A biochemical multi-item liquid quality control product containing glycocholic acid and its preparation method

Abstract

The present invention belongs to the technical field of biochemical multi-item liquid quality control products containing glycocholic acid, and specifically relates to a biochemical multi-item liquid quality control product containing glycocholic acid and a preparation method thereof. The liquid quality control product uses human serum as a matrix and contains the following components: non-analytes, including 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); analytes, including glycocholic acid, total bile acid, and other biochemical markers. In response to the shortcomings of the prior art, the present invention uses human serum as a matrix and solves the problems of glycocholic acid being easily oxidized and degraded, cross-reacting with total bile acid, and insufficient stability of the liquid quality control product by adding a mixed anti-interference agent of acetylcysteine ​​and cyclodextrin, thereby realizing a method for long-term stable and accurate detection of liquid quality control products containing CG and multiple biochemical markers.

Description

Technical Field

[0001] The present invention mainly relates to the technical field of biochemical multi-item liquid quality control products containing glycocholic acid, and specifically relates to a biochemical multi-item 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 amide bond between bile acid and glycine. After metabolism by hepatocytes, CG is approximately 95% reabsorbed through the enterohepatic circulation. Under normal circumstances, its peripheral blood concentration is very low, approximately 1.3±0.8 mg / L. CG is considered an important biomarker for hepatobiliary diseases: serum CG levels in patients with primary liver cancer can exceed 20 times the normal value and are more sensitive than alpha-fetoprotein; in patients with cirrhosis, elevated CG levels are closely associated with pathological progression; in hepatitis, CG can reflect hepatocellular damage earlier, with higher sensitivity and specificity than traditional markers such as ALT / AST. In patients with intrahepatic cholestasis of pregnancy (ICP), serum CG levels can increase 10- to 100-fold and are directly associated with the risk of fetal distress and preterm birth. Therefore, it has been clinically adopted as a core indicator for ICP screening. In addition, CG also has significant value in the early warning of alcoholic liver damage and the diagnosis of biliary obstruction (such as cholelithiasis and jaundice). The CG level in patients with obstructive liver disease can increase 10 to 20 times.

[0003] The stability of CG is influenced by its molecular structural properties: the three hydroxyl groups of its bile acid core are easily oxidized to keto or carboxylic acid groups; the amide bond is susceptible to hydrolysis in acidic or alkaline environments or high humidity; and the carboxylic acid group binds to metal ions and catalyzes its degradation. Furthermore, the amphiphilic nature of CG can trigger micellar aggregation, while heterologous matrices (such as bovine serum) can accelerate its degradation. Repeated freeze-thaw cycles or storage at high humidity can damage its structure, while photooxidation and hydrolysis continue to occur during long-term storage.

[0004] Current technologies have improved stability to some extent through the use of complex stabilizers (such as sugars or chelating agents) and storage at low temperatures and in the dark. However, batch variability and multi-component compatibility still require further optimization. Furthermore, when CG is added to a composite biochemical quality control product containing bile acids as a test item, cross-reactions may occur due to the structural similarities between the two (such as the shared bile acid core), resulting in a falsely elevated CG test value.

[0005] The aforementioned stability challenges and interference from total bile acids further exacerbate the difficulty in developing complex biochemical quality control products containing CG. At the same time, current CG-containing quality control products on the market still face multiple technical shortcomings: First, structural analogs such as total bile acids interfere with CG detection, resulting in CG detection values ​​that are too high to meet the established target range, making it difficult to meet clinical needs. Second, CG's unique amphiphilic structure places stringent requirements on the matrix of the quality control product. Heterogeneous matrices not only accelerate CG degradation, but also introduce other substances that easily induce matrix effects such as nonspecific binding or enzyme inhibition, resulting in significant deviations from clinical sample detection and affecting the reliability of laboratory quality control.

[0006] Therefore, there is an urgent need to develop liquid composite biochemical quality control products with little interference to CG detection, using standardized human serum matrix and having good batch-to-batch stability. This will fill the shortcomings of existing products in 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] In response to the shortcomings of the existing technology, one of the objectives of the present invention is to provide a biochemical multi-point liquid quality control product containing glycocholic acid and a preparation method thereof. Using human serum as the matrix, by adding a mixed anti-interference agent of acetylcysteine ​​and cyclodextrin, the problems of easy oxidative degradation of glycocholic acid (CG), cross-reaction with total bile acid (TBA), and insufficient stability of the liquid quality control product are solved, thereby realizing a method for long-term stable and accurate detection of liquid quality control products containing CG and multiple biochemical markers.

[0008] The present invention is the first to address the matrix effect interference problem of biochemical multi-item liquid quality control products containing glycocholic acid and total bile acid, especially the problem of stability of glycocholic acid and interference detection of structural analogs, and provides the following ideas: stabilize the structure of glycine, enhance its specific epitope exposure, and mask its shared steroid ring structure. The core of the present invention is to provide a specific anti-interference agent in this specific quality control product composition. By mixing acetylcysteine ​​and cyclodextrin as anti-interference agents, the anti-interference ability of biochemical multi-item liquid quality control products containing glycocholic acid and total bile acid is significantly improved, and the uniformity effect is significantly improved. Especially when bile acid and glycocholic acid are mixed for detection, the CG structure can remain stable and can be efficiently detected to avoid cross-reactions caused by bile acid and glycocholic acid having the same steroid ring structure during detection, resulting in false positive or false negative results.

[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, when it comes to the oxidative degradation and cross-reactivity of small molecules such as bile acids, traditional stabilizers have obvious limitations:

[0010] Those skilled in the art typically prefer EDTA as a metal ion chelator or vitamin C / E as a free radical scavenger. However, EDTA only inhibits metal-catalyzed oxidation and cannot resolve the conformational isomerization of CG. Vitamin C / E itself is easily oxidized and ineffective in liquid environments, and may even react chemically with CG.

[0011] Although NAC is widely used as an expectorant or detoxifier in the medical field, and the antioxidant effect of its sulfhydryl group (-SH) is well known, its application in biochemical quality control products has the following technical biases:

[0012] 1. High concentrations of NAC (>5% w / v) may damage the structures of other protein analytes (such as enzymes and antibodies) in quality control products through sulfhydryl-disulfide bond exchange reactions. Those skilled in the art generally avoid using it in multi-parameter quality control products.

[0013] 2. The amphiphilic structure of NAC (acetyl + mercaptopropionic acid) may change the surface tension of the human serum matrix, causing aggregation or precipitation of lipoprotein analytes (such as sdLDL), affecting the uniformity of test results.

[0014] 3. Regarding the structural similarity between CG and TBA, existing technologies mainly address it through antibody modification (such as screening for highly specific monoclonal antibodies) or chemical blockers (such as adding excess structural analogs to compete for binding sites), but these methods are costly and may introduce new interfering substances.

[0015] Therefore, those skilled in the art have not yet realized that cross-reactivity rates can be indirectly reduced by stabilizing the molecular conformation of CG rather than directly blocking antibody binding. To address this technical problem, the core of the present invention is the introduction of a mixed anti-interference agent, 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 its sulfhydryl group. Furthermore, through hydrophobic interactions between the acetyl group and the CG steroid nucleus and hydrogen bonding between the carboxylic acid group and the CG hydroxyl group, it inhibits oxidative degradation, metal-catalyzed damage, and conformational isomerization of CG, thereby stabilizing its molecular structure. On the other hand, cyclodextrin specifically encompasses the steroid nucleus of CG through its hydrophobic cavity, masking the Si-Lea epitope shared with total bile acids while exposing the glycine side chain-specific epitope of CG, enhancing specific binding with antibodies during detection and thus reducing cross-reactivity. The combined effect of these two agents ensures that CG maintains structural stability in the liquid quality control product while enabling accurate detection, synergistically resolving the challenges of CG stability and detection interference.

[0016] Specifically, the technical solutions adopted by the present invention are as follows:

[0017] In a first aspect, the present invention provides a biochemical multi-item liquid quality control product containing glycocholic acid, wherein the liquid quality control product is based on human serum and contains the following components:

[0018] Non-analytes include a buffer, a mixed anti-interference agent, a stabilizer, a surfactant, and a preservative, wherein 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);

[0019] Analytes include glycocholic acid, total bile acid and other biochemical markers.

[0020] In the present invention, human serum is used as a matrix to simulate the protein composition (such as albumin, globulin) and ionic environment (pH, salt concentration) of clinical samples, reducing nonspecific binding or enzyme activity inhibition caused by heterologous matrices (such as bovine serum).

[0021] In the present invention, the addition of acetylcysteine ​​to the quality control products containing glycocholic acid and total bile acid can significantly improve their stability, mainly through the following multi-dimensional mechanism of action:

[0022] (1) Eliminate reactive oxygen species: The sulfhydryl group (-SH) of acetylcysteine ​​can directly neutralize oxidative substances such as hydroxyl radicals and hydrogen peroxide, blocking the oxidative cleavage of the glycocholic acid nucleus (such as 7α-hydroxyl, C-24 side chain).

[0023] (2) Inhibition of Fenton reaction: Acetylcysteine ​​and Fe 2+ 、Cu 2+ It can form stable chelates with transition metal ions, blocking the hydrogen bonds produced by metal catalysis and reducing the rate of iron-mediated glycocholic acid oxidation.

[0024] (3) Hydrophobic interaction and hydrogen bond formation: The acetyl group of acetylcysteine ​​binds to the glycocholic acid nucleus through van der Waals forces, reducing conformational isomerization caused by molecular thermal motion. At the same time, the carboxylic acid group forms a hydrogen bond with the C-12 hydroxyl group, enhancing the rigidity of the steroid skeleton.

[0025] In summary, acetylcysteine ​​effectively overcomes key stability issues such as oxidative degradation and conformational changes of glycocholic acid in quality control products through synergistic effects such as anti-oxidation, molecular stabilization, and environmental regulation, providing technical support for the precise quality control of bile acid biomarkers.

[0026] The present invention adds cyclodextrin to form a "stable-anti-interference complex" on the basis of adding acetylcysteine. Acetylcysteine ​​is responsible for maintaining the stability of the structure of glycocholic acid, and cyclodextrin is responsible for coating the steroid nucleus of glycocholic acid and exposing the glycine side chain of glycocholic acid, thereby enhancing its specific binding with the antibody in the test kit during detection and reducing the cross-reaction of bile acids.

[0027] In some achievable embodiments, the mass ratio of acetylcysteine ​​to cyclodextrin is 0.1:1-1:1.

[0028] In some achievable embodiments, the cyclodextrin is selected from one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. Preferably, the cyclodextrin is β-cyclodextrin, which includes the CG steroid nucleus via a hydrophobic cavity, masking the Si-Lea epitope and exposing the LSTa epitope on the glycine side chain, thereby reducing cross-reactivity with TBA.

[0029] In some embodiments, the stabilizer includes:

[0030] A carbohydrate stabilizer, selected from at least one of sucrose, trehalose and mannitol, preferably trehalose, which inhibits water loss and ice crystal damage through a hydroxyl hydrogen bond network; its concentration in the quality control product is 0.1%-4% (w / v).

[0031] The protein stabilizer is 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).

[0032] In some achievable embodiments, the buffer is selected from at least one of HEPES buffer, MES buffer, PIPES buffer, and Tris-HCl buffer, preferably HEPES buffer, and has a concentration of 20-100 mM and a pH of 6.0-8.0.

[0033] And / or, the surfactant is selected from at least one of Tween 20 and Triton 100, preferably Tween 20.

[0034] And / or, the preservative is at least one selected from sodium azide, Proclin 300, Proclin 950, and gentamicin, preferably Proclin 300.

[0035] And / or, the pH value of the liquid quality control product is in the range of 6.0-8.0.

[0036] In some feasible embodiments, 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 the enzyme analyte, ensuring the compatibility of multi-project detection.

[0037] In some achievable embodiments, other biochemical markers include at least one of homocysteine, β-hydroxybutyrate, glutamate dehydrogenase, β2-microglobulin, C-reactive protein, aspartate aminotransferase, glutathione reductase, 5'-nucleotidase, glycated albumin, glycated serum protein, small dense low-density lipoprotein cholesterol, lipoprotein (a), apolipoprotein E, retinol-binding protein, cystatin C, creatine kinase isoenzyme, α-fucosidase and adenosine deaminase to meet the requirements of multi-project joint quality control.

[0038] When glycocholic acid is co-tested with total bile acid, acetylcysteine ​​stabilizes the conformation of glycocholic acid, while cyclodextrin coats the steroid nucleus of glycocholic acid and exposes the glycine side chain of glycocholic acid, thereby enhancing its specific binding to the antibody in the test kit and reducing the cross-reaction of bile acids.

[0039] In a second aspect, the present invention further provides a method for preparing a biochemical multi-item liquid quality control product containing glycocholic acid, which is used to prepare the above-mentioned liquid quality control product, and the preparation method comprises the following steps:

[0040] (1) Matrix preparation: Take infectious disease negative human serum, add buffer, acetylcysteine ​​and cyclodextrin mixed anti-interference agent, stabilizer and preservative in sequence, and stir to dissolve; add acetylcysteine ​​to the serum matrix first to ensure that it preferentially binds to the glycocholic acid molecules to form a stable complex.

[0041] (2) pH adjustment: Use acid and base reagents to adjust the system pH to 6.0-8.0;

[0042] (3) Analyte addition: Add glycocholic acid, total bile acid and other biochemical markers according to the set concentration and stir until completely dissolved;

[0043] (4) Filtration and packaging: Sterilize through a 0.2-0.45 μm filter membrane, and store frozen after packaging.

[0044] In some achievable approaches, the prepared liquid quality control product has a shelf life of ≥3 years, with CG concentration fluctuation ≤±5% after 38 months of storage. The 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. By adding a mixed anti-interference agent (acetylcysteine ​​+ cyclodextrin), the glycocholic acid and bile acid components in the quality control product can be stably stored for 38 months and still be effectively detected, with minimal interference and measured value fluctuations within the allowable range.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) Significantly improved stability: The sulfhydryl group of acetylcysteine ​​scavenges reactive oxygen species, chelates metal ions, and forms hydrophobic interactions and hydrogen bonds with CG, thereby inhibiting the oxidation, hydrolysis, and conformational isomerization of CG. At the same time, cyclodextrin inclusion of the CG steroid nucleus further enhances the structural rigidity, so that after the quality control product is stored at -20°C for 38 months, the CG concentration fluctuation 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.

[0047] (2) Effective inhibition of cross-reaction: NAC stabilizes the conformation of CG and reduces the exposure of the steroid ring structure shared by it and total bile acid. Cyclodextrin selectively encapsulates the steroid nucleus of CG and exposes the glycine side chain specific epitope LSTa, blocking the nonspecific binding of the antibody to the shared Si-Lea epitope, reducing the cross-reaction rate between CG and TBA from 15%-20% in traditional methods to below 5%, significantly improving the detection accuracy.

[0048] (3) Excellent matrix compatibility and homogeneity: Human serum matrix is ​​used to simulate the protein composition and ion environment of clinical samples, reducing nonspecific binding caused by heterogeneous matrices. At the same time, the mixed anti-interference agent (NAC and cyclodextrin mixed anti-interference agent) has no significant interaction with other biochemical markers in the quality control product (such as HCY, sdLDL, etc.), making the intra-bottle coefficient of variation (CV intra-bottle) of each analyte ≤3%, and the inter-bottle coefficient of variation (CV inter-bottle) ≤3%. The homogeneity is significantly better than that of traditional heterogeneous matrix quality control products.

[0049] (4) Improved operational convenience: The quality control product is in the form of frozen liquid and does not require a reconstitution step, thus avoiding the human error introduced by the reconstitution of freeze-dried quality control products. The detection efficiency is significantly improved, while reducing the laboratory manpower and time costs, making it more suitable for clinical rapid detection needs.

[0050] (5) Excellent synergistic stability of multiple components: The mixed anti-interference agent is optimized through precise concentration. 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 biochemical markers, meeting the needs of clinical multi-project joint quality control, and solving the problem of poor compatibility of multiple components of traditional composite quality control products.

[0051] The present invention will be explained in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a graphical representation of the results of the stability experiment of the analytes TBA and CG in the liquid quality control product at level 3 in Example 1 of the present invention. The horizontal axis represents the time span of 0-38 months, and the vertical axis represents the corresponding concentrations of the analytes TBA and CG. DETAILED DESCRIPTION

[0053] To facilitate understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, 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 content disclosed in the present invention more thorough and comprehensive.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used by those skilled in the art to which the present invention pertains. The terminology used in the specification of the present invention is for the purpose of describing specific embodiments and is 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.

[0055] Example 1: The present invention provides a method for preparing a biochemical multi-item liquid quality control product containing glycocholic acid, comprising the following steps:

[0056] (1) Matrix preparation: Take infectious disease negative human serum, add 50mM HEPES buffer, 2% w / v mixed anti-interference agent, 4% w / v trehalose, 2% w / v antifreeze glycoprotein and 1mL / L ProClin300 in sequence, and stir to dissolve. The mass ratio of acetylcysteine ​​to cyclodextrin in the mixed anti-interference agent is 1:1.

[0057] (2) pH adjustment: Use acid and base reagents to adjust the system pH to 7.5;

[0058] (3) Analyte addition: Add glycocholic acid, total bile acid, and other biochemical markers at different concentrations as set in Table 1 and stir until completely dissolved;

[0059] (4) Filtration and packaging: Sterilize through a 0.2 μm filter membrane, and store frozen after packaging.

[0060] Table 1 shows the analyte concentrations at level 3

[0061] 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

[0062] Perform an analytical performance evaluation experiment on a liquid control at level 3: measure each of 10 vials of the liquid control at level 3 three times on an appropriate immunoassay analyzer, and calculate the intra-vial coefficient of variation (CV intra-vial) and the inter-vial coefficient of variation (CV inter-vial).

[0063] Table 2 shows the analytical performance test results of the quality control products at different concentration levels after adding 2% mixed anti-interference agent

[0064] Level 3 TBA B2MG CRP mAST GR CV bottle 0.6% 1.4% 1.5% 1.5% 0.7% CV Bottle Room 0.3% 0.9% 0.5% 0.8% 1.1% Project Name 5'NT GA GSP D3H sdLDL CV bottle 1.0% 0.9% 1.2% 1.0% 0.8% CV Bottle Room 0.4% 0.9% 0.5% 1.1% 0.4% Project Name LPa APOE RBP CysC CK-MB CV bottle 1.1% 0.9% 1.5% 0.7% 0.8% CV Bottle Room 0.9% 0.4% 0.8% 1.1% 1.2% Project Name HCY AFU CG ADA GLDH CV bottle 1.3% 0.7% 0.7% 0.8% 0.9% CV Bottle Room 1.2% 0.8% 0.5% 1.4% 1.2%

[0065] As shown in Tables 1 and 2 above, the CVs of the analytes of the quality control products at concentration level 3 in this example were all less than 1.5%, indicating good homogeneity.

[0066] The biochemical multi-item liquid quality control product with the analyte concentration at level 3 was stored at -20°C or below, and two bottles were taken out for testing at 0, 6, 12, 18, 24, 30, 36, and 38 months, respectively. Each bottle was tested 3 times and the average value was taken. The t-test method was used to test the significance of the difference, and the difference in the test results was required to be insignificant.

[0067] Table 3 Stability evaluation results of multiple biochemical liquid quality control products

[0068] 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 6th month 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 30th month 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 differences 1358 1358 1358 1358 1358 square root of the sum of squared differences 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 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 5'-nucleotidase - 5'NT Glycated albumin - GA Glycated serum protein (fructosamine) - GSP D-3 Hydroxybutyrate (β-Hydroxybutyrate) – 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 6th month 32.43 506.83 3.66 1.29 0.85 12th month 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 30th month 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 differences 1358 1358 1358 1358 1358 square root of the sum of squared differences 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 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 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 6th month 452.17 10.57 86.23 3.05 65 12th month 451.83 10.59 85.6 3.05 64.8 Month 18 452 10.54 85.85 3.06 65.15 Month 24 451 10.53 86.28 3.06 65.25 30th month 452.33 10.56 85.8 3.07 64.77 Month 36 452.33 10.59 86 3.04 65.25 Month 38 453.17 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 differences 1358 1358 1358 1358 1358 square root of the sum of squared differences 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 Month 0 37.05 16.78 2.19 12.35 30.05 6th month 36.28 16.52 2.14 12.15 29.58 12th month 36.27 16.43 2.15 12.13 29.53 Month 18 36.47 16.4 2.14 12.17 29.58 Month 24 36.35 16.52 2.15 12.1 29.57 30th month 36.42 16.45 2.14 12.15 29.57 Month 36 36.42 16.43 2.15 12.12 29.53 Month 38 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 differences 1358 1358 1358 1358 1358 square root of the sum of squared differences 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

[0069] As shown in Table 3, the CG concentration decreased from 2.19 μg / mL to 2.16 μg / mL, with a fluctuation of ≤0.03 μg / mL, and the difference was not significant by t-test. The concentration fluctuations of other markers (such as TBA and GR) at each time point were all less than 3%, and trend analysis showed no statistical significance. Therefore, the mixed anti-interference agent in the examples of the present invention ensures that the quality control product maintains high stability during long-term storage, with a shelf life of up to three years, meeting the long-term quality control needs of clinical practice.

[0070] Example 2: This embodiment of the present invention provides a method for preparing a biochemical multi-item liquid quality control product containing glycocholic acid. The basic steps are consistent with those of Example 1, except that the concentration of the mixed anti-interference agent in this embodiment is 1% (w / v), and the mass ratio of acetylcysteine ​​and cyclodextrin in the mixed anti-interference agent is 0.1:1.

[0071] The analytical performance evaluation experiment was conducted on the liquid quality control products of the analytes at level 3 in Table 1: 10 bottles of liquid quality control product at level 3 were measured three times per bottle on an applicable immunoassay analyzer. The intra-bottle coefficient of variation (CV intra-bottle) and the inter-bottle coefficient of variation (CV inter-bottle) were calculated.

[0072] Table 4 shows the analytical performance test results of the quality control products at different concentration levels after adding 1% mixed anti-interference agent

[0073] Level 3 TBA B2MG CRP mAST GR CV bottle 0.6% 1.0% 0.4% 1.1% 0.5% CV Bottle Room 1.2% 0.5% 0.5% 1.1% 1.2% Project Name 5'NT GA GSP D3H sdLDL CV bottle 1.1% 0.2% 0.5% 0.5% 0.3% CV Bottle Room 0.8% 0.6% 1.0% 0.8% 0.1% Project Name LPa APOE RBP CysC CK-MB CV bottle 0.5% 1.1% 0.9% 0.8% 0.9% CV Bottle Room 0.4% 0.3% 0.6% 0.2% 0.6% Project Name HCY AFU CG ADA GLDH CV bottle 0.2% 0.7% 1.1% 1.1% 0.9% CV Bottle Room 0.2% 0.2% 0.3% 0.3% 0.6%

[0074] As shown in Table 4 above, the CVs of the quality control analytes at different concentration levels in this example were all less than 1.5%, indicating good uniformity.

[0075] Example 3: This embodiment of the present invention provides a method for preparing a biochemical multi-item liquid quality control product containing glycocholic acid. The basic steps are consistent with those of Example 1, except that the concentration of the mixed anti-interference agent in this embodiment is 0.1% (w / v), and the mass ratio of acetylcysteine ​​and cyclodextrin in the mixed anti-interference agent is 0.5:1.

[0076] The analytical performance evaluation experiment was conducted on the liquid quality control products of the analytes at level 3 in Table 1: 10 bottles of liquid quality control product at level 3 were measured three times per bottle on an applicable immunoassay analyzer. The intra-bottle coefficient of variation (CV intra-bottle) and the inter-bottle coefficient of variation (CV inter-bottle) were calculated.

[0077] Table 5 shows the analytical performance test results of the quality control products at different concentration levels after adding 0.1% mixed anti-interference agent

[0078] Level 3 TBA B2MG CRP mAST GR CV bottle 0.8% 0.2% 1.1% 0.8% 0.9% CV Bottle Room 0.3% 0.1% 1.1% 0.2% 0.6% Project Name 5'NT GA GSP D3H sdLDL CV bottle 0.9% 1.0% 0.5% 0.1% 0.5% CV Bottle Room 0.1% 0.4% 0.7% 0.9% 0.1% Project Name LPa APOE RBP CysC CK-MB CV bottle 0.3% 0.6% 0.5% 0.2% 1.0% CV Bottle Room 0.8% 0.4% 1.1% 0.4% 1.1% Project Name HCY AFU CG ADA GLDH CV bottle 1.0% 0.2% 0.9% 0.9% 0.4% CV Bottle Room 1.1% 0.2% 0.4% 0.6% 0.9%

[0079] As shown in Table 5, the CVs for the quality control analytes at different concentration levels in this example all did not exceed 1.5%, demonstrating good homogeneity. The intra-vial CV for CG was 0.9% and the inter-vial CV was 0.4%, both meeting the homogeneity requirement (≤1.5%), but slightly higher than the CV values ​​in Example 1. For other markers, such as TBA (intra-vial CV = 0.8%) and HCY (inter-vial CV = 1.1%), the homogeneity approached that of the optimal concentration group, although some indicators showed slightly higher dispersion.

[0080] Comparative Example 1: In this comparative example, no mixed anti-interference agent was added. The following substances were added to human serum, repeatedly stirred and dissolved:

[0081] Non-analyte: 50 mM HEPES buffer, 4% trehalose, 2% antifreeze glycoprotein, 0.5% Tween 20 and 1 mL / L ProClin 300, adjusted to pH 7.5. Analytes: See Table 1 Level 3 for details.

[0082] Table 6 shows the effect of not adding acetylcysteine ​​on the homogeneity of CG biochemical multi-product liquid quality control products.

[0083] Level 3 TBA B2MG CRP mAST GR CV bottle 7.9% 1.7% 1.0% 1.2% 2.8% CV Bottle Room 6.4% 2.6% 0.6% 1.0% 1.7% Project Name 5'NT GA GSP D3H sdLDL CV bottle 1.2% 2.6% 1.6% 2.6% 1.7% CV Bottle Room 1.9% 1.9% 2.3% 1.9% 1.3% Project Name LPa APOE RBP CysC CK-MB CV bottle 2.4% 1.6% 2.1% 2.1% 2.1% CV Bottle Room 2.3% 0.6% 0.7% 0.7% 1.7% Project Name HCY AFU CG ADA GLDH CV bottle 2.6% 2.2% 8.7% 1.8% 1.2% CV Bottle Room 2.0% 3.0% 7.0% 2.5% 1.4%

[0084] As shown in Table 6, the CV within the vial for CG was 8.7% and the CV between vials was 7.0%, significantly higher than those in Example 1. Other markers, such as TBA, had a CV within the vial of 7.9% and a CV between vials of 6.4%, demonstrating significant lack of uniformity. This indicates that the lack of a mixed anti-interference agent significantly degrades the stability of CG and its multiple components, confirming the necessity of NAC and cyclodextrin for degradation inhibition.

[0085] Comparative Example 2: In this comparative example, 0.01% acetylcysteine ​​was added to human serum, and the following substances were added, repeatedly stirred, and dissolved:

[0086] Non-analyte: 50 mM HEPES buffer, 4% trehalose, 4% antifreeze glycoprotein, 0.5% Tween 20, 1 mL / L ProClin 300, and 0.01% acetylcysteine, adjusted to pH 7.5. Analytes: See Table 1, Level 3 for details.

[0087] Table 7 shows the effect of adding 0.01% acetylcysteine ​​on the homogeneity of CG biochemical multi-product liquid quality control products.

[0088] Level 3 TBA B2MG CRP mAST GR CV bottle 4.8% 1.5% 1.3% 1.4% 1.6% CV Bottle Room 3.5% 1.1% 1.5% 1.8% 2.0% Project Name 5'NT GA GSP D3H sdLDL CV bottle 1.8% 1.7% 1.6% 1.7% 1.5% CV Bottle Room 1.1% 1.3% 1.8% 1.4% 1.6% Project Name LPa APOE RBP CysC CK-MB CV bottle 1.4% 1.1% 1.1% 1.0% 1.8% CV Bottle Room 1.6% 1.6% 1.7% 1.6% 1.5% Project Name HCY AFU CG ADA GLDH CV bottle 1.2% 1.1% 5.2% 1.4% 1.8% CV Bottle Room 1.2% 1.5% 4.3% 1.4% 1.4%

[0089] As shown in Table 7, while the addition of 0.01% acetylcysteine ​​has a modest effect on the uniformity of the CG-containing control sample, its effect on the uniformity of the CG component within the control sample is very limited. The CV within the bottle for CG is 5.2%, and the CV between bottles is 4.3%. While this is an improvement over Comparative Example 1, it still falls short of acceptable performance. Low-concentration NAC has insufficient antioxidant capacity and cannot effectively inhibit the multiple degradation pathways of CG.

[0090] Comparative Example 3: In this comparative example, 10% mixed anti-interference agent was added, wherein the mass ratio of acetylcysteine ​​to cyclodextrin in the mixed anti-interference agent was 1:1. The following substances were added to human serum, repeatedly stirred and dissolved:

[0091] 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, adjusted to pH 7.5. Analytes: See Table 1, Level 3 for details.

[0092] Table 8 shows the effect of adding 10% mixed anti-interference agent on the homogeneity of CG biochemical multi-product liquid quality control products

[0093] Level 3 TBA B2MG CRP mAST GR CV bottle 8.6% 0.8% 0.6% 0.8% 0.9% CV Bottle Room 7.4% 0.9% 0.6% 1.1% 0.7% Project Name 5'NT GA GSP D3H sdLDL CV bottle 1.1% 0.6% 0.9% 0.8% 1.1% CV Bottle Room 1.0% 1.1% 0.6% 0.8% 0.9% Project Name LPa APOE RBP CysC CK-MB CV bottle 1.1% 0.5% 0.7% 0.9% 0.7% CV Bottle Room 0.9% 0.8% 1.1% 0.8% 0.8% Project Name HCY AFU CG ADA GLDH CV bottle 0.5% 0.7% 0.6% 0.8% 0.9% CV Bottle Room 1.1% 0.9% 1.0% 0.5% 0.8%

[0094] As shown in the table above, excessive addition of the mixed anti-interference agent resulted in excessive cyclodextrin coating of the bile acid steroid nucleus, preventing effective bile acid recognition by the antibodies in the kit. The CG assay value deviation rate exceeded 10%, due to excessive cyclodextrin coating of the steroid nucleus, which prevented the antibody from recognizing the LSTa epitope. Lipoprotein analytes (such as sdLDL) exhibited aggregation, with a CV between vials of 1.1%, exceeding normal levels. Therefore, excessive concentrations disrupted the exposure of the CG conformation, demonstrating the need for precise concentration optimization of the mixed anti-interference agent.

[0095] Comparative Example 4: In this comparative example, 2% EDTA was added to human serum, and the following substances were added, repeatedly stirred and dissolved:

[0096] 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, adjusted to pH 7.5. Analytes: See Table 1, Level 3 for details.

[0097] Table 9 shows the effect of adding 2% EDTA on the homogeneity of multiple liquid quality control products containing CG biochemicals

[0098] Level 3 TBA B2MG CRP mAST GR CV bottle 2.5% 1.9% 2.1% 2.2% 2.6% CV Bottle Room 2.1% 2.5% 1.8% 1.6% 2.7% Project Name 5'NT GA GSP D3H sdLDL CV bottle 1.7% 2.4% 1.7% 2.3% 2.3% CV Bottle Room 2.2% 2.2% 1.7% 1.7% 1.8% Project Name LPa APOE RBP CysC CK-MB CV bottle 1.8% 1.8% 2.0% 2.3% 2.6% CV Bottle Room 2.3% 2.2% 2.3% 2.3% 2.1% Project Name HCY AFU CG ADA GLDH CV bottle 2.4% 1.7% 2.2% 1.7% 2.0% CV Bottle Room 2.5% 2.4% 2.0% 1.8% 2.5%

[0099] As shown in the table above, the effect of adding 2% EDTA on the homogeneity of the CG-containing quality control product was less pronounced than in Example 1. The CV within the vial of CG was 2.2%, and the CV between vials was 2.0%, both significantly higher than in Example 1. Cross-reactivity was significant because EDTA only chelated metal ions and failed to address conformational isomerization and epitope exposure. This demonstrates that single-mechanism stabilizers (such as EDTA) are unable to overcome existing technological bottlenecks, and the multifaceted effects of mixed anti-interference agents are irreplaceable.

[0100] Any numerical value cited herein includes all values ​​of the lower and upper values ​​in increments of one unit from the lower limit to the upper limit, and there is an interval of at least two units between any lower value and any higher value. For example, if the value of the quantity of a component or a process variable (such as temperature, pressure, time, etc.) is set forth to be from 1 to 90, preferably from 20 to 80, more preferably from 30 to 70, the purpose is to illustrate that values ​​such as 15 to 85, 22 to 68, 43 to 51, 30 to 32 are also explicitly listed in this specification. For values ​​less than 1, one unit is appropriately considered to be 0.0001, 0.001, 0.01, 0.1. These are merely examples that are intended to be clearly expressed, and it can be considered that all possible combinations of the numerical values ​​listed between the minimum and maximum values ​​are explicitly set forth in this specification in a similar manner.

[0101] Unless otherwise indicated, all ranges include the endpoints and all numbers between the endpoints. When used with a range, "about" or "approximately" applies 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.

[0102] It should be understood that the above description is for illustration and not for limitation. Many embodiments and many applications beyond the examples provided will be apparent to those skilled in the art upon reading the above description. Therefore, the scope of the present teachings should not be determined with reference to the above description, but rather with reference to the appended claims and the full scope of equivalents to which such claims are entitled. For the purpose of comprehensiveness, all articles and references, including disclosures of patent applications and publications, are incorporated herein by reference. The omission of any aspect of the subject matter disclosed herein from the foregoing claims is not intended to be a disclaimer of such subject matter, nor should it be assumed that the inventors did not consider such subject matter to be part of the disclosed inventive subject matter.

[0103] The above description of the present invention is exemplified in conjunction with the accompanying drawings. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned method. 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 scope of protection of the present invention.

Claims

1. A biochemical multi-item liquid quality control product containing glycocholic acid, characterized in that: This liquid control is based on human serum and contains the following components: Non-analytes include a buffer, a mixed anti-interference agent, a stabilizer, a surfactant, and a preservative, wherein the mixed anti-interference agent is a mixture of acetylcysteine ​​and cyclodextrin, the mass ratio of acetylcysteine ​​to cyclodextrin is 0.1:1-1:1, and the concentration of the mixed anti-interference agent in the quality control product is 0.1%-2% (w / v), and the cyclodextrin is selected from one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin; The buffer is selected from at least one of HEPES buffer, MES buffer, PIPES buffer, and Tris-HCl buffer, and the concentration of the buffer is 20-100 mM; The stabilizer is selected from at least one of a carbohydrate stabilizer and a protein stabilizer, and the concentration of the carbohydrate stabilizer in the quality control product is 0.1%-4% (w / v), and the concentration of the protein stabilizer in the quality control product is 0.1%-2% (w / v); The preservative is selected from at least one of sodium azide, Proclin 300, Proclin 950, and gentamicin, and the surfactant is selected from at least one of Tween 20 and Triton 100; Analytes include glycocholic acid, total bile acid and other biochemical markers, wherein the other biochemical markers include at least one of homocysteine, β-hydroxybutyrate, glutamate dehydrogenase, β2-microglobulin, C-reactive protein, aspartate aminotransferase, glutathione reductase, 5'-nucleotidase, glycated albumin, glycated serum protein, small dense low-density lipoprotein cholesterol, lipoprotein (a), apolipoprotein E, retinol-binding protein, cystatin C, creatine kinase isoenzyme, α-fucosidase and adenosine deaminase.

2. The biochemical multi-item liquid quality control product containing glycocholic acid according to claim 1, characterized in that: The carbohydrate stabilizer is selected from at least one of sucrose, trehalose and mannitol; The protein stabilizer is selected from at least one of casein, ovalbumin and antifreeze glycoprotein.

3. The biochemical multi-item liquid quality control product containing glycocholic acid according to claim 1, characterized in that: The pH value range of the biochemical multi-item liquid quality control product is 6.0-8.

0.

4. The biochemical multi-item liquid quality control product containing glycocholic acid according to claim 1, characterized in that: The buffer is HEPES buffer with a pH of 6.0-8.

0.

5. 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 an antifreeze glycoprotein; And / or, the surfactant is Tween 20; And / or, the preservative is ProClin300.

6. A method for preparing a biochemical multi-item liquid quality control product containing glycocholic acid, for preparing the biochemical multi-item liquid quality control product according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: (1) Matrix preparation: Take infectious disease-negative human serum, add buffer, acetylcysteine, stabilizer and preservative in sequence, and stir to dissolve; (2) pH adjustment: Use acid and base reagents to adjust the system pH to 6.0-8.0; (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 packaging: Sterilize through a 0.2-0.45 μm filter membrane, and store frozen after packaging.

7. The method for preparing the biochemical multi-item liquid quality control product containing glycocholic acid according to claim 6, characterized in that: The shelf life of the prepared liquid quality control product is ≥3 years, and the CG concentration fluctuation is ≤±5% after 38 months of storage; And / or, the intra-bottle coefficient of variation of each analyte in the biochemical multi-liquid quality control product is ≤3%, and the inter-bottle coefficient of variation (CV inter-bottle) is ≤3%, meeting the long-term clinical quality control requirements.

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