Myocardial marker substance control containing brain natriuretic peptide type b stable antigen and preparation method thereof

Abstract

The application discloses a myocardial marker control product containing B-type brain natriuretic peptide stable antigen and a preparation method thereof. The control product contains B-type brain natriuretic peptide stable antigen, and the B-type brain natriuretic peptide stable antigen contains a cyclic BNP polypeptide. The amino acid sequence of the cyclic BNP polypeptide is shown as SEQ ID NO:1. The preparation method comprises the following steps: S1, designing the B-type brain natriuretic peptide stable antigen, coupling the cyclic polypeptide with a carrier protein, and purifying to obtain the B-type brain natriuretic peptide stable antigen, which is recorded as BNP-Protector antigen; and S2, adding the prepared BNP-Protector antigen into a myocardial and inflammatory marker liquid control product containing BNP according to a proportion, and using the BNP-Protector antigen to stabilize or protect the BNP activity in the control product. The BNP-Protector antigen is used as a stabilizer of the myocardial marker control product, and the BNP-Protector antigen has high stability and high detection accuracy.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a control sample of a myocardial marker containing stable antigen of type B brain natriuretic peptide and its preparation method. Background Technology

[0002] Brain natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP), members of the natriuretic peptide (NP) family, are key biomarkers for the clinical diagnosis and assessment of heart failure. They are released into the bloodstream in equimolar proportions after enzymatic hydrolysis of a common precursor protein (proBNP), primarily originating from the heart, with smaller amounts expressed in extracardiac tissues such as the brain and lungs. Although homologous, their biological characteristics and clinical applications differ significantly: BNP is a biologically active hormone that exerts natriuretic, diuretic, vasodilatory, and neuroendocrine system-regulating effects, but its in vivo half-life is extremely short (approximately 20 minutes), and its in vitro stability is poor, easily affected by various factors; while NT-proBNP has no biological activity, a longer half-life, and is relatively stable in vitro, but its blood concentration is significantly affected by age and renal function.

[0003] When BNP (B-type natriuretic peptide) is used as an analyte in quality control products, some in vitro diagnostic reagent manufacturers, despite using human matrix serum to simulate the real clinical sample environment and avoiding the rapid degradation problems common in whole blood and plasma, still face a series of unique stability challenges. The core issue stems from the physicochemical properties of the BNP molecule itself: BNP has a small molecular weight (approximately 3.5 kDa) and is a hapten lacking immunogenicity. Sites in its molecular structure that are easily targeted by proteases (such as the N-terminal serine and proline residues) may still undergo slow degradation even in a serum matrix, leading to a gradual loss of immunoreactivity. More importantly, the morphological heterogeneity of BNP in quality control products is a critical issue. In addition to intact BNP (1-32), the human circulation contains its precursor proBNP and various degradation fragments (such as BNP3-32). Commercial quality control products cannot fully simulate this complex mixture, and differences in antibody recognition epitopes between different manufacturers (some target the cyclic structure, while others target the C-terminus or N-terminus) lead to inconsistent detection responses to these variants, resulting in significant differences of 15%-50% between different platforms. Furthermore, the oligomerization tendency of proBNP can mask antibody-binding epitopes, further interfering with detection results.

[0004] To address these challenges, conventional solutions primarily focus on three aspects: antibody design, quality control preparation, and detection process optimization. At the antibody design level, priority is given to antibody epitopes that recognize relatively stable regions of the BNP molecule. Studies have shown that the BNP 6-26 region (including its cyclic structure) is a good choice, helping to reduce the impact of degradation fragments and improve consistency across different platforms. At the quality control preparation level, manufacturers attempt to add protease inhibitors (such as aprotinin) to the quality control to stabilize the BNP molecule and slow down the degradation rate; simultaneously, they strive to mimic the natural proportions of BNP, proBNP, and their fragments in the human body to improve the interoperability of the quality control with actual clinical samples. At the detection process level, laboratories must strictly adhere to the manufacturer's recommended storage conditions for the quality control (usually requiring deep cryogenic freezing) and reconstitution procedures to maximize the stability of BNP.

[0005] However, the above solutions still have many limitations. First, the slow degradation of BNP in quality control products is difficult to completely inhibit. Even with the addition of protease inhibitors, the degradation process is only slowed down, not stopped. This means that the target value of the quality control product will drift over time, placing higher demands on the verification of the stability period of the quality control product after opening. Second, the lack of internationally recognized primary reference materials for BNP and standardized reference measurement procedures makes it difficult to trace the calibration of different testing systems to the same standard, which is one of the root causes of interlaboratory variability. Finally, the commutability problem between quality control products and real clinical samples cannot be completely solved. Although human serum matrix is ​​used, stabilizers, preservatives, etc., added during the manufacturing process to maintain stability and prevent microbial growth may change the properties of the matrix, causing the BNP immunoreactivity in the quality control product to be inconsistent with that in patient samples, which may introduce imperceptible biases on some testing platforms. These factors collectively limit the effectiveness of BNP quality control products in ensuring the accuracy and comparability of test results.

[0006] Therefore, there is an urgent need to develop a stable and highly accurate liquid quality control product containing BNP, a marker of myocardial and inflammatory function. Summary of the Invention

[0007] This invention provides a control sample of myocardial markers containing stable B-type brain natriuretic peptide antigen and its preparation method. The BNP-Protector antigen is used as a stabilizer for the control sample of myocardial markers, which has strong stability and high detection accuracy.

[0008] To address the aforementioned technical problems, the first aspect of this invention is to provide a myocardial marker control product containing a stable B-type brain natriuretic peptide antigen, wherein the control product includes the stable B-type brain natriuretic peptide antigen, the stable B-type brain natriuretic peptide antigen including a cyclic BNP polypeptide, and the amino acid sequence of the cyclic BNP polypeptide is shown in SEQ ID NO: 1.

[0009] Where SEQ ID NO:1 is:

[0010] H-Lys-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-Asp-OH.

[0011] Preferably, the quality control product further includes a matrix solution, which is a biological buffer matrix selected from any one or more of phosphate buffer, Tris-HCl buffer, and HEPES buffer, and the pH value of the matrix solution is 6.0-8.0.

[0012] Preferably, the quality control product further comprises at least one of a stabilizer, a preservative, and an osmotic pressure regulator; the stabilizer is selected from one or more of a protein stabilizer, bovine serum albumin, and sucrose; the preservative is selected from sodium azide and / or ProClin300; and the osmotic pressure regulator is selected from one or more of sodium chloride, potassium chloride, and glycerol.

[0013] Preferably, the stable antigen of type B brain natriuretic peptide is a conjugate of a cyclic BNP polypeptide and a carrier protein, wherein the carrier protein is selected from one or more of keyhole hemocyanin (KLH), bovine serum albumin (BSA), and ovalbumin (OVA).

[0014] A second aspect of this invention is to provide a method for preparing a myocardial marker control containing a stable antigen of type B brain natriuretic peptide, the specific steps of which are:

[0015] S1: Design a stable antigen of type B brain natriuretic peptide (BNP), then couple it with a carrier protein via a cyclic polypeptide, and purify it to obtain the stable antigen of type B brain natriuretic peptide (BNP), denoted as BNP-Protector antigen.

[0016] S2: The BNP-Protector antigen obtained in step S1 is added to the liquid quality control product containing BNP myocardial and inflammatory markers in proportion to stabilize or protect the BNP activity in the quality control product.

[0017] Preferably, the specific steps of step S1 are as follows:

[0018] S11 Synthesis of Cyclic Peptides: Linear precursor peptides were prepared by solid-phase synthesis, followed by cyclization in the liquid phase, deprotection and purification to obtain cyclic BNP peptides.

[0019] S12 Carrier protein coupling: The cyclic BNP polypeptide obtained in step S11 is activated and then coupled with the pretreated carrier protein. After purification, the BNP-Protector antigen is obtained.

[0020] Preferably, in step S2, the concentration of BNP-Protector antigen added to the liquid quality control material containing BNP myocardial and inflammatory markers is 0.1 mg / mL to 2 mg / mL.

[0021] Preferably, the specific steps of step S11 are as follows:

[0022] S111 Resin Selection and Solid-Phase Synthesis: Fmoc-protected Rink amide resin was selected as the solid-phase support. The target BNP peptide sequence for cyclization was designed, and amino acids that can be used for cyclization were introduced into the target BNP peptide sequence. An orthogonal protection strategy was adopted during synthesis, and the linear peptide resin was obtained by sequentially linking amino acids through Fmoc-SPPS cycling.

[0023] S112 Cycling Pretreatment: Selectively deprotect the linear peptide resin obtained in step S111, selectively remove the orthogonal protecting group Mtt ​​of the Lys side chain, then cleave the linear peptide from the resin, and precipitate to obtain a partially protected linear precursor peptide.

[0024] S113 Liquid-phase cyclization reaction: A partially protected linear precursor peptide is reacted with a condensing agent, HOAt, and the base DIPEA to generate a cyclic peptide.

[0025] S114 final deprotection and purification: Add final cleavage reagent to remove all remaining side chain protecting groups, then collect the crude cyclic peptide by precipitation and centrifugation, and then purify to obtain cyclic BNP peptide.

[0026] Preferably, in step S111, an orthogonal protection strategy is adopted to protect the Lys side chain with orthogonal protection bases Mtt or Dde during synthesis, while the Asp side chain is protected with conventional side chain protection base OtBu.

[0027] Preferably, in step S111, amino acids are sequentially linked according to the standard Fmoc-SPPS cycle, specifically: washing with DMF, removing the N-terminal Fmoc protecting group with 20% piperidine / DMF, washing with DMF again, then performing the coupling reaction, finally washing with DMF, and monitoring the coupling efficiency by ninhydrin detection until coupling is complete, to obtain a linear peptide resin.

[0028] Preferably, the removal of the N-terminal Fmoc protecting group using 20% ​​piperidine / DMF in step S111 is performed in two separate steps:

[0029] First treatment: Add 20% piperidine / DMF, soak and react for 10 minutes, then drain the waste liquid;

[0030] Second treatment: Add fresh 20% piperidine / DMF again, continue the reaction for 10 minutes, and then dry.

[0031] Preferably, the coupling reaction in step S111 specifically involves dissolving 4 equivalents of Fmoc-amino acid, 3.9 equivalents of HBTU, and 8 equivalents of DIPEA in DMF and reacting for 45 minutes. During the reaction, samples are taken periodically to detect the coupling efficiency using ninhydrin until the coupling is complete.

[0032] Preferably, the specific steps of the pre-circulation treatment in step S112 are as follows:

[0033] S1121 Selective Deprotection: After the linear peptide resin synthesis is completed, 1% TFA / DCM is used to selectively remove the orthogonal protecting group Mtt ​​of the Lys side chain, exposing the side chain amino groups, while the main chain and other side chain protecting groups remain unchanged.

[0034] S1122 cleavage of linear precursor peptides: The linear peptide with intact side-chain protecting groups (except for the deprotected Lys side chain) is cleaved from the resin using a mild cleavage reagent TFA / water (volume ratio of 95:5), and then precipitated to obtain a partially protected linear precursor peptide.

[0035] Preferably, the specific steps of the liquid phase cyclization in step S113 are as follows:

[0036] S1131: Dissolve the partially protected linear precursor peptide in DMF or a DMF / DCM mixed solvent;

[0037] S1132: Add condensing agents HATU (3 equivalents) and HOAt (3 equivalents) and base DIPEA (6 equivalents).

[0038] S1133: Under inert gas protection, the reaction is stirred at room temperature or low temperature, and the reaction process is monitored by HPLC until the linear precursor peptide is completely consumed and a cyclic peptide is generated.

[0039] In some embodiments, the specific process of monitoring the reaction process using HPLC chromatography is as follows:

[0040] The round-bottom flask containing the reaction solution was placed under nitrogen protection and the reaction was continuously stirred at 0°C. The reaction progress was monitored by periodic sampling and analytical high-performance liquid chromatography (HPLC).

[0041] The chromatographic conditions are as follows:

[0042] Chromatographic analysis was performed using an Agilent 1260 high-performance liquid chromatography system equipped with a diode array detector (DAD). A Waters XBridge BEH C18 column (4.6 × 250 mm, 5 μm, 130 Å) was used, with the column temperature set at 30–40 °C. The mobile phase composition was: (A) ultrapure water (containing 0.1% trifluoroacetic acid, v / v); (B) acetonitrile (containing 0.1% trifluoroacetic acid, v / v). The elution gradient was set to linearly increase the volume fraction of mobile phase B from 10% to 60% over 30 minutes at a flow rate of 1.0 mL / min. The detection wavelength was 214 nm, and the injection volume was 20 μL.

[0043] Sampling and pretreatment are as follows:

[0044] At preset time points of 0, 1, 3, 6, and 12 hours, 10 μL of the reaction solution was drawn using a microsyringe. The sample was immediately diluted with a 0.1% TFA aqueous solution pre-cooled in an ice bath to terminate the reaction, and then filtered through a 0.22 μm nylon membrane for analysis.

[0045] Preferably, the specific steps of the final deprotection and purification in step S114 are as follows:

[0046] S1141: After the cyclization reaction is complete, add the final cleavage reagent TFA / TIS / water (volume ratio 95:2.5:2.5) to remove all remaining side chain protecting groups;

[0047] S1142: Precipitate the product with cold diethyl ether and collect the crude cyclic peptide by centrifugation;

[0048] S1143: The crude cyclic peptide was purified by preparative reversed-phase HPLC. The chromatographic conditions were: C18 column, mobile phase of water and acetonitrile containing 0.1% TFA, and gradient elution. The main peak was collected and lyophilized to obtain the purified cyclic BNP peptide.

[0049] Preferably, the specific steps of step S12 are as follows:

[0050] S121 Carrier Protein Pretreatment: The carrier proteins (keyhole hemocyanin KLH or bovine serum albumin BSA) were dissolved in PBS buffer, reduced by DTT, and the final concentration was adjusted to 5 mg / mL. Keyhole hemocyanin KLH was used to immunize animals to prepare antibodies, while BSA was used as the coating antigen in the detection stage.

[0051] S122 peptide activation: The purified cyclic BNP peptide was dissolved in DMSO, and the cross-linking agent Sulfo-SMCC was added. The reaction was carried out at room temperature for 30-60 minutes to derive maleimide groups from the peptide ends.

[0052] S123 Coupling reaction: The activated cyclic BNP peptide was added dropwise to the carrier protein solution treated with reducing agent DTT in step S121, and the reaction was carried out by slow stirring at 4°C for 12 hours.

[0053] S124 Purification and Identification: The reaction mixture was dialyzed to remove unreacted cyclic BNP peptides and small molecule impurities, yielding the BNP-Protector antigen.

[0054] Preferably, in step S122, the molar ratio of the cyclic BNP peptide to the crosslinking agent Sulfo-SMCC is 1:10, and in step S123, the molar ratio of the cyclic BNP peptide to the carrier protein is 20:1.

[0055] Preferably, the concentration of the PBS buffer in step S121 is 0.1M and the pH value is 7.4.

[0056] Preferably, in step S124, dialysis is performed at 4°C with PBS for 24-48 hours.

[0057] Specifically, the obtained reaction mixture solution was transferred into a pretreated dialysis bag with a molecular weight cutoff of 14 kDa, sealed at both ends with dialysis clips, and immersed in PBS buffer (pH 7.2-7.4, concentration 0.01-0.1 mol / L, optionally containing 0.02% NaN3 for antibacterial purposes) pre-cooled to 4°C. The sample volume: dialysis solution volume ≥ 1:100. The entire dialysis process was carried out under magnetic stirring (200-400 rpm) at 4°C, and fresh pre-cooled PBS was replaced every 6–12 hours, for a total of 3–5 times.

[0058] A third aspect of this invention is to provide the application of BNP-Protector antigen in BNP quality control products. The BNP-Protector antigen serves as a stabilizer for the BNP quality control products.

[0059] Using the above technical solution, this invention designs and synthesizes a enzyme capable of specifically recognizing and binding to the key cleavage site (Met) in the B-type natriuretic peptide (BNP) molecule. 4 -Val 5 and Arg 17 -Ile 18 The BNP-Protector antigen is a cyclic polypeptide antigen. This antigen competitively inhibits the hydrolysis of these sites by neutral endopeptidase (NEP) through steric hindrance, thereby protecting BNP from degradation. The antigen design is based on the following principles:

[0060] (1) Precise epitope localization: Based on the protease digestion characteristics of BNP, the designed polypeptide sequence needs to precisely cover Met. 4-Val 5 and Arg 17 -Ile 18 Two sites are designed for easy NEP cleavage, ensuring a spatial conformation highly similar to the corresponding region of natural BNP and easier binding to endopeptidases compared to BNP (the cyclic polypeptide designed in this patent has less steric hindrance than the cyclic structure of BNP, thus exhibiting stronger binding affinity to endopeptidases). The polypeptide length is designed to be 25-32 amino acids to ensure the inclusion of key cleavage sites and flanking sequences necessary to maintain a stable conformation.

[0061] (2) Carrier protein conjugation: To enhance immunogenicity and facilitate subsequent detection, the synthetic peptide is conjugated to a carrier protein (such as keyhole hemocyanin KLH or bovine serum albumin BSA) via a heterobifunctional cross-linking agent (such as Sulfo-SMCC). KLH is used to immunize animals to prepare antibodies, while BSA is used as the coating antigen in the detection stage.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] (1) Adding BNP-Protector antigen as a stabilizer to BNP-containing quality control products can effectively improve the stability of BNP in the quality control products and enhance its detection specificity; at the same time, it has excellent compatibility and can be compatible with a variety of reagents.

[0064] (2) The BNP-Protector antigen-stabilized BNP quality control material improved the storage time of BNP-containing quality control material. It can still maintain good antigen activity and detection consistency after 6 days in an environment of 2-8℃.

[0065] (3) Using BNP-Protector antigen as a stabilizer not only reduces the risk of false negatives caused by BNP degradation, but also improves the accuracy of clinical test results, providing more precise quality control support for early screening and dynamic monitoring of diseases such as heart failure. Attached Figure Description

[0066] Figure 1 The chromatogram of the stable B-type brain natriuretic peptide antigen in the myocardial marker control product containing stable B-type brain natriuretic peptide antigen of the present invention is shown.

[0067] Figure 2 This is a schematic diagram illustrating the stability monitoring of the myocardial marker control product containing stable antigen of type B brain natriuretic peptide according to the present invention.

[0068] Figure 3 This is a schematic diagram illustrating the stability monitoring of quality control products with different amounts of the B-type brain natriuretic peptide stable antigen of the present invention. Detailed Implementation

[0069] The specific embodiments of the present invention will be described in detail below, but the present invention is not limited thereto.

[0070] Unless otherwise specified, the preparation methods and use conditions used in the following examples are all conventional methods; the reagent materials used in the following examples can be obtained from commercial sources unless otherwise specified.

[0071] To avoid excessive unnecessary details, the well-known structures or functions will not be described in detail in the following examples. The approximate language used in the following examples can be used for quantitative expressions, indicating that certain changes in quantity are allowed without changing the basic function. Unless otherwise defined, the technical and scientific terms used in the following examples have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0072] Noun Explanation:

[0073] BNP: B-type natriuretic peptide;

[0074] NT-ProBNP: N-terminal pro-brain natriuretic peptide;

[0075] CK-MB: Creatine kinase isoenzyme;

[0076] MYO: Myoglobin;

[0077] cTnI: High-sensitivity cardiac troponin I;

[0078] cTnT: High-sensitivity cardiac troponin T;

[0079] Base DIPEA: Diisopropylethylamine;

[0080] HOAt: Full name is 1-Hydroxy-7-azabenzotriazole, Chinese name is 1-hydroxy-7-azabenzotriazole;

[0081] DMF: Dimethylformamide;

[0082] DCM: Dichloromethane;

[0083] TFA: Trifluoroacetic acid;

[0084] DMSO: Dimethyl sulfoxide;

[0085] HBTU: O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0086] DIPEA: Full name is N,N-Diisopropylethylamine; Chinese name is diisopropylethylamine;

[0087] Sulfo-SMCC: Sodium salt of sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate;

[0088] PBS buffer: Phosphate-buffered saline.

[0089] Unless otherwise specified, all reagents used in this invention are commercially available reagents commonly used by those skilled in the art.

[0090] Example 1: A myocardial marker control containing stable B-type brain natriuretic peptide antigen, wherein the control contains stable B-type brain natriuretic peptide antigen, wherein the stable B-type brain natriuretic peptide antigen contains a cyclic BNP polypeptide, and the amino acid sequence of the cyclic BNP polypeptide is shown in SEQ ID NO: 1;

[0091] The quality control material also includes a matrix solution, which is a biological buffer matrix selected from any one or more of phosphate buffer, Tris-HCl buffer, and HEPES buffer, and the pH value of the matrix solution is 6.0~8.0.

[0092] The quality control product further includes at least one of the following additives: stabilizer, preservative, and osmotic pressure regulator; the stabilizer is selected from one or more of protein stabilizers, bovine serum albumin, and sucrose; the preservative is selected from sodium azide and / or ProClin300; and the osmotic pressure regulator is selected from one or more of sodium chloride, potassium chloride, and glycerol.

[0093] Among them, SEQ ID NO: 1:

[0094] H-Lys-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-Asp-OH; where the side chains of the first lysine (Lys) and the last aspartic acid (Asp) are used for subsequent cyclization. In this invention, the first lysine (Lys) and the last aspartine (Asp) are responsible for the further cyclization of the linear peptide resin (linear BNP polypeptide) to obtain a cyclic BNP polypeptide, which is then used to couple with a carrier protein to finally obtain the BNP-Protector antigen. Alternatively, an antigen sequence without the first lysine (Lys) and last aspartine (Asp) is a linear peptide resin. Compared to the "BNP-Protector antigen" obtained by directly coupling the linear peptide resin (linear BNP polypeptide) with the carrier protein, the cyclic BNP polypeptide and the carrier protein have less steric hindrance and a higher affinity for endopeptidases that cause BNP instability. This reduces the degradation of BNP by endopeptidases in the quality control material and prolongs the stability of BNP in the quality control material. Essentially, the BNP-Protector antigen synthesized in this invention replaces the "BNP" in the quality control material in reacting with the endopeptidases.

[0095] In some embodiments, the stable antigen of type B brain natriuretic peptide is a conjugate of a cyclic BNP polypeptide and a carrier protein, wherein the carrier protein is keyhole hemocyanin (KLH).

[0096] Accordingly, the preparation method of the myocardial marker control product containing stable B-type brain natriuretic peptide antigen includes the following specific steps:

[0097] S1: Design a stable antigen of type B brain natriuretic peptide (BNP), then couple it with a carrier protein via a cyclic polypeptide, and purify it to obtain the stable antigen of type B brain natriuretic peptide (BNP), denoted as BNP-Protector antigen.

[0098] The specific steps of step S1 are as follows:

[0099] S11 Synthesis of Cyclic Peptides: Linear precursor peptides were prepared by solid-phase synthesis, followed by cyclization in the liquid phase, deprotection and purification to obtain cyclic BNP peptides.

[0100] The specific steps of step S11 are as follows:

[0101] S111 Resin Selection and Solid-Phase Synthesis: Fmoc-protected Rink amide resin was selected as the solid-phase support. A target BNP peptide sequence for cyclization was designed, and amino acids suitable for cyclization were introduced into the target BNP fragment sequence (e.g., lysine (Lys) and aspartic acid (Asp) or glutamic acid (Glu) were introduced at the ends of the sequence or at specific positions). An orthogonal protection strategy was employed during synthesis, and the linear peptide resin was obtained by sequentially linking amino acids via Fmoc-SPPS cycling.

[0102] In step S111, an orthogonal protection strategy is adopted. During synthesis, the Lys side chain is protected by orthogonal protection bases Mtt or Dde, while the Asp side chain is protected by conventional side chain protection base OtBu.

[0103] In step S111, amino acids are sequentially linked according to the standard Fmoc-SPPS cycle, specifically: washing with DMF, removing the N-terminal Fmoc protecting group with 20% piperidine / DMF, washing with DMF again, then performing the coupling reaction, finally washing with DMF, and monitoring the coupling efficiency by ninhydrin detection until coupling is complete, to obtain a linear peptide resin.

[0104] In step S111, the removal of the N-terminal Fmoc protecting group using 20% ​​piperidine / DMF is specifically performed in two steps:

[0105] First treatment: Add 20% piperidine / DMF, soak and react for 10 minutes, then drain the waste liquid;

[0106] Second treatment: Add fresh 20% piperidine / DMF again, continue the reaction for 10 minutes, and then dry.

[0107] In some embodiments, the coupling reaction in step S111 specifically involves dissolving 4 equivalents of Fmoc-amino acid, 3.9 equivalents of HBTU, and 8 equivalents of DIPEA in DMF and reacting for 45 minutes. During the reaction, samples are taken periodically to detect the coupling efficiency using ninhydrin until the coupling is complete.

[0108] S112 Cycling Pretreatment: Selectively deprotect the linear peptide resin obtained in step S111, selectively remove the orthogonal protecting group Mtt ​​of the Lys side chain, then cleave the linear peptide from the resin, and precipitate to obtain a partially protected linear precursor peptide.

[0109] The specific steps of the pre-cyclization treatment in step S112 are as follows:

[0110] S1121 Selective Deprotection: After the linear peptide resin synthesis is completed, 1% TFA / DCM is used to selectively remove the orthogonal protecting group Mtt ​​of the Lys side chain, exposing the side chain amino groups, while the main chain and other side chain protecting groups remain unchanged.

[0111] S1122 cleavage of linear precursor peptides: The linear peptide with intact side-chain protecting groups (except for the deprotected Lys side chain) is cleaved from the resin using a mild cleavage reagent TFA / water (volume ratio of 95:5), and then precipitated to obtain a partially protected linear precursor peptide.

[0112] S113 Liquid-phase cyclization reaction: A partially protected linear precursor peptide is reacted with a condensing agent, HOAt, and the base DIPEA to generate a cyclic peptide.

[0113] The specific steps of the liquid phase cyclization in step S113 are as follows:

[0114] S1131: Dissolve the partially protected linear precursor peptide in DMF or a DMF / DCM mixed solvent;

[0115] S1132: Add condensing agents HATU (3 equivalents) and HOAt (3 equivalents) and base DIPEA (6 equivalents).

[0116] S1133: Under inert gas protection, the reaction is stirred at room temperature or low temperature, and the reaction process is monitored by HPLC until the linear precursor peptide is completely consumed and a cyclic peptide is generated.

[0117] S114 final deprotection and purification: Add final cleavage reagent to remove all remaining side chain protecting groups, then collect the crude cyclic peptide by precipitation and centrifugation, and then purify to obtain cyclic BNP peptide;

[0118] The specific steps of the final deprotection and purification in step S114 are as follows:

[0119] S1141: After the cyclization reaction is complete, add the final cleavage reagent TFA / TIS / water (volume ratio 95:2.5:2.5) to remove all remaining side chain protecting groups;

[0120] S1142: Precipitate the product with cold diethyl ether and collect the crude cyclic peptide by centrifugation;

[0121] S1143: The crude cyclic peptide was purified by preparative reversed-phase HPLC. The chromatographic conditions were: C18 column, mobile phase of water and acetonitrile containing 0.1% TFA, and gradient elution. The main peak was collected and lyophilized to obtain the purified cyclic BNP peptide.

[0122] S12 Carrier protein coupling: The cyclic BNP polypeptide obtained in step S11 is activated and then coupled with the pretreated carrier protein. After purification, the BNP-Protector antigen is obtained.

[0123] The specific steps of step S12 are as follows:

[0124] S121 Carrier Protein Pretreatment: The carrier proteins (keyhole hemocyanin KLH or bovine serum albumin BSA) were dissolved in PBS buffer (0.1M, pH 7.4), and DTT was added for reduction. The final concentration was adjusted to 5 mg / mL. Keyhole hemocyanin KLH was used to immunize animals to prepare antibodies, while BSA was used as the coating antigen in the detection stage.

[0125] S122 peptide activation: The purified cyclic BNP peptide was dissolved in DMSO, and the cross-linking agent Sulfo-SMCC was added. The molar ratio of cyclic BNP peptide to cross-linking agent Sulfo-SMCC was 1:10. The reaction was carried out at room temperature for 30 minutes to derive maleimide groups from the peptide ends.

[0126] S123 Coupling reaction: The activated cyclic BNP peptide was added dropwise to the carrier protein solution treated with reducing agent DTT in step S121. The molar ratio of cyclic BNP peptide to carrier protein was 20:1. The reaction was carried out by slow stirring at 4°C for 12 hours.

[0127] S124 Purification and Identification: Unreacted cyclic BNP peptides and small molecule impurities were removed by dialysis (dialysis with PBS at 4°C for 24 hours) to obtain BNP-Protector antigen. The prepared BNP-Protector antigen was used as a stabilizer in BNP quality control materials. The BNP-Protector antigen was analyzed by reversed-phase HPLC, and the HPLC chromatogram is shown below. Figure 1 As shown.

[0128] The chromatographic conditions are as follows:

[0129] Instrument: Agilent 1260 Infinity II high-performance liquid chromatography system, equipped with a diode array detector (DAD).

[0130] Column: Waters XBridge® BEH C18 column (4.6 mm × 250 mm, 5 μm, pore size 130 Å); Column temperature: 40 °C.

[0131] Mobile phase: Phase A is ultrapure water (containing 0.1% trifluoroacetic acid, v / v), and Phase B is acetonitrile (containing 0.1% trifluoroacetic acid, v / v).

[0132] A linear gradient elution mode was used, with a flow rate of 1.0 mL / min, a detection wavelength of 214 nm, and a single injection volume of 20 μL. The gradient elution procedure is as follows:

[0133] From 0 to 5 minutes, the proportion of mobile phase A decreased linearly from 90% to 70%.

[0134] Within 5–25 minutes, the proportion of mobile phase A decreased linearly from 70% to 40%.

[0135] Over 25–30 minutes, the proportion of mobile phase A decreased linearly from 40% to 10%.

[0136] For 30–35 minutes, maintain the column at a mobile phase A ratio of 10% to equilibrate it.

[0137] S2: The BNP-Protector antigen obtained in step S1 is added to the liquid quality control product containing BNP myocardial and inflammatory markers in proportion to stabilize or protect the BNP activity in the quality control product.

[0138] In step S2, the concentration of BNP-Protector antigen added to the liquid quality control material containing BNP myocardial and inflammatory markers is 0.1 mg / mL to 2 mg / mL.

[0139] Example 2: Exploring the performance of liquid quality control products containing myocardial standard at different concentrations of analytes.

[0140] (1) Preparation of liquid quality control samples of myocardial standard with different concentrations of analytes. The specific steps for preparing 40 mL of quality control dilution solution are as follows:

[0141] Add 0.2221 g of disodium hydrogen phosphate dodecahydrate and 0.0593 g of sodium dihydrogen phosphate dihydrate to 5 mL of pure water and stir until completely dissolved. Weigh 0.04 g of protein stabilizer and 0.04 g of synthesized BNP-Protector antigen, add them to the buffer solution, and stir to dissolve. Add 0.04 mL of ProClin 300 preservative to the solution and mix gently; then bring the volume to 20 mL with pure water. Add 20 mL of human serum matrix solution to the solution, mix slowly, add the analyte raw materials according to Table 1, and aliquot and store.

[0142] Table 1. Analytes, raw materials, and concentrations

[0143] BNP pg / mL 71 427 3191 NT-ProBNP pg / mL 118 512 4030 CK-MB ng / mL 7.2 15.3 41 MYO ng / mL 23.4 64.7 240 cTnI pg / mL 11.8 58.1 865 cTnT pg / mL 12 98 796

[0144] The above three levels of concentration of myocardial biomarkers liquid quality control samples were analyzed and their performance evaluated.

[0145] Ten vials of each of the three concentrations of myocardial marker liquid quality control samples were taken, and each vial was tested three times on a suitable immunoassay analyzer. The intra-vial coefficient of variation (CV) and inter-vial coefficient of variation (CV) were calculated. The test results are shown in Table 2.

[0146] Table 2. Analytical performance test results of liquid quality control samples of three different concentrations of myocardial markers

[0147] CV bottle 5.2% 3.0% 5.6% 3.9% 4.6% 4.3% CV Bottle Room 3.4% 4.0% 3.9% 3.1% 6.1% 6.5% Level 2 BNP NT-ProBNP CK-MB MYO cTnI cTnT CV bottle 6.0% 5.7% 5.1% 6.6% 4.9% 3.1% CV Bottle Room 5.9% 3.9% 3.3% 5.6% 4.8% 4.6% Level 3 BNP NT-ProBNP CK-MB MYO cTnI cTnT CV bottle 4.8% 6.8% 6.0% 5.3% 3.0% 5.3% CV Bottle Room 5.1% 6.6% 5.9% 4.3% 3.1% 5.2%

[0148] As can be seen from the results in Table 2, even for the same analyte at different concentration levels, the maximum CV does not exceed 7%, which indicates that the myocardial biomarker liquid quality control of the present invention has good homogeneity.

[0149] Comparative Example: The difference from Example 2 is that BNP-Protector antigen was not added to the quality control sample. The specific steps for preparing 40 mL of quality control sample dilution are as follows:

[0150] Add 0.2221 g of disodium hydrogen phosphate dodecahydrate and 0.0593 g of sodium dihydrogen phosphate dihydrate to 5 mL of pure water and stir until completely dissolved. Weigh 0.04 g of protein stabilizer and add it to the buffer solution, stirring to dissolve. Add 0.04 mL of ProClin 300 preservative to the solution and mix gently. Then, bring the volume to 20 mL with pure water. Add 20 mL of human serum matrix solution to the solution, mix slowly, add the analyte raw materials according to Table 1, and aliquot and store.

[0151] The above three levels of concentration of myocardial biomarkers liquid quality control samples were analyzed and their performance evaluated.

[0152] Ten vials of each of the three concentrations of myocardial biomarkers in liquid form were taken, and each vial was tested three times on a suitable immunoassay analyzer. The intra-vial coefficient of variation (CV) and inter-vial coefficient of variation (CV) were calculated. The test results are shown in Table 3.

[0153] Table 3. Analytical performance test results of liquid quality control products for myocardial markers without BNP-Protector antigen.

[0154] CV bottle 11.2% 5.8% 6.2% 6.8% 4.4% 6.5% CV Bottle Room 12.9% 3.5% 4.0% 6.2% 3.0% 5.2% Level 2 BNP NT-ProBNP CK-MB MYO cTnI cTnT CV bottle 12.1% 6.0% 5.5% 3.7% 3.4% 6.8% CV Bottle Room 11.8% 4.7% 5.4% 5.0% 3.3% 5.4% Level 3 BNP NT-ProBNP CK-MB MYO cTnI cTnT CV bottle 11.0% 6.2% 4.2% 4.1% 5.4% 4.7% CV Bottle Room 11.7% 3.9% 6.0% 3.7% 5.0% 4.3%

[0155] As shown in Table 3, Example 2 is superior to the comparative example (the CV of BNP measurement is significantly increased). This indicates that the quality control material with added BNP-Protector antigen significantly improves the stability of BNP in liquid quality control materials, effectively reducing the coefficient of variation during detection, especially exhibiting better repeatability and consistency at different concentration levels. This demonstrates that the BNP-Protector antigen plays a crucial role in maintaining the conformational integrity of BNP molecules and preventing their degradation, thereby ensuring the reliability and accuracy of the quality control material in clinical testing.

[0156] Example 3: The difference from Example 2 is that the amount of BNP-Protector antigen added is lower than the lower limit. The specific steps for preparing 40 mL of quality control diluent are as follows:

[0157] Add 0.2221 g of disodium hydrogen phosphate dodecahydrate and 0.0593 g of sodium dihydrogen phosphate dihydrate to 5 mL of pure water and stir until completely dissolved. Weigh 0.04 g of protein stabilizer and 0.001 g of synthesized BNP-Protector antigen, add them to the buffer solution, and stir to dissolve. Add 0.04 mL of ProClin 300 preservative to the solution and mix gently. Then, bring the volume to 20 mL with pure water. Add 20 mL of human serum matrix solution to the solution, mix slowly, add the analyte raw materials according to Table 1, and aliquot and store.

[0158] The above three levels of concentration of myocardial biomarkers liquid quality control samples were analyzed and their performance evaluated.

[0159] Ten vials of liquid quality control samples of myocardial markers at three different concentrations were taken, and each vial was tested three times on a suitable immunoassay analyzer. The intra-vial coefficient of variation (CV) and inter-vial coefficient of variation (CV) were calculated. The test results are shown in Table 4.

[0160] Table 4 Analytical performance test results of liquid quality control products for myocardial markers

[0161] CV bottle 11.4% 6.8% 3.8% 6.2% 4.9% 6.8% CV Bottle Room 11.3% 4.2% 5.9% 3.6% 5.7% 4.3% Level 2 BNP NT-ProBNP CK-MB MYO cTnI cTnT CV bottle 11.7% 5.1% 3.1% 3.6% 6.1% 4.6% CV Bottle Room 11.6% 5.3% 3.2% 6.7% 5.5% 5.6% Level 3 BNP NT-ProBNP CK-MB MYO cTnI cTnT CV bottle 10.8% 5.3% 4.8% 5.5% 6.0% 6.9% CV Bottle Room 10.0% 3.9% 6.3% 5.4% 3.8% 3.4%

[0162] As can be seen from the test results in Table 4, Example 2 is superior to Example 3 (the CV of BNP measurement is increased). This indicates that if the amount of BNP-Protector antigen added is too small, it cannot effectively stabilize BNP in the quality control sample, leading to the easy degradation or conformational changes of BNP in the quality control sample, thus increasing the volatility of the test results. This result shows that the amount of BNP-Protector antigen added needs to be maintained within the effective concentration range to fully protect the stability of BNP molecules and ensure that the quality control sample maintains good analytical performance during storage and use.

[0163] Example 4: The difference from Example 2 is that the amount of BNP-Protector antigen added is higher than the upper limit. The specific steps for preparing 40 mL of quality control diluent are as follows:

[0164] Add 0.2221 g of disodium hydrogen phosphate dodecahydrate and 0.0593 g of sodium dihydrogen phosphate dihydrate to 5 mL of pure water and stir until completely dissolved. Weigh 0.04 g of protein stabilizer and 0.2 g of synthesized BNP-Protector antigen, add them to the buffer solution, and stir to dissolve. Add 0.04 mL of ProClin 300 preservative to the solution and mix gently. Then, bring the volume to 20 mL with pure water. Add 20 mL of human serum matrix solution to the solution, mix slowly, and then add the analytes according to Table 1 and aliquot for storage.

[0165] The above three levels of concentration of myocardial biomarkers liquid quality control samples were analyzed and their performance evaluated.

[0166] Ten vials of each of the three concentrations of myocardial biomarkers in liquid quality control were taken, and each vial was tested three times on a suitable immunoassay analyzer. The intra-vial coefficient of variation (CV) and inter-vial coefficient of variation (CV) were calculated. The test results are shown in Table 5.

[0167] Table 5 Analytical performance test results of liquid quality control products for myocardial markers

[0168] CV bottle 9.9% 4.7% 4.3% 6.7% 3.8% 5.9% CV Bottle Room 9.9% 5.2% 6.8% 6.2% 3.7% 3.7% Level 2 BNP NT-ProBNP CK-MB MYO cTnI cTnT CV bottle 9.5% 4.9% 6.5% 5.4% 3.2% 6.7% CV Bottle Room 8.9% 4.0% 4.6% 3.3% 3.8% 3.8% Level 3 BNP NT-ProBNP CK-MB MYO cTnI cTnT CV bottle 9.2% 4.2% 3.0% 3.0% 4.0% 3.7% CV Bottle Room 9.7% 6.4% 4.2% 3.4% 4.8% 5.2%

[0169] As shown in Table 5, Example 2 is superior to Example 4 (the CV of BNP measurement is increased). This indicates that adding too much BNP-Protector antigen may lead to excessively high protein concentrations in the system, causing non-specific interactions or steric hindrance effects, which in turn interfere with the binding of BNP to its detection antibody, resulting in unstable detection signals and thus increasing the coefficient of variation. Furthermore, excessive BNP-Protector antigen may alter the physicochemical properties of the quality control matrix, affecting the stability or detection performance of other myocardial biomarkers. Therefore, the concentration of added BNP-Protector antigen must be strictly controlled within the range of 0.1 mg / mL to 2 mg / mL to effectively protect BNP activity.

[0170] Example 5: The effect of adding BNP-Protector antigen on the stability of BNP in liquid quality control samples of myocardial markers was investigated using diluents of different analytes prepared in Example 2. Stability experiments were conducted on the above three concentrations of liquid quality control samples of myocardial markers, specifically as follows:

[0171] Seven vials were opened, reconstituted, and stored at 2–8°C protected from light for 6 days. One vial was randomly selected on days 1, 2, 3, 4, 5, and 6 for testing. Each vial was measured three times on a suitable immunoassay analyzer, and the mean was calculated. Results are as follows: Figure 2 As shown in the figure. The results indicate that BNP in the liquid quality control of myocardial markers can be stably stored for 6 days at 2-8°C after the addition of BNP-Protector antigen.

[0172] Example 6: The effect of adding BNP-Protector antigen on the stability of BNP in the liquid quality control of myocardial markers was investigated using the quality control diluents of different analytes prepared in Example 2. Unlike Example 5, BNP-Protector antigen was not added to the three concentrations of the liquid quality control of myocardial markers. Stability experiments were conducted on the above three concentrations of the liquid quality control of myocardial markers, specifically as follows:

[0173] Seven vials were opened, reconstituted, and stored at 2-8℃ protected from light for 6 days. One vial was randomly selected on days 1, 2, 3, 4, 5, and 6 for testing. Each vial was measured three times on a suitable immunoassay analyzer, and the mean was calculated. Results are as follows: Figure 3 As shown in the figure, the results indicate that the BNP levels in the three concentrations of the myocardial marker liquid quality control without added BNP-Protector antigen showed a gradual decreasing trend during the monitoring period, indicating significantly reduced stability.

[0174] For those skilled in the art, the specific embodiments are merely illustrative descriptions of the present invention. Obviously, the specific implementation of the present invention is not limited to the above-described manner. Any non-substantial improvements made using the inventive concept and technical solution, such as changing the mass of a substance or a reaction parameter, or directly applying the inventive concept and technical solution to other situations without modification, are all within the protection scope of the present invention.

Claims

1. A myocardial marker control product containing stable B-type brain natriuretic peptide antigen, characterized in that, The quality control material includes a stable B-type brain natriuretic peptide antigen, which includes a cyclic BNP polypeptide, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The myocardial marker control product containing stable B-type brain natriuretic peptide antigen according to claim 1, characterized in that, The quality control material also includes a matrix solution, which is a biological buffer matrix selected from any one or more of phosphate buffer, Tris-HCl buffer, and HEPES buffer, and the pH value of the matrix solution is 6.0-8.

0.

3. A method for preparing a myocardial marker control product containing stable B-type brain natriuretic peptide antigen, characterized in that, The specific steps are as follows: S1: Design a stable antigen of type B brain natriuretic peptide (BNP), then couple it with a carrier protein via a cyclic polypeptide, and purify it to obtain the stable antigen of type B brain natriuretic peptide (BNP), denoted as BNP-Protector antigen. S2: The BNP-Protector antigen obtained in step S1 is added to the liquid quality control containing BNP myocardial and inflammatory markers in proportion to stabilize or protect the activity of BNP in the quality control.

4. The preparation method according to claim 3, characterized in that, The specific steps of step S1 are as follows: S11 Synthesis of Cyclic Peptides: Linear precursor peptides were prepared by solid-phase synthesis, followed by cyclization in the liquid phase, deprotection and purification to obtain cyclic BNP peptides. S12 Carrier protein coupling: The cyclic BNP polypeptide obtained in step S11 is activated and then coupled with the pretreated carrier protein. After purification, the BNP-Protector antigen is obtained.

5. The preparation method according to claim 4, characterized in that, In step S2, the concentration of BNP-Protector antigen added to the liquid quality control material containing BNP myocardial and inflammatory markers is 0.1 mg / mL to 2 mg / mL.

6. The preparation method according to claim 4, characterized in that, The specific steps of step S11 are as follows: S111 Resin Selection and Solid-Phase Synthesis: Fmoc-protected Rink amide resin was selected as the solid-phase support. The target BNP peptide sequence was designed, and amino acids that can be used for cyclization were introduced into the target BNP peptide sequence. An orthogonal protection strategy was adopted during synthesis, and the linear peptide resin was obtained by sequentially linking amino acids through Fmoc-SPPS cycling. S112 Cycling Pretreatment: Selectively deprotect the linear peptide resin obtained in step S111, selectively remove the orthogonal protecting group Mtt ​​of the Lys side chain, then cut the linear peptide from the resin, and precipitate to obtain a partially protected linear precursor peptide. S113 Liquid-phase cyclization reaction: A partially protected linear precursor peptide is reacted with a condensing agent, HOAt, and the base DIPEA to generate a cyclic peptide. S114 final deprotection and purification: Add final cleavage reagent to remove all remaining side chain protecting groups, then collect the crude cyclic peptide by precipitation and centrifugation, and then purify to obtain cyclic BNP peptide.

7. The preparation method according to claim 6, characterized in that, In step S111, an orthogonal protection strategy is adopted. During synthesis, the Lys side chain is protected by orthogonal protection bases Mtt or Dde, and the Asp side chain is protected by side chain protection base OtBu.

8. The preparation method according to claim 7, characterized in that, In step S111, amino acids are sequentially linked according to the standard Fmoc-SPPS cycle, specifically: washing with DMF, removing the N-terminal Fmoc protecting group with 20% piperidine / DMF, washing with DMF again, then performing the coupling reaction, finally washing with DMF, and monitoring the coupling efficiency by ninhydrin detection until coupling is complete, to obtain a linear peptide resin.

9. The preparation method according to claim 4, characterized in that, The specific steps of step S12 are as follows: S121 carrier protein pretreatment: Dissolve the carrier protein in PBS buffer, add reducing agent DTT for reduction, and adjust the final concentration to 5 mg / mL; S122 peptide activation: The purified cyclic BNP peptide is dissolved in DMSO, a cross-linking agent is added, and the reaction is carried out at room temperature for 30-60 minutes to derive maleimide groups from the peptide ends. S123 Coupling reaction: The activated cyclic BNP peptide was added dropwise to the carrier protein solution treated with reducing agent DTT in step S121, and the reaction was carried out by slow stirring at 4°C for 12 hours. S124 Purification and Identification: The reaction mixture was dialyzed to remove unreacted cyclic BNP peptides and impurities, yielding the BNP-Protector antigen.

10. The preparation method according to claim 9, characterized in that, In step S122, the molar ratio of cyclic BNP peptide to crosslinking agent is 1:10, and in step S123, the molar ratio of cyclic BNP peptide to carrier protein is 20:1.

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