A buffer solution and its use

Abstract

The application discloses a kind of buffer and its application.The application can reduce the sample matrix difference caused by different anticoagulants by optimizing the buffer system of NfL detection component, so that the same subject's EDTA anticoagulant plasma and sodium citrate anticoagulant plasma obtain good consistency of determination results.Meanwhile, the buffer system presents good gradient response to different concentrations of calibrators, and can distinguish Alzheimer's disease patients from normal samples, indicating that it maintains good detection performance while improving the compatibility of different anticoagulant plasma.The application can improve the accuracy and comparability of NfL detection results, and expand the application range of the detection system to different anticoagulant plasma samples, with wide market application scenarios.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a buffer solution and its applications. Background Technology

[0002] Plasma is a commonly used biological sample in clinical testing and in vitro diagnostics. To prevent blood clotting outside the body, an anticoagulant is usually added to blood collection tubes, followed by centrifugation to obtain plasma. Ethylenediaminetetraacetic acid (EDTA) and its salts, as well as sodium citrate, are commonly used anticoagulants, both primarily inhibiting the coagulation process by chelating calcium ions in the blood. However, EDTA and sodium citrate differ in chemical composition, form of addition, and concentration, and sodium citrate anticoagulant can dilute blood samples. Therefore, plasma prepared using the two anticoagulants may differ in metal ion state, ion composition, pH environment, and degree of sample dilution.

[0003] When detecting target analytes in plasma, the results are affected not only by the actual concentration of the target analyte but also by the sample matrix. Differences in the degree of metal ion complexation, ionic composition, pH environment, and sample dilution caused by different anticoagulants can affect molecular interactions, reaction equilibrium, or signal generation processes in the detection system, leading to discrepancies in the measurement results of the same target analyte in sodium citrate-anticoagulated plasma and EDTA-anticoagulated plasma. Therefore, a detection system established and validated for one type of anticoagulated plasma may not guarantee consistent results when directly used for another type of anticoagulated plasma.

[0004] In current practice, separate detection conditions, calibration systems, or conversion relationships can be established for different anticoagulated plasmas, or detection reagents can be limited to only one type of anticoagulated plasma. However, establishing and validating detection conditions separately increases the complexity of reagent development, quality control, and clinical use. Empirical conversion relationships may not adequately compensate for matrix differences between different samples, while limiting sample types reduces the applicability of the detection method. Therefore, how to reduce the impact of matrix differences between sodium citrate anticoagulated plasma and EDTA anticoagulated plasma on the determination results of target analytes, and how to make the same detection system compatible with both types of anticoagulated plasma and improve the consistency of their determination results, is a technical problem that needs to be solved in this field. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a buffer solution and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a buffer composition for reducing target detection bias between different anticoagulated plasma samples, the buffer composition comprising: 4-hydroxyethylpiperazine ethanesulfonic acid, sodium chloride, lauryl alcohol polyoxyethylene ether, bovine serum albumin, glycerol, and Proclin-300.

[0007] A second aspect of the present invention provides a kit for reducing target detection bias between different anticoagulated plasma samples, the kit comprising the buffer composition described in the first aspect of the present invention.

[0008] A third aspect of the present invention provides a detection system, the detection system comprising: (1) The buffer composition described in the first aspect of the present invention, or the kit described in the second aspect of the present invention; and (2) Testing instruments.

[0009] A fourth aspect of the present invention provides a method for reducing target detection bias among plasma samples with different anticoagulants, the method comprising: The target-specific binding reagent is dissolved using the buffer composition described in the first aspect of the present invention to obtain the detection working solution; Different anticoagulated plasma samples were tested using the test working solution.

[0010] A fifth aspect of the present invention provides a method for detecting a target in a plasma sample, the method comprising: (1) Magnetic beads coated with target-specific binding reagents; (2) Acridinium ester-labeled target-specific binding reagents; (3) Add the buffer composition described in the first aspect of the present invention to obtain the detection working solution; (4) Use the detection working solution to test different anticoagulated plasma samples.

[0011] A sixth aspect of the invention provides the use of the buffer composition described in the first aspect of the invention in the preparation of a kit for reducing target detection bias between sodium citrate-anticoagulated plasma samples and EDTA-anticoagulated plasma samples.

[0012] The seventh aspect of the present invention provides the use of the buffer composition of the first aspect of the present invention, the kit of the second aspect of the present invention, or the detection system of the third aspect of the present invention in the in vitro detection of neurofilament light chain proteins in plasma.

[0013] Advantages and beneficial effects of the present invention: This application optimizes the buffer system for NfL detection components, reducing sample matrix differences caused by different anticoagulants and achieving consistent measurement results between EDTA-anticoagulated plasma and sodium citrate-anticoagulated plasma from the same subject. Simultaneously, this buffer system exhibits a good gradient response to different concentrations of calibrators and can distinguish between samples from Alzheimer's patients and healthy individuals, demonstrating that it maintains good detection performance while improving compatibility with different anticoagulated plasmas. This application can improve the accuracy and comparability of NfL detection results and expand the applicability of the detection system to different anticoagulated plasma samples, thus having broad market application prospects. Detailed Implementation

[0014] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0015] The present invention provides a buffer composition for reducing target detection bias between different anticoagulated plasma samples, the buffer composition comprising (buffer 4): including 4-hydroxyethylpiperazine ethanesulfonic acid, sodium chloride, lauryl alcohol polyoxyethylene ether, bovine serum albumin, glycerol, and Proclin-300.

[0016] In some embodiments, the different anticoagulated plasma samples are sodium citrate anticoagulated plasma samples and EDTA anticoagulated plasma samples.

[0017] In some embodiments, the target is neurofilament light chain protein (NfL).

[0018] In some embodiments, the detection methods include, but are not limited to, immunoassay, chemiluminescence assay, electrochemiluminescence assay, fluorescence assay, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, chromatography, colorimetry, turbidity assay, or nucleic acid amplification assay.

[0019] In a specific implementation, the detection is an immune detection.

[0020] The present invention provides a kit for reducing target detection bias between different anticoagulated plasma samples, the kit comprising the above-described buffer composition.

[0021] In some embodiments, the different anticoagulated plasma samples are sodium citrate anticoagulated plasma samples and EDTA anticoagulated plasma samples.

[0022] In some embodiments, the kit further includes a solid support. The solid support is a solid support material used to immobilize bioactive molecules (such as antibodies and antigens). The solid support includes, but is not limited to, microplates, magnetic beads, latex particles, microfluidic chips, and test strips.

[0023] In some embodiments, the kit further comprises a target-specific binding reagent. The target-specific binding reagent is a reagent capable of specifically recognizing and binding to the analyte (target analyte). The target-specific binding reagent includes, but is not limited to, antibodies, aptamers, receptors, and ligands.

[0024] In some embodiments, the kit further includes calibrators and / or quality control samples. The calibrators are standard substances containing known concentrations of a target, used to establish a standard curve (calibration curve) of "signal value-concentration". When testing a sample, the concentration is calculated by substituting the sample signal value into the standard curve. Quality control samples are standard substances containing known target concentrations, used to monitor the normal operation of the detection system. Quality control samples typically have three concentration levels: high, medium, and low. The quality control samples are tested simultaneously with each sample test to determine if the results are within acceptable ranges.

[0025] In some embodiments, the calibrators and / or quality control materials are sodium citrate anticoagulated plasma matrix and / or EDTA anticoagulated plasma matrix containing known concentrations of the target.

[0026] In some embodiments, the kit also includes reagents for processing samples.

[0027] In some embodiments, the kit also includes instructions for use.

[0028] This invention provides a detection system, the detection system comprising: (1) the above-described buffer composition, or the above-described kit; and (2) Testing instruments.

[0029] In some embodiments, the detection instruments include, but are not limited to, chemiluminescence immunoassay analyzers, electrochemiluminescence immunoassay analyzers, enzyme-linked immunosorbent assay (ELISA) readers, fluorescence analyzers, mass spectrometers, chromatographs, PCR instruments, and fully automated biochemical analyzers.

[0030] In a preferred embodiment, the detection instrument is selected from an immunoassay analyzer (including a chemiluminescence immunoassay analyzer and an electrochemiluminescence immunoassay analyzer).

[0031] This invention provides a method for reducing target detection bias among plasma samples with different anticoagulants, the method comprising: The target-specific binding reagent was dissolved using the above-mentioned buffer composition to obtain the detection working solution; Different anticoagulated plasma samples were tested using the test working solution.

[0032] In some embodiments, the different anticoagulated plasma samples are sodium citrate anticoagulated plasma samples and EDTA anticoagulated plasma samples.

[0033] In some embodiments, the target-specific binding agent includes, but is not limited to, antibodies, aptamers, receptors, and ligands.

[0034] In a preferred embodiment, the target-specific binding agent is an antibody against the target.

[0035] In some embodiments, the antibody is an acridinium ester-labeled antibody. The acridinium ester (AE) is a key chemiluminescent label in immunoassay methods.

[0036] In some embodiments, the target is neurofilament light chain protein.

[0037] This invention provides a method for detecting a target in a plasma sample, the method comprising: (1) Magnetic beads coated with target-specific binding reagents; (2) Acridinium ester-labeled target-specific binding reagents; (3) Add the above buffer composition to obtain the detection working solution; (4) Use the detection working solution to test different anticoagulated plasma samples.

[0038] In some embodiments, the different anticoagulated plasma samples are sodium citrate anticoagulated plasma samples and EDTA anticoagulated plasma samples.

[0039] In some embodiments, the target-specific binding agent is an antibody against the target.

[0040] In some embodiments, the antibody is an acridine ester-labeled antibody.

[0041] In some embodiments, the target is neurofilament light chain protein.

[0042] This invention provides the application of the above-described buffer composition in the preparation of a kit for reducing target detection bias between sodium citrate-anticoagulated plasma samples and EDTA-anticoagulated plasma samples.

[0043] The present invention provides the application of the above-described buffer composition, the above-described kit, or the above-described detection system in the in vitro detection of neurofilament light chain proteins in plasma.

[0044] In some embodiments, the detection of neurofilament light chain protein in plasma is a non-diagnostic detection of neurofilament light chain protein in plasma.

[0045] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0046] Example I. Experimental Methods 1. Preparation of reagent components 1.1 Preparation of magnetic bead-conjugated antibodies Magnetic beads (manufacturer: MBL, catalog number: MagneSphere MS160 / Carboxyl) were washed three times with Beijing Kaixiang Hongkang's self-developed cleaning solution (formulation: Trizma-alkali (manufacturer: Sigma, catalog number: V900483), sodium chloride (manufacturer: Shanghai Testing, catalog number: 10019360), and Tween 20 (manufacturer: GPC, catalog number: AP900)) and set aside. Subsequently, the carboxyl groups on the surface of the magnetic beads were activated with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride). After activation, an antibody (manufacturer: Aibosheng (Beijing) Science and Technology Co., Ltd., catalog number: KC-683) was added, and the mixture was incubated at room temperature for 2 hours. After incubation, an appropriate amount of glycine solution was added to block any remaining activated carboxyl groups. Finally, the streptavidin magnetic beads were washed three more times with the same cleaning solution to obtain the final product.

[0047] 1.2 Preparation of acridinium ester-labeled antibodies Antibody II (manufacturer: Iboson (Beijing) Science & Technology Co., Ltd., catalog number: KC-495) was purified using a PD-10 desalting column. Before the column change, the buffer was 0.02M PBS buffer + 0.1% sodium azide; after the change, it was 0.02M PBS to remove interference from sodium azide in the coupling reaction. After the column change, NSP-DMAE-NHS acridine ester solution was added to the antibody, and the reaction was carried out at room temperature for 2 hours. After the reaction, an appropriate amount of glycine solution was added to block excess NSP-DMAE-NHS acridine ester. Finally, the labeled antibody was purified again using a PD-10 desalting column to remove excess free NSP-DMAE-NHS acridine ester. The purified antibody was then ready for use.

[0048] 2. Preparation of buffer system 2.1. Preparation of control buffer Weigh 1000g of purified water into a beaker. Weigh out HEPES (4-hydroxyethylpiperazine ethanesulfonic acid, manufacturer: Sigma, item number: H3375), NaCl (sodium chloride, manufacturer: Shanghai Testing, item number: 10019360), Tween-20 (GPC, item number: AP900), bovine serum albumin (BSA, manufacturer: Sigma, item number: V900933), and Proclin-300 (preservative, manufacturer: Sigma, item number: 48914-U) and pour them into the beaker. Mix thoroughly with a magnetic stirrer.

[0049] 2.2. Preparation of Buffer 1 Weigh 1000g of purified water and place it in a beaker. Weigh 2-morpholinoethanesulfonic acid (MES, manufacturer: Sigma, item number: V900336), NaCl (sodium chloride), Tween 20, bovine serum albumin (BSA), trehalose (manufacturer: Wako, item number: 206-18455), and Proclin-300 (preservative) into the beaker and mix thoroughly using a magnetic stirrer.

[0050] 2.3. Preparation of Buffer 2 Weigh 1000g of purified water and place it in a beaker. Weigh out Tris-HCl (manufacturer: VWR, item number: 0234-1KG), NaCl (sodium chloride), bovine serum albumin (BSA), newborn calf serum (manufacturer: Zhejiang Tianhang, item number: 40212-8615), and Proclin-300 (preservative) and pour them into the beaker. Mix thoroughly with a magnetic stirrer.

[0051] 2.4. Preparation of Buffer 3 Weigh 1000g of purified water and place it in a beaker. Weigh out tris(hydroxymethyl)aminomethane (Tris (manufacturer: Sigma, catalog number: V900483)), bovine serum albumin (BSA), trehalose, casein (Casein (manufacturer: Sigma, catalog number: C8654)), and Proclin-300 (preservative) and pour them into the beaker. Mix thoroughly with a magnetic stirrer.

[0052] 2.5. Preparation of Buffer 4 Weigh 1000g of purified water and place it in a beaker. Weigh HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), NaCl (sodium chloride), lauryl alcohol polyoxyethylene ether (manufacturer: VWR, item number: 0217-1KG), bovine serum albumin (BSA), glycerin (manufacturer: Beijing Solarbio Technology Co., Ltd., item number: G8190), and Proclin-300 (preservative) into the beaker and mix thoroughly using a magnetic stirrer.

[0053] 2.6. Preparation of Buffer 5 Weigh 1000g of purified water and place it in a beaker. Weigh out Na2HPO4 (manufacturer: Aladdin, item number: S112446), NaH2PO4·2H2O (manufacturer: Shanghai Trial, item number: 20040718), NaCl, bovine serum albumin (BSA), sucrose (manufacturer: Shanghai Trial, item number: 10021463), and Proclin-300 (preservative) and pour them into the beaker. Mix thoroughly with a magnetic stirrer.

[0054] All buffer formulations are shown in Table 1.

[0055] Table 1 Buffer Formulation

[0056] 3. Preparation of working solution 3.1 Preparation of working solution for magnetic bead antibody conjugate Add the conjugated magnetic bead antibody conjugate to the control buffer to prepare a working solution of 0.2-0.4 mg / ml magnetic bead antibody conjugate.

[0057] 3.2 Preparation of working solution for acridine ester labeled antibody After coupling, the acrid ester-labeled antibody was added to control buffer, buffer 1, buffer 2, buffer 3, buffer 4, and buffer 5 to prepare a 0.1-1 μg / mL acrid ester-labeled antibody working solution.

[0058] 4. Testing Using the working solutions prepared in 3.1 and 3.2, sodium citrate anticoagulated plasma and EDTA anticoagulated plasma of the same person were tested separately. It was preferred to test combinations of different sample types with similar measured values. Then, more AD / NC samples were tested, and the combination of buffer systems that could significantly distinguish AD / NC patients was preferred.

[0059] II. Experimental Results Table 2. Results of anticoagulant plasma tests in different anticoagulant samples using different buffer solutions.

[0060] Table 3. Validation results of buffer 4 anticoagulant plasma.

[0061] The control buffer was used as the initial buffer, and five other buffers were screened. Samples were obtained from the same individual, and plasma was obtained using two different anticoagulants. Table 2 shows that the measured values ​​of the two different sample types were similar under buffer system 4. A good gradient was also observed for different concentrations of the calibrator (manufacturer: Beijing Bio-Sens Biotechnology Co., Ltd., catalog number: bs-41217P).

[0062] Validation of Buffer 4 (Table 3): Consistent measurements were observed for samples with two different anticoagulations. Simultaneously, tests were conducted using 4 samples from Alzheimer's disease (AD) and 4 from healthy individuals (NC) (collected in August 2025 at Xuanwu Hospital, Capital Medical University), showing inter-group differences. Buffer 4 was ultimately selected as the component buffer for the NfL acridinium ester-labeled antibody working solution.

[0063] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A buffer composition for reducing target detection bias among different anticoagulated plasma samples, characterized in that, The buffer composition comprises: 4-hydroxyethylpiperazine ethanesulfonic acid, sodium chloride, lauryl alcohol polyoxyethylene ether, bovine serum albumin, glycerol, and Proclin-300; Preferably, the different anticoagulated plasma samples are sodium citrate anticoagulated plasma samples and EDTA anticoagulated plasma samples; Preferably, the target is a neurofilament light chain protein; Preferably, the detection is an immune detection.

2. A kit for reducing target detection bias among different anticoagulated plasma samples, characterized in that, The kit comprises the buffer composition of claim 1; Preferably, the different anticoagulated plasma samples are sodium citrate anticoagulated plasma samples and EDTA anticoagulated plasma samples.

3. The reagent kit according to claim 2, characterized in that, The kit also includes a solid-phase support; Preferably, the solid support is selected from one or more of microplates, magnetic beads, latex particles, microfluidic chips, and test strips.

4. The reagent kit according to claim 2, characterized in that, The kit also contains a target-specific binding reagent; Preferably, the target-specific binding reagent is selected from one or more of antibodies, aptamers, receptors, and ligands.

5. The reagent kit according to claim 2, characterized in that, The kit also includes calibrators and / or quality control products; Preferably, the calibrators and / or quality control materials are sodium citrate anticoagulated plasma matrix and / or EDTA anticoagulated plasma matrix containing known concentrations of the target. Preferably, the kit further includes reagents for processing samples.

6. A detection system, characterized in that, The detection system includes: (1) The buffer composition of claim 1, or the kit of any one of claims 2-5; and (2) Testing instruments; Preferably, the detection instrument is selected from an immunoassay analyzer.

7. A method for reducing target detection bias among plasma samples with different anticoagulants, characterized in that, The method includes: The target-specific binding reagent is dissolved using the buffer composition of claim 1 to obtain the detection working solution; Different anticoagulated plasma samples were tested using the test working solution; Preferably, the different anticoagulated plasma samples are sodium citrate anticoagulated plasma samples and EDTA anticoagulated plasma samples; Preferably, the target-specific binding agent is an antibody against the target; Preferably, the antibody is an acridinium ester-labeled antibody; Preferably, the target is neurofilament light chain protein.

8. A method for detecting a target in a plasma sample, characterized in that, The method includes: (1) Magnetic beads coated with target-specific binding reagents; (2) Acridinium ester-labeled target-specific binding reagents; (3) Add the buffer composition according to claim 1 to obtain the detection working solution; (4) Use the detection working solution to test different anticoagulated plasma samples; Preferably, the different anticoagulated plasma samples are sodium citrate anticoagulated plasma samples and EDTA anticoagulated plasma samples; Preferably, the target-specific binding agent is an antibody against the target; Preferably, the antibody is an acridinium ester-labeled antibody; Preferably, the target is neurofilament light chain protein.

9. The use of the buffer composition of claim 1 in the preparation of a kit for reducing target detection bias between sodium citrate-anticoagulated plasma samples and EDTA-anticoagulated plasma samples.

10. The use of the buffer composition of claim 1, the kit of any one of claims 2-5, or the detection system of claim 6 in the in vitro detection of neurofilament light chain proteins in plasma.

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