A kit for chemiluminescence detection of FGF21

Abstract

This invention relates to the field of in vitro diagnostic technology, and in particular to a chemiluminescence immunoassay kit for detecting FGF21. The kit includes a solid support, a label, a sample diluent, and a chemiluminescent substrate solution. The solid support is a magnetic microsphere with Protein A / G bound to its surface, and an anti-FGF21 capture antibody is immobilized on the microsphere via Protein A / G. The label is a conjugate of the anti-FGF21 detection antibody and polymeric horseradish peroxidase. The chemiluminescent substrate solution contains luminol and hydrogen peroxide, and the sample diluent contains a zwitterionic surfactant and a cyclodextrin derivative. This invention utilizes Protein A / G-mediated antibody fixation to ensure sufficient exposure of the antigen-binding fragment of the capture antibody, effectively improving the capture efficiency of low-concentration FGF21. Simultaneously, the use of polymeric horseradish peroxidase as a label enables signal cascade amplification, and the synergistic effect of both meets the requirements for ultrasensitive detection.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and in particular to a kit for detecting FGF21 using chemiluminescence immunoassay. Background Technology

[0002] FGF21 is an important metabolic regulatory hormone secreted by the liver. It is significantly elevated in patients with non-alcoholic fatty liver disease, type 2 diabetes, and obesity, and has become a biomarker of clinical interest. Currently, the mainstream detection method is magnetic particle chemiluminescence immunoassay (CLIA).

[0003] Patent (CN202410852849.5) discloses an FGF21 detection kit. Although this method achieves automated detection, it faces the following technical obstacles in practical applications: its acridil ester label has poor stability, is sensitive to light and heat, and is easily hydrolyzed, resulting in large batch-to-batch variability (CV>8%) and high cost; the antibody is randomly coupled to the surface of magnetic beads, and some antigen binding sites are masked, resulting in insufficient capture efficiency of low-concentration FGF21, limiting the improvement of sensitivity (LOD approximately 15 pg / mL); complex samples cause severe interference, and non-specific adsorption is significant in high-lipid and hemolyzed samples, affecting the accuracy of the results. Summary of the Invention

[0004] The present invention aims to at least solve the technical problems existing in the prior art. To this end, the present invention proposes a kit for detecting FGF21 by chemiluminescence, which has good stability, high sensitivity, strong anti-interference, and compatibility with existing chemiluminescence platforms.

[0005] A chemiluminescence assay kit for detecting FGF21 according to some embodiments of the present invention includes a solid support, a label, a sample diluent, and a chemiluminescence substrate solution. The solid support is a magnetic microsphere with Protein A / G bound to its surface, and an anti-FGF21 capture antibody is immobilized on the magnetic microsphere via Protein A / G. The label is a conjugate of an anti-FGF21 detection antibody and polymerized horseradish peroxidase. The chemiluminescence substrate solution contains luminol and hydrogen peroxide, and the sample diluent contains an amphoteric surfactant and a cyclodextrin derivative. The method for preparing the Protein A / G magnetic microspheres is as follows: preparing surface carboxylated magnetic microspheres, activating the magnetic microspheres and coupling them with Protein A / G to obtain Protein A / G modified magnetic beads, and binding the anti-FGF21 capture antibody to the Protein A / G modified magnetic beads.

[0006] A chemiluminescence assay kit for detecting FGF21 according to some embodiments of the present invention has at least the following beneficial effects: This invention utilizes Protein A / G-mediated antibody fixation to ensure full exposure of the antigen-binding fragments of the captured antibody, effectively improving the capture efficiency of low-concentration FGF21. Simultaneously, it employs polymerized horseradish peroxidase as a label to achieve cascaded signal amplification. The synergistic effect of these two technologies lowers the detection limit to 1.2 pg / mL, an improvement of more than an order of magnitude compared to existing technologies, meeting the requirements for ultrasensitive detection. The introduction of zwitterionic surfactants and cyclodextrin derivatives into the sample diluent effectively shields against interfering substances such as high triglycerides, bilirubin, and hemoglobin. It abandons the light- and heat-sensitive acridine ester labeling method, instead using a stable PolyHRP system. Combined with lyophilized calibrators and an optimized buffer system, the reagents maintain stability for over 12 months at 4°C. The entire detection process is compatible with mainstream fully automated chemiluminescence analyzers, requiring no equipment modification and facilitating clinical promotion and industrial application.

[0007] According to some embodiments of the present invention, a chemiluminescence assay kit for detecting FGF21 is provided, wherein the concentration of the magnetic microspheres is 20–60 mg / L.

[0008] According to some embodiments of the present invention, a chemiluminescence assay kit for detecting FGF21 is provided, wherein the concentration of the marker is 0.1–0.5 mg / L.

[0009] According to some embodiments of the present invention, a chemiluminescence assay kit for detecting FGF21 is provided, wherein the magnetic microspheres have an iron oxide core and a polymer shell.

[0010] According to some embodiments of the present invention, a chemiluminescence assay kit for detecting FGF21 is provided, wherein the polymer shell is polylactic acid.

[0011] According to some embodiments of the present invention, a chemiluminescence assay kit for detecting FGF21 is provided, wherein the anti-FGF21 capture antibody binds to Protein A / G via its Fc fragment.

[0012] According to some embodiments of the present invention, a chemiluminescence assay kit for detecting FGF21 is provided, wherein the polymeric horseradish peroxidase is in polymeric form, and each molecule contains multiple horseradish peroxidase units.

[0013] According to some embodiments of the present invention, a chemiluminescence assay kit for detecting FGF21 is provided, wherein the zwitterionic surfactant is SB3-10 with a concentration of 0.05–0.15 wt%; and the cyclodextrin derivative is hydroxypropyl-β-cyclodextrin with a concentration of 0.1–0.2 wt%.

[0014] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Detailed Implementation

[0015] In the description of this invention, it should be understood that the directional descriptions, such as up, down, left, right, front, and back, are only for the purpose of describing this invention and simplifying the description, and do not indicate or imply that the module or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0016] In the description of this invention, the use of "first" and "second" is for the purpose of distinguishing technical features only, and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or the order of the technical features indicated.

[0017] In the description of this invention, unless otherwise explicitly defined, terms such as "set up," "install," and "connect" should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0018] Example 1: The preparation steps of Protein A / G modified magnetic microspheres are as follows: Step S1: Preparation of carboxylated Fe3O4@PLA magnetic beads: 100 mg of Fe3O4 nanoparticles were dispersed in 50 mL of chloroform; Dissolve 200 mg PLA (Mw = 10,000) in water, inject into 500 mL of 0.5% PVA aqueous solution, and emulsify at high speed; evaporate chloroform, collect microspheres by centrifugation; hydrolyze the ester bonds on the PLA surface with 0.1 M NaOH to introduce carboxyl groups, and obtain carboxylated magnetic beads.

[0019] Step S2: Protein A / G Coupling Take 10 mg of carboxylated magnetic beads, perform magnetic separation, and wash three times with 0.02 M pH 7.4 Tris-NaCl; add 1 mL of 0.05 M pH 6.0 MES buffer, then add 100 μL each of 10 mg / mL LEDC and 12 mg / mL NHS, and shake at room temperature for 30 min; perform magnetic separation, discard the supernatant, add 1 mL of 0.1 mg / mL His-Protein A / G fusion protein solution (dissolved in MES), and shake at room temperature for 2 h; perform magnetic separation, and wash three times with blocking buffer (0.05 M Tris-NaCl, pH 7.4, 1% BSA).

[0020] Step S3: Capture antibody fixation Add 0.2 mg / mL of anti-FGF21 capture antibody (recognizing the N-terminal epitope), bind at 4°C for 2 h; block with BSA, resuspend in blocking buffer, and store at 4°C.

[0021] The preparation of PolyHRP-detected antibody markers is as follows: Material: PolyHRP (ThermoFisher, Cat#20243), with an average of 40 HRP molecules; anti-FGF21 detection antibody (recognizes C-terminal epitope, IgG1 type).

[0022] Step T1: Antibody-PolyHRP conjugation Dissolve 1 mg PolyHRP in 0.5 mL of 0.1 M NaHCO3 (pH 8.5); add 0.2 mg SMCC (dissolved in DMF), and react at room temperature in the dark for 1 h; remove unreacted SMCC using a desalting column; add 0.3 mg of detection antibody, and react at 4 °C for 12 h; stop the reaction by adding 10 mM glycine; purify by dialysis, and stabilize with 1% BSA to obtain the PolyHRP-antibody complex.

[0023] Assembly and detection process of the reagent kit of this invention Group allocation system: Sample dilution buffer: 100 mM PBS, 5 mM MTCEP, 0.1 wt% SB3-10, 0.15 wt% hydroxypropyl-β-cyclodextrin, 1 wt% BSA; Chemiluminescent substrate solution: 1 mM luminol, 0.3% H2O2, 0.1 M Tris-HCl (pH 8.5); Calibrator: Lyophilized powder containing 5% trehalose, reconstituted at concentrations of 0, 5, 20, 80, 320, and 1280 pg / mL.

[0024] Testing process (IncreCareShinei1910): Add 50 μL of Protein A / G magnetic beads (40 mg / L) to the reaction vessel; add 50 μL of sample and incubate at 37°C for 10 min; perform magnetic separation and wash; add 50 μL of LyHRP-antibody (0.2 mg / L) and incubate at 37°C for 10 min; perform magnetic separation and wash. Add 200 μL of substrate solution and immediately read the RLU value.

[0025] The concentration of the magnetic microspheres is 20–60 mg / L, and the concentration of the label is 0.1–0.5 mg / L.

[0026] Comparative Example 1: The kit includes magnetic microspheres with FGF21-specific antibody conjugated to their surface, FGF21-specific antibody containing acridinium ester label, sample diluent, luminescence pre-excitation solution, excitation solution, and FGF21 calibrators at different concentrations. The sample diluent comprises the following ingredients: 100 mM PBS buffer, 4 mM tris(2-hydroxyethyl)phosphine TCEP, 0.1 wt% Tween 20, 0.1 wt% Triton X-100, 0.1 wt% dodecyl glucoside, and 0.5 wt% BSA.

[0027] A method for preparing magnetic microspheres with FGF21-specific antibodies conjugated to their surface includes the following steps: S1. Take 10 mg of magnetic microspheres, magnetically separate for 1 min, remove the supernatant, and wash three times with 0.02 mol / L pH 7.4 Tris-NaCl buffer. S2. Prepare 10 mg / mL EDC and 12 mg / mL NHS solutions using 0.05 mol / L pH 6.0 MES buffer, and take 100 μL of each solution into the magnetic microspheres cleaned in step S1, and shake to react for 30 min. S3. Separate the magnetic microspheres using a magnet, discard the supernatant, add 200 μL of 0.05 mol / L pH 6.0 MES buffer and 3 μL of FGF21 specific antibody solution, and shake at room temperature for 1 h. S4. Use a magnet to separate the magnetic microspheres, discard the supernatant, add 300 μL of blocking solution A, mix and shake, then magnetically separate and discard the supernatant again. Repeat this process three times, then add 3 mL of blocking solution A and shake at room temperature for 0.5 h to obtain the magnetic microspheres with FGF21 specific antibody coupled to the surface.

[0028] The magnetic microspheres are a core-shell structure formed by iron oxide as the core layer and polylactic acid polymer as the shell layer.

[0029] In step S4, the blocking solution A comprises the following components: 0.05 mol / L Tris-NaCl buffer at pH 7.4, 1% BSA, 0.01% ProClin-300, 5% sucrose, and 0.3% ascorbic acid.

[0030] Comparative Example 2: Replace only the markers with regular HRP, without PolyHRP, without Protein A / G. Magnetic beads: carboxylated Fe3O4@PLA, same as comparative example 1; Label: HRP-labeled detection antibody, HRP:antibody = 4:1; The remaining components are the same as in Example 1 of this invention; Luminescent substrate: luminol + H2O2.

[0031] Comparative Example 3: Only Protein A / G magnetic beads are used, but the labeling agent is still acridine ester. Magnetic beads: Protein A / G modified Fe3O4@PLA, same as in Example 1 of this invention; Labels: acridinium ester-labeled antibody, same as control group 1; others same as control group 1.

[0032] Test method descriptions for each performance indicator: 1. Detection Limit (LOD) Measurements were performed according to the CLSI EP17-A2 guideline: Prepare a blank matrix (such as a mixture of serum from healthy individuals) that does not contain FGF21. Repeat the test on blank samples ≥20 times and record the chemiluminescence signal value (RLU). Calculate the mean (Xˉblank) and standard deviation (SDblank) of the blank signal. LOD is defined as: LOD = (Xˉblank + 3×SDblank − Xˉblank) / Standard Curve Slope = Slope 3×SDblank The standard curve was obtained by fitting with known concentrations of FGF21 calibrators (0–1280 pg / mL).

[0033] 2. Recovery Rate Refer to CLSI EP06-A and EP09-A3 methods: Take three concentration levels of FGF21 calibrator (pg / mL, 80 pg / mL, and 500 pg / mL), respectively, and add them to normal serum and interfering samples (such as high-lipid serum with triglycerides of 8 mmol / L and hemolyzed serum with a hemolysis index of HI=400); each sample is tested three times, and the average measured concentration is calculated; the recovery rate (%) is calculated according to the following formula: Recovery rate (%) = (Measured concentration - Background concentration) / Added concentration × 100% Acceptance criteria: The recovery rate should be in the range of 85%–115%, and can be relaxed to 80%–120% for low concentrations.

[0034] 3. Precision (intra-batch coefficient of variation, CV) According to CLSIEP05-A3 guidelines: Quality control samples were selected at three concentration levels: low (~20 pg / mL), medium (~200 pg / mL), and high (~1000 pg / mL). Perform 10 consecutive tests in the same run; Calculate the mean (Xˉ) and standard deviation (SD) of the 10 results; The formula for calculating the intra-batch CV (%) is: CV(%) = SD / Xˉ × 100% Requirements: CV ≤ 5%, preferably ≤ 3%.

[0035] 4. Stability (accelerated stability at 4℃) Refer to the "Technical Guidelines for Stability Studies of In Vitro Diagnostic Reagents": Place each component of the kit (magnetic beads, label, diluent, substrate solution) according to the actual storage conditions (4±2℃); Samples were taken at 0, 1, 3, 6, 9, and 12 months. Using the same batch of calibrators and quality control samples, the signal values ​​of samples with medium concentrations (~200 pg / mL) were detected on the same instrument; with the initial signal value as 100%, the relative activity retention rate at each time point was calculated; Judgment criteria: A signal retention rate of ≥90% is considered stable.

[0036] 5. Performance of high triglyceride samples (8 mmol / L) Human serum samples with a triglyceride concentration of 8 mmol / L were prepared, and FGF21 standard was added to a final concentration of 5 pg / mL. The interfering sample was detected using the kit of this invention, while a normal serum sample with the same concentration was detected as a control. The recovery rate (%) of the high-lipid sample was calculated based on the value obtained from the normal sample to evaluate the anti-lipid interference ability.

[0037] Based on the testing methods for the above performance indicators, the following results were obtained: The present invention has the following advantages: Significantly improved detection sensitivity: Protein A / G-mediated antibody fixation ensures full exposure of the antigen-binding fragment (Fab) of the capture antibody, effectively improving the capture efficiency of low-concentration FGF21; simultaneously, polymeric horseradish peroxidase (PolyHRP) is used as a label to achieve cascaded signal amplification. The synergistic effect of these two technologies lowers the limit of detection (LOD) to 1.2 pg / mL, an improvement of more than an order of magnitude compared to existing technologies (approximately 15 pg / mL), meeting the requirements for ultrasensitive detection.

[0038] Excellent anti-interference ability: The introduction of zwitterionic surfactants (such as SB3-10) and cyclodextrin derivatives (such as hydroxypropyl-β-cyclodextrin) into the sample diluent can effectively shield interfering substances such as high triglycerides, bilirubin, and hemoglobin. In high-lipid samples with triglyceride concentrations as high as 8 mmol / L, the recovery rate of FGF21 remained stable at over 97%, significantly better than existing kits (recovery rate of approximately 76%).

[0039] Good stability and repeatability: Abandoning light- and heat-sensitive acridine ester labels, a stable PolyHRP system is used in conjunction with lyophilized calibrators and an optimized buffer system, so that the reagents are stable for more than 12 months at 4°C, with an intra-batch coefficient of variation (CV) ≤2.3% and significantly improved inter-batch consistency.

[0040] Lower costs and strong platform compatibility: PolyHRP is a commercially available and mature raw material, with a lower cost than imported acridine esters; the entire detection process is compatible with mainstream fully automated chemiluminescence analyzers (such as Phytoluminescence, New Industries, Mindray, etc.), requiring no equipment modification, which facilitates clinical promotion and industrial application.

[0041] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A kit for the chemiluminescence detection of FGF21, characterized in that, The apparatus includes a solid support, a label, a sample diluent, and a chemiluminescent substrate solution. The solid support is a magnetic microsphere with Protein A / G bound to its surface, and an anti-FGF21 capture antibody is immobilized on the magnetic microsphere via Protein A / G. The label is a conjugate of an anti-FGF21 detection antibody and polymerized horseradish peroxidase. The chemiluminescent substrate solution contains luminol and hydrogen peroxide, and the sample diluent contains an amphoteric surfactant and a cyclodextrin derivative. The method for preparing the Protein A / G magnetic microspheres is as follows: preparing surface carboxylated magnetic microspheres, activating the magnetic microspheres and coupling them with Protein A / G to obtain Protein A / G modified magnetic beads, and binding the anti-FGF21 capture antibody to the Protein A / G modified magnetic beads.

2. The kit for detecting FGF21 by chemiluminescence immunoassay according to claim 1, characterized in that: The concentration of the magnetic microspheres is 20–60 mg / L.

3. The kit for detecting FGF21 by chemiluminescence immunoassay according to claim 1, characterized in that: The concentration of the marker is 0.1–0.5 mg / L.

4. The kit for detecting FGF21 by chemiluminescence immunoassay according to claim 1, characterized in that: The magnetic microspheres have an iron oxide core and a polymer shell.

5. The kit for detecting FGF21 by chemiluminescence immunoassay according to claim 4, characterized in that: The polymer shell is polylactic acid.

6. The kit for detecting FGF21 by chemiluminescence immunoassay according to claim 1, characterized in that: The anti-FGF21 capture antibody binds to Protein A / G via its Fc segment.

7. The kit for detecting FGF21 by chemiluminescence immunoassay according to claim 1, characterized in that: The polymeric horseradish peroxidase is in polymeric form, with each molecule containing multiple horseradish peroxidase units.

8. The kit for detecting FGF21 by chemiluminescence immunoassay according to claim 1, characterized in that: The zwitterionic surfactant is SB3-10, with a concentration of 0.05–0.15 wt%; the cyclodextrin derivative is hydroxypropyl-β-cyclodextrin, with a concentration of 0.1–0.2 wt%.

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