A kit for determining the content of 8-isoprostaglandin F2a in urine and a preparation method thereof

Abstract

The present disclosure relates to the field of immunoassay, in particular to a kit for determining the content of 8-isoprostane F2a in urine and a preparation method thereof. The present disclosure provides a kit for detecting the content of 8-isoprostane F2a in urine with high sensitivity, accurate results, simple operation and strong anti-interference ability, and a preparation method thereof, which provides a better detection scheme for the clinical and scientific research fields.

Description

Technical Field

[0001] This disclosure relates to the field of immunoassay, specifically to a kit for determining the content of 8-isoprostaglandin F2α in urine and its preparation method. Background Technology

[0002] Oxidative stress in aging is a pathological state in which the body, under pathological or external stimuli, generates free radicals such as reactive oxygen species (ROS) at a rate exceeding the body's own antioxidant defense system's ability to scavenge them, leading to an imbalance in redox equilibrium. Because highly reactive oxygen species such as superoxide anions and hydroxyl radicals have extremely short half-lives and extremely low concentrations in the body, they cannot be directly and quantitatively detected in situ. Currently, in clinical practice, the level of oxidative damage in the body is usually reflected indirectly by detecting DNA oxidation products (such as 8-OHdG), protein carbonylation products, or lipid peroxidation end products. Among these, F2-isoprostanes, especially 8-isoprostanes F2α (8-iso-PGF2α), generated from arachidonic acid in cell membrane phospholipids via a non-enzymatic peroxidation pathway, are widely recognized as the "gold standard" biomarker for evaluating oxidative stress damage in the body because they are synthesized independently of the cyclooxygenase pathway, are stable in body fluids for a long time, and exhibit a good dose-response relationship with the degree of oxidative stress. The non-invasive collection and stable expression characteristics of 8-iso-PGF2α in urine make it an important detection indicator for screening and epidemiological studies of oxidative stress-related diseases.

[0003] Currently, the main methods for quantitative analysis of 8-isoprostaglandin F2α in urine include enzyme-linked immunosorbent assay (ELISA), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While LC-MS / MS offers high sensitivity and selectivity, the complex urine matrix and numerous coexisting structural analogs necessitate cumbersome pretreatment steps such as solid-phase extraction or liquid-liquid extraction for purification and concentration. This process is complex and prone to human error, and matrix effects can also interfere with quantitative results. Commercially available ELISA kits are relatively easy to use, but they are susceptible to antibody cross-reactivity, resulting in poor sensitivity and batch-to-batch reproducibility, and inconsistent results with mass spectrometry.

[0004] Chemiluminescence immunoassay (CLIA) combines highly sensitive chemiluminescence assays with highly specific immunoreactions, offering advantages such as high sensitivity, wide detection range, simple and rapid operation, and stable labeling. Combining magnetic microparticle separation technology with chemiluminescence, leveraging the large specific surface area and uniformity of magnetic microparticles, can further improve reaction efficiency and specificity, reduce non-specific binding, and achieve automated detection.

[0005] Therefore, there is an urgent need to develop a method for quantifying urinary 8-isoprostaglandin F2α that is highly sensitive, specific, easy to operate, quick and suitable for automated clinical detection. Summary of the Invention

[0006] This disclosure aims to provide a rapid, accurate, and convenient kit for determining the content of 8-isoprostaglandin F2α in urine and its preparation method.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted in this disclosure is as follows: In one aspect, this disclosure provides a kit for detecting the content of 8-isoprostaglandin F2α in urine, comprising streptavidin-labeled anti-8-isoprostaglandin F2α polyclonal antibody, alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen, analytical buffer, calibrators, and quality control materials.

[0008] In some embodiments, the method for preparing the streptavidin-labeled anti-8-isoprostaglandin F2α polyclonal antibody is as follows: Dissolve the anti-8-isoprostaglandin F2α polyclonal antibody in PBS buffer; The anti-8-isoprostaglandin F2α polyclonal antibody was mixed with biotin in a certain proportion and reacted at room temperature in the dark for 30 minutes. Free biotin was removed by dialysis or desalting column to obtain biotin-labeled anti-8-isoprostaglandin F2α polyclonal antibody; Commercial streptavidin-labeled paramagnetic nanoparticles were taken, thoroughly shaken and mixed, and then magnetically separated and washed. The biotin-labeled anti-8-isoprostaglandin F2α polyclonal antibody prepared above was added to the washed streptavidin magnetic beads, mixed well, and incubated to allow the biotin-labeled antibody to fully bind to the streptavidin magnetic beads. After incubation, the magnetic beads were resuspended in phosphate buffer containing 0.1% BSA. The ligated magnetic beads were then resuspended to the required concentration in phosphate buffer containing 0.1% BSA to obtain the streptavidin-labeled anti-8-isoprostaglandin F2α polyclonal antibody.

[0009] In a preferred embodiment, the pH of the PBS buffer is 7.4.

[0010] In a preferred embodiment, the concentration of the anti-8-isoprostaglandin F2α polyclonal antibody is 1 mg / mL.

[0011] In some embodiments, the molar ratio of the anti-8-isoprostaglandin F2α polyclonal antibody to biotin is 1:20.

[0012] In some implementations, the incubation time is 30 minutes.

[0013] In some embodiments, the alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen is prepared as follows: Alkaline phosphatase is activated by adding an activating agent; The activated alkaline phosphatase was mixed with 8-isoprostaglandin F2α antigen in a certain ratio, reacted at room temperature for 2 hours, and the enzyme-labeled antigen was purified by filtration chromatography. The alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen was obtained by diluting it to the working concentration with phosphate buffer containing 1% BSA.

[0014] In some implementations, the activated alkaline phosphatase is mixed with 8-isoprostaglandin F2α antigen at a molar ratio of 1:1.2.

[0015] In some embodiments, the analysis buffer contains 50 mM Tris, 0.15 M NaCl, 0.1% Tween-20, 1% BSA, and 10% fetal bovine serum.

[0016] In some implementations, preparing the analytical buffer includes the following steps: It is prepared using a buffer solution containing Tris and filtered through a filter membrane.

[0017] In a preferred embodiment, the pore size of the filter membrane is 0.22 μm.

[0018] In some implementations, the 8-isoprostaglandin F2α standard is serially diluted to a series of concentrations using calibration buffer, aliquoted and lyophilized or stored in liquid form as a calibrator and quality control sample.

[0019] Secondly, this disclosure provides the application of the kit described in the first aspect of this disclosure, the kit being used for the detection of 8-isoprostaglandin F2α levels in urine.

[0020] This disclosure has the following advantages: Compared with existing technologies, this disclosure provides a kit and its preparation method for detecting the content of 8-isoprostaglandin F2α in urine, which has high sensitivity, accurate results, simple operation and strong anti-interference ability, providing a better detection solution for clinical and scientific research fields. Detailed Implementation

[0021] Definitions and Explanations To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. In this disclosure, unless otherwise stated, the scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Furthermore, the cell and tissue culture, microbiology-related terms, and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this disclosure, definitions and explanations of relevant terms are provided below. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. It should also be understood that the terminology used in this application is for describing specific embodiments only and is not intended to be limiting.

[0022] Unless otherwise expressly stated, the terms “a,” “an,” and “the” as used in this specification and the appended claims cover one or more types.

[0023] As used herein, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0024] In the description herein, references to “some embodiments,” “some implementations,” or “some implementation schemes” describe a subset of all possible embodiments. However, it is understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0025] In a first aspect, this disclosure provides a kit for determining the content of 8-isoprostaglandin F2α in urine, comprising streptavidin-labeled anti-8-isoprostaglandin F2α polyclonal antibody, alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen, analytical buffer, calibrators, and quality control materials. Wherein: -The streptavidin magnetic bead-labeled anti-8-isoprostaglandin F2α polyclonal antibody is used to capture 8-isoprostaglandin F2α in the test sample; - The alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen is used to compete with 8-isoprostaglandin F2α in the test sample for binding to the antibody on the magnetic beads; - The calibrators and quality control products contain 8-isoprostaglandin F2α standards at known concentrations.

[0026] Preferably, the analytical buffer contains bovine serum albumin or calf serum to reduce nonspecific adsorption.

[0027] In some embodiments, the method for preparing the streptavidin-labeled anti-8-isoprostaglandin F2α polyclonal antibody is as follows: Dissolve the anti-8-isoprostaglandin F2α polyclonal antibody in PBS buffer; The anti-8-isoprostaglandin F2α polyclonal antibody was mixed with biotin in a certain proportion and reacted at room temperature in the dark for 30 minutes. Free biotin was removed by dialysis or desalting column to obtain biotin-labeled anti-8-isoprostaglandin F2α polyclonal antibody; Commercial streptavidin-labeled paramagnetic nanoparticles were taken, thoroughly shaken and mixed, and then magnetically separated and washed. The biotin-labeled anti-8-isoprostaglandin F2α polyclonal antibody prepared above was added to the washed streptavidin magnetic beads, mixed well, and incubated to allow the biotin-labeled antibody to fully bind to the streptavidin magnetic beads. After incubation, the magnetic beads were resuspended in phosphate buffer containing 0.1% BSA. The ligated magnetic beads were then resuspended to the required concentration in phosphate buffer containing 0.1% BSA to obtain the streptavidin-labeled anti-8-isoprostaglandin F2α polyclonal antibody.

[0028] In a preferred embodiment, the pH of the PBS buffer is 7.4.

[0029] In a preferred embodiment, the concentration of the anti-8-isoprostaglandin F2α polyclonal antibody is 1 mg / mL.

[0030] In some embodiments, the molar ratio of the anti-8-isoprostaglandin F2α polyclonal antibody to biotin is 1:20.

[0031] In some implementations, the incubation time is 30 minutes.

[0032] In some embodiments, the alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen is prepared as follows: Alkaline phosphatase is activated by adding an activating agent; The activated alkaline phosphatase was mixed with 8-isoprostaglandin F2α antigen in a certain ratio, reacted at room temperature for 2 hours, and the enzyme-labeled antigen was purified by filtration chromatography. The alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen was obtained by diluting it to the working concentration with phosphate buffer containing 1% BSA.

[0033] In some implementations, the activated alkaline phosphatase is mixed with 8-isoprostaglandin F2α antigen at a molar ratio of 1:1.2.

[0034] In some embodiments, the analysis buffer contains 50 mM Tris, 0.15 M NaCl, 0.1% Tween-20, 1% BSA, and 10% fetal bovine serum.

[0035] In some implementations, preparing the analytical buffer includes the following steps: It is prepared using a buffer solution containing Tris and filtered through a filter membrane.

[0036] In a preferred embodiment, the pore size of the filter membrane is 0.22 μm.

[0037] In some implementations, the 8-isoprostaglandin F2α standard is serially diluted to a series of concentrations using calibration buffer, aliquoted and lyophilized or stored in liquid form as a calibrator and quality control sample.

[0038] Secondly, this disclosure provides the application of the kit described in the first aspect of this disclosure, the kit being used for the detection of 8-isoprostaglandin F2α levels in urine.

[0039] This kit employs a competitive assay principle. Anti-8-isoprostaglandin F2α polyclonal antibody is pre-linked to the surface of magnetic beads via a biotin-streptavidin system, forming an antibody-magnetic bead complex. 8-isoprostaglandin F2α in the test sample competes with alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen for binding to the antibody on the magnetic beads. After magnetic separation and washing, a chemiluminescent substrate is added, and the intensity of the luminescence signal is detected. Higher concentrations of 8-isoprostaglandin F2α in the sample result in less enzyme-labeled antigen binding to the magnetic beads and a weaker luminescence signal; conversely, lower concentrations result in lower concentrations. The concentration in the sample can be calculated using a standard curve established with calibrators.

[0040] This reagent kit can be used with the fully automated chemiluminescence analyzers (LA2000, LA3000) from Sichuan Wovente Biotechnology Co., Ltd. to achieve high-throughput, automated detection.

[0041] Example To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions in the embodiments of this disclosure are described clearly and completely. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. The following is merely a further description of this disclosure, and the scope of protection of this disclosure is not limited thereto. In the embodiments and experimental examples of this disclosure, the technical means or methods, etc., not specifically described, are conventional technical means or methods in the art. Unless otherwise specified, the materials, reagents, experimental equipment, etc., used in the embodiments and experimental examples of this disclosure can all be obtained commercially.

[0042] Example 1: Preparation of the reagent kit 1.1 Preparation of Biotin-Labeled Anti-8-Isoprostaglandin F2α Polyclonal Antibody Anti-8-isoprostaglandin F2α polyclonal antibody was dissolved in PBS buffer (pH 7.4) at a concentration of 1 mg / mL. Activated biotin was added at a molar ratio of antibody to biotin of 1:20, and the reaction was carried out at room temperature in the dark for 30 minutes. Free biotin was removed by dialysis or a desalting column to obtain biotin-labeled anti-8-isoprostaglandin F2α polyclonal antibody. An equal volume of glycerol was added, and the mixture was aliquoted and stored at -20°C.

[0043] 1.2 Preparation of streptavidin magnetic beads coated with anti-8-isoprostaglandin F2α polyclonal antibody Commercially available streptavidin-labeled paramagnetic nanoparticles were thoroughly shaken and mixed. An appropriate amount of the magnetic bead suspension was placed in a centrifuge tube. The centrifuge tube was placed on a magnetic rack and allowed to stand for 1 minute until the magnetic beads were completely aggregated. The supernatant was then carefully removed. The magnetic beads were resuspended in phosphate buffer (pH 7.4) containing 0.1% BSA. The mixture was then magnetically separated and washed again. This washing process was repeated twice to remove preservatives and protectants.

[0044] The biotin-labeled anti-8-isoprostaglandin F2α polyclonal antibody prepared above was added to the washed streptavidin magnetic beads and gently mixed by pipetting. The mixture was then placed on a rotary mixer and incubated slowly at room temperature for 30 minutes to allow the biotin-labeled antibody to fully bind to the streptavidin magnetic beads.

[0045] After incubation, place the centrifuge tubes on a magnetic rack and let them stand for 1 minute until the magnetic beads are completely aggregated. Remove the supernatant. Resuspend the magnetic beads in phosphate buffer containing 0.1% BSA, and wash three times with magnetic separation to remove unbound free biotin-labeled antibody. Finally, resuspend the ligated magnetic beads to the desired concentration in phosphate buffer containing 0.1% BSA and store at 2-8°C for later use.

[0046] 1.3 Preparation of alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen Take alkaline phosphatase (ALP) and add an activator (such as SMCC) to activate it.

[0047] The activated ALP was mixed with 8-isoprostaglandin F2α antigen at a molar ratio of 1:1.2 and reacted at room temperature for 2 hours. The enzyme-labeled antigen was then purified by filtration chromatography.

[0048] Dilute to working concentration with phosphate buffer containing 1% BSA and store at 2-8°C.

[0049] 1.4 Preparation of analytical buffer Prepare a buffer solution containing 50 mM Tris, 0.15 M NaCl, 0.1% Tween-20, 1% BSA and 10% fetal bovine serum, adjust the pH to 7.4, and filter it through a 0.22 μm filter membrane for later use.

[0050] 1.5 Preparation of calibrators and quality control samples The 8-isoprostaglandin F2α standard was serially diluted to a series of concentrations using calibration buffer (containing 0.5% BSA), aliquoted and lyophilized or stored in liquid form as calibrators and quality control samples.

[0051] Example 2: Reagent Kit Performance Evaluation 2.1 Reaction Mode: Take 50 μL of calibrator or urine sample to be tested, add 50 μL of streptavidin-labeled anti-8-isoprostaglandin F2α polyclonal antibody, then add 50 μL of analysis buffer, and incubate at 37°C for 10 minutes.

[0052] Add 50 μL of alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen and incubate at 37°C for 10 minutes.

[0053] After magnetic separation, wash three times with cleaning solution.

[0054] Add 100 μL of chemiluminescent substrate solution, incubate at 37°C for 5 minutes, and then detect the luminescence intensity (RLU) on a fully automated chemiluminescence analyzer.

[0055] 2.2 Blank Limit The assay was repeated 20 times with zero calibrator, and the mean RLU value and standard deviation were calculated. The concentration value corresponding to the mean RLU minus twice the standard deviation is the blank limit. The results showed that the blank limit of the kit was less than 5.00 pg / mL.

[0056] 2.3 Linear Range High-value samples were diluted proportionally to five concentrations, and each concentration was measured three times. A linear regression was performed between the average measured concentration and the theoretical concentration, with a correlation coefficient r ≥ 0.9990. The results indicate that the kit exhibits good linearity in the range of 10.00 pg / mL to 2000.00 pg / mL.

[0057] 2.4 Precision The test was repeated 10 times each using quality control samples at low and high levels, and the average value was calculated. ), standard deviation (SD), and coefficient of variation (CV). The calculation formula is: In the formula: CV — Coefficient of Variation; SD — Standard deviation of 10 measurements; --The average value of 10 measurements The results showed that the CV of both the low and high quality control samples was less than 5%, indicating that the kit had good precision.

[0058] 2.5 Accuracy Test Using the company's reference sample as a sample, repeat the measurement three times according to the instructions, and calculate the relative deviation of the measured values.

[0059] 2.6. Reagent thermal stability The reagent kit was stored in a 37℃ incubator for 3, 6, and 9 days, and then its performance was verified. Two quality control samples (low and high levels) were used, and each was measured three times. The deviations of the measured values ​​from the initial values ​​at each time point were calculated. The results showed that the deviations of the measured values ​​at each time point were within ±10%, and there was no significant attenuation in the luminescence signal intensity, indicating that the reagent kit can be stably stored at 37℃ for at least 9 days and has good thermal stability.

[0060] In summary, the kit provided in this disclosure employs a competitive detection method using streptavidin magnetic beads-labeled anti-8-isoprostaglandin F2α polyclonal antibody and enzyme-labeled antigen, combined with magnetic microparticle chemiluminescence technology, to achieve rapid, sensitive, and accurate detection of 8-isoprostaglandin F2α in urine. It is easy to operate, suitable for automated analysis, and has good application prospects.

[0061] The foregoing description of specific exemplary embodiments of this disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit this disclosure to the precise form disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of this disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of this disclosure, as well as various different choices and variations. The scope of this disclosure is intended to be defined by the claims and their equivalents.

Claims

1. A kit for detecting the content of 8-isoprostaglandin F2α in urine, characterized in that, This includes streptavidin-labeled anti-8-isoprostaglandin F2α polyclonal antibody, alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen, analytical buffer, calibrators, and quality control products.

2. The reagent kit according to claim 1, characterized in that, The method for preparing the streptavidin-labeled anti-8-isoprostaglandin F2α polyclonal antibody is as follows: Dissolve the anti-8-isoprostaglandin F2α polyclonal antibody in PBS buffer; The anti-8-isoprostaglandin F2α polyclonal antibody was mixed with biotin in a certain proportion and reacted at room temperature in the dark for 30 minutes. Free biotin was removed by dialysis or desalting column to obtain biotin-labeled anti-8-isoprostaglandin F2α polyclonal antibody; Commercial streptavidin-labeled paramagnetic nanoparticles were taken, thoroughly shaken and mixed, and then magnetically separated and washed. The biotin-labeled anti-8-isoprostaglandin F2α polyclonal antibody prepared above was added to the washed streptavidin magnetic beads, mixed well, and incubated to allow the biotin-labeled antibody to fully bind to the streptavidin magnetic beads. After incubation, the magnetic beads were resuspended in phosphate buffer containing 0.1% BSA. The ligated magnetic beads were then resuspended in phosphate buffer containing 0.1% BSA to the required concentration to obtain the streptavidin-labeled anti-8-isoprostaglandin F2α polyclonal antibody. Preferably, the pH of the PBS buffer is 7.4; Preferably, the concentration of the anti-8-isoprostaglandin F2α polyclonal antibody is 1 mg / mL.

3. The reagent kit according to claim 2, characterized in that, The molar ratio of the anti-8-isoprostaglandin F2α polyclonal antibody to biotin is 1:

20.

4. The reagent kit according to claim 2, characterized in that, The incubation time is 30 minutes.

5. The reagent kit according to claim 1, characterized in that, The method for preparing the alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen is as follows: Alkaline phosphatase is activated by adding an activating agent; The activated alkaline phosphatase was mixed with 8-isoprostaglandin F2α antigen in a certain ratio, reacted at room temperature for 2 hours, and the enzyme-labeled antigen was purified by filtration chromatography. The alkaline phosphatase-labeled 8-isoprostaglandin F2α antigen was obtained by diluting it to the working concentration with phosphate buffer containing 1% BSA.

6. The reagent kit according to claim 5, characterized in that, The activated alkaline phosphatase was mixed with 8-isoprostaglandin F2α antigen at a molar ratio of 1:1.

2.

7. The kit according to any one of claims 1-6, characterized in that, The analytical buffer contains 50 mM Tris, 0.15 M NaCl, 0.1% Tween-20, 1% BSA and 10% fetal bovine serum.

8. The reagent kit according to claim 7, characterized in that, The preparation of the analytical buffer includes the following steps: It is prepared using a buffer solution containing Tris and then filtered through a filter membrane. Preferably, the pore size of the filter membrane is 0.22 μm.

9. The kit according to any one of claims 1-8, characterized in that, The 8-isoprostaglandin F2α standard was serially diluted to a series of concentrations using calibration buffer, aliquoted and lyophilized or stored in liquid form as calibrators and quality control products.

10. The application of the reagent kit according to any one of claims 1-9, characterized in that, The kit is used to detect the content of 8-isoprostaglandin F2α in urine.

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