from 20 to 80, more preferably from 30 to 70, it is intended that values such as 15 to 85, 22 to 68, 43 to 51, 30 to 32, etc. are also expressly recited in this specification. For values less than 1, a unit is suitably considered to be 0.0001, 0.001, 0.01, 0.1. These are merely examples of what is intended to be specified, and all possible combinations of numerical values recited between the lowest value and the highest value are to be considered to be expressly stated in this specification in a similar manner.

[0066] Unless otherwise indicated, all ranges include the endpoints and all numbers therebetween. The term “about” or “approximate” when used in connection with a range is applicable to both endpoints of the range. Thus, “about 20 to 30” is intended to cover “about 20 to about 30”, including at least the specified endpoints.

[0067] It should be understood that the above description is for illustrative purposes and not for purposes of limitation. Many embodiments and many applications other than the examples provided will be apparent to those skilled in the art upon reading the above description. Accordingly, the scope of the present teachings should not be determined with reference to the above description, but should instead be determined with reference to the appended claims and the full scope of equivalents to which such claims are entitled. For the sake of completeness, all articles and references, including patent applications and publications, are incorporated herein by reference. The omission of any aspect of the subject matter disclosed herein in the foregoing claims is not a disclaimer of such subject matter, nor should it be assumed that the inventor did not consider such subject matter to be part of the disclosed inventive subject matter.

[0068] The above description of the present invention has been presented by way of example, and it is obvious that the specific implementation of the present invention is not limited by the above methods. As long as such non-substantial improvements are made using the method concept and technical solution of the present invention, or the concept and technical solution of the present invention are directly applied to other occasions without improvement, they are all within the protection scope of the present invention.

Claims

1. A biochemical multi-item liquid quality control product containing glycocholic acid, characterized in that, This liquid quality control product uses human serum as the matrix and contains the following components: Non-analytes, including buffer, mixed anti-interference agent, stabilizer, surfactant and preservative. The mixed anti-interference agent is a mixture of acetylcysteine and cyclodextrin, and the concentration of the mixed anti-interference agent in the quality control product is 0.1%-2% (w / v); Analytes, including glycocholic acid, total bile acid and other biochemical markers.

2. The biochemical multi-item liquid quality control product containing glycocholic acid according to claim 1, wherein The mass ratio of acetylcysteine to cyclodextrin is 0.1:1 - 1:1; And / or, the cyclodextrin is selected from one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin.

3. The liquid quality control product according to claim 1, wherein The stabilizer includes: Carbohydrate stabilizer, and the carbohydrate stabilizer is selected from at least one of sucrose, trehalose and mannitol; Protein stabilizer, and the protein stabilizer is selected from at least one of casein, ovalbumin and antifreeze glycoprotein.

4. The biochemical multi-item liquid quality control product containing glycocholic acid according to claim 3, characterized in that, The concentration of the carbohydrate stabilizer in the quality control product is 0.1%-4% (w / v); And / or, the concentration of the protein stabilizer in the quality control product is 0.1%-2% (w / v).

5. The biochemical multi-item liquid quality control product containing glycocholic acid according to claim 1, wherein The buffer is selected from at least one of HEPES buffer, MES buffer, PIPES buffer, Tris-HCl buffer; And / or, the surfactant is selected from at least one of Tween 20 and Triton X-100; And / or, the preservative is selected from at least one of sodium azide, Proclin 300, Proclin 950, gentamicin; And / or, the pH value range of the liquid quality control product is 6.0 - 8.

0.

6. The biochemical multi - item liquid quality control product containing glycocholic acid according to claim 1, wherein, The buffer is HEPES buffer, with a concentration of 20 - 100 mM and a pH of 6.0 - 8.

0.

7. The biochemical multi-item liquid quality control product containing glycocholic acid according to claim 1, characterized in that, The concentration of the HEPES buffer is 50 mM and its pH is 7.5; And / or, the carbohydrate stabilizer is trehalose; And / or, the protein stabilizer is antifreeze glycoprotein; And / or, the surfactant is Tween 20; And / or, the preservative is Proclin 300.

8. The biochemical multi - item liquid quality control product containing glycocholic acid according to claim 1, characterized in that, The other biochemical markers include at least one of homocysteine, β-hydroxybutyric acid, glutamate dehydrogenase, β2-microglobulin, C-reactive protein, aspartate aminotransferase, glutathione reductase, 5'-nucleotidase, glycated albumin, glycated serum protein, small and dense low-density lipoprotein cholesterol, lipoprotein (a), apolipoprotein E, retinol-binding protein, cystatin C, creatine kinase isoenzyme, α-fucosidase and adenosine deaminase.

9. A method for preparing a biochemical multi - item liquid quality control product containing glycocholic acid, which is used to prepare the liquid quality control product according to any one of claims 1 - 8, characterized in that, The preparation method includes the following steps: (1) Matrix preparation: Take human serum negative for infectious diseases, and sequentially add buffer, acetylcysteine, stabilizer and preservative, and stir to dissolve; (2) pH adjustment: Adjust the pH of the system to 6.0 - 8.0 with acid-base reagents; (3) Analyte addition: Add glycocholic acid, total bile acid and other biochemical markers according to the set concentration, and stir until completely dissolved; (4) Filtration and sub-packaging: Sterilize through a 0.2 - 0.45 μm filter membrane, and freeze and store after sub-packaging.

10. The preparation method of the biochemical multi-item liquid quality control product containing glycocholic acid according to claim 9, wherein, The shelf life of the prepared liquid quality control product is ≥3 years, and the concentration fluctuation of CG after 38 months of storage is ≤±5%; And / or, the coefficient of variation (CV) of each analyte in the liquid quality control product is ≤3% within a vial and ≤3% between vials, meeting the long-term clinical quality control requirements.

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