Adiponectin detection method based on chemiluminescence

Abstract

The present application relates to the technical field of in vitro diagnosis, in particular to a chemiluminescence-based adiponectin detection method, which uses MQ silicon resin-coated Fe3O4 magnetic microspheres as a solid-phase carrier, captures adiponectin in a sample through a double-antibody sandwich immunoreaction, and uses HRP enzyme-labeled detection antibodies to combine with a luminol-phenolic enhanced chemiluminescence system for signal detection. The key lies in significantly reducing non-specific background by optimizing washing conditions and substrate formula, and regulating luminescence kinetics to adapt to the reading window of mainstream chemiluminescence instruments. The present application uses HRP enzyme-catalyzed cascade amplification and an optimized luminol enhancement system, and the detection lower limit is better than that of conventional methods, meeting the needs of early metabolic risk screening for ultra-low concentration detection, the linear detection range covers 0.01 mg / L to 100 mg / L, effectively avoiding the common hook effect in high-concentration samples, and the signal-to-noise ratio is greatly improved, ensuring the reliability of low-value sample detection.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and in particular to a method for detecting adiponectin based on chemiluminescence. Background Technology

[0002] Adiponectin is an important metabolic regulatory protein secreted by adipocytes. Decreased serum adiponectin levels are closely associated with metabolic diseases such as type 2 diabetes, insulin resistance, and atherosclerosis, and it has become a key biomarker for clinical assessment of cardiovascular and metabolic risks. Currently, chemiluminescent immunoassay (CLIA) has become the mainstream technology for adiponectin detection due to its high sensitivity and automation compatibility.

[0003] Existing chemiluminescence detection methods for adiponectin primarily employ magnetic microparticles as a solid-phase carrier, capturing the target antigen through a double-antibody sandwich mechanism and relying on chemiluminescent labels to generate a detectable signal. However, current technologies still face the following challenges: First, some commonly used chemiluminescence systems are limited by the luminescence efficiency of the label itself, resulting in insufficient detection sensitivity and difficulty in accurately measuring physiologically relevant concentrations as low as 0.05 mg / L, thus limiting their application in early disease screening.

[0004] Secondly, in the detection of high-concentration samples, some methods are prone to the "hook effect," which means that the signal decreases due to excessive antigen, resulting in severely low results and affecting clinical judgment.

[0005] Furthermore, although enzyme-labeled amplification systems, such as horseradish peroxidase (HRP), can theoretically enhance signal intensity, when combined with luminol-enhanced chemiluminescence systems and applied to magnetic microparticle platforms, the background signal often increases significantly and the signal-to-noise ratio deteriorates due to non-specific adsorption or incomplete washing, making it difficult to meet the precision requirements of clinical testing.

[0006] Furthermore, the signal acquisition programs of mainstream chemiluminescence analyzers are mostly optimized for transient flash luminescence systems. However, the luminescence kinetics of luminol-type glow discharge systems are different. If the reaction conditions and reading timing are not adjusted accordingly, it may lead to incomplete signal capture or poor repeatability. Summary of the Invention

[0007] This invention aims to at least address the technical problems existing in the prior art. To this end, this invention proposes an adiponectin detection method based on chemiluminescence, which features high sensitivity, low background, wide linear range, and compatibility with mainstream chemiluminescence analyzers, thus overcoming the technical obstacles to the application of the HRP-luminol system in heterogeneous magnetic microparticle platforms.

[0008] A method for detecting adiponectin based on chemiluminescence according to some embodiments of the present invention includes the following steps: S1. Provide magnetic microspheres with an MQ silicone resin layer on their surface, wherein the magnetic microspheres have Fe3O4 as the core, the molar ratio of monofunctional siloxane units M to tetrafunctional siloxane units Q in the MQ silicone resin is 1:2 to 1:5, and the average particle size of the microspheres is 0.8 to 1.5 μm. S2. The anti-adiponectin capture antibody is immobilized on the surface of the magnetic microspheres to form immunomagnetic beads; S3. Mix the human serum or plasma sample to be tested with the immunomagnetic beads and incubate to allow adiponectin to bind with the capture antibody; S4. After magnetic separation and washing, add horseradish peroxidase (HRP)-labeled anti-adiponectin detection antibody and incubate to form a sandwich immune complex. S5. After magnetic separation and washing again, a chemiluminescent substrate solution is added, wherein the chemiluminescent substrate solution contains luminol, an enhancer and hydrogen peroxide, and the enhancer is a phenolic compound; S6. Detect the intensity of the generated chemiluminescence signal and calculate the concentration of adiponectin in the sample to be tested based on the standard curve.

[0009] A method for detecting adiponectin based on chemiluminescence according to some embodiments of the present invention has at least the following beneficial effects: This invention employs an HRP-enzyme-catalyzed cascade amplification and an optimized luminol enhancement system, achieving a lower limit of detection (LoD) superior to conventional methods. This meets the needs of early metabolic risk screening for ultra-low concentration detection, with a linear detection range covering 0.01 mg / L to 100 mg / L. It effectively avoids the "hook effect" commonly found in high-concentration samples, ensuring accurate results. It is particularly suitable for high-value sample detection in obese patients or those with severe metabolic disorders. By rationally selecting phenolic enhancers and combining them with a thorough magnetic bead washing step, the non-specific background signal is controlled below 200 RLU, significantly improving the signal-to-noise ratio and ensuring the reliability of low-value sample detection.

[0010] According to some embodiments of the present invention, a method for detecting adiponectin based on chemiluminescence is provided, wherein a buffer solution containing a nonionic surfactant is used for washing in both the washing step S4 and the washing step S5.

[0011] According to some embodiments of the present invention, a method for detecting adiponectin based on chemiluminescence is provided, wherein the nonionic surfactant is Tween-20, and its concentration in the washing buffer is 0.15 to 0.30 wt%.

[0012] According to some embodiments of the present invention, an adiponectin detection method based on chemiluminescence is provided, wherein the enhancer is selected from p-iodophenol, 4-hydroxyphenylacetic acid, or vanillic acid.

[0013] According to some embodiments of the present invention, an adiponectin detection method based on chemiluminescence is provided, wherein the enhancer is p-iodophenol, and the concentration of p-iodophenol in the chemiluminescent substrate solution is 0.15–0.25 mM.

[0014] According to some embodiments of the present invention, in an adiponectin detection method based on chemiluminescence, the incubation temperature in steps S3 and S4 is 37±1℃, the incubation time in S3 is 10-20 minutes, and the incubation time in S4 is 8-15 minutes.

[0015] According to some embodiments of the present invention, a method for detecting adiponectin based on chemiluminescence is provided, in step S6, the intensity of the chemiluminescence signal is detected within 0.3 to 1.0 seconds after the chemiluminescence substrate solution is added.

[0016] According to some embodiments of the present invention, a method for detecting adiponectin based on chemiluminescence is provided, wherein the concentration range of the standard curve covers 0.01 mg / L to 100 mg / L.

[0017] 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

[0018] 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.

[0019] 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.

[0020] 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.

[0021] Example 1: Preparation of immunomagnetic beads: Fe3O4 microspheres (average particle size 1.2 μm) were dispersed in anhydrous ethanol, and MQ silicone resin precursor (M:Q molar ratio = 1:3) was added. The mixture was hydrolyzed and condensed at 60 °C for 6 h, and centrifuged and washed to obtain MQ-coated magnetic beads. The beads were then treated with 3-aminopropyltriethoxysilane (APTES) to introduce amino groups, and then reacted with glutaraldehyde to generate aldehyde groups. Streptavidin (5 mg / mL) was dissolved in 0.1 M NaHCO3 (pH 8.5), mixed with the magnetic beads, activated by EDC / NHS, and coupled at 4 °C for 12 h. After blocking, biotinylated anti-adiponectin capture antibody (CloneADI-1, 10 μg / mg magnetic beads) was added, and the mixture was incubated at room temperature for 1 h to obtain immunomagnetic beads.

[0022] HRP-labeled antibody: Dissolve 10 mg of HRP in 1 mL of 0.1 M NaIO4, react at 4 °C in the dark for 30 min, add an equal volume of ethylene glycol to terminate the reaction, and after dialysis, mix with anti-adiponectin detection antibody (CloneADI-2) at a molar ratio of 4:1, couple at 4 °C for 18 h, and purify with Sephadex G-25 to obtain HRP-ADI-2.

[0023] Washing condition screening: Comparison of Tween-20 concentrations (0.05%, 0.15%, 0.30%, 0.50%) revealed that the lowest background (RLU < 200) was observed at 0.15–0.30% without affecting specific binding. Enhancer screening: The signal-to-noise ratio (SNR) of p-iodophenol, 4-hydroxyphenylacetic acid (4-HPA), and vanillic acid in the range of 0.1–0.5 mM was tested. The results showed that the SNR was highest at 0.15–0.25 mM p-iodophenol. Luminescence dynamics test: The luminescence curve was detected using the new industry Maglumi2000Plus. The signal peaked at 0.4 seconds and decayed to 50% after 1 second. The optimal reading window was determined to be 0.3–1.0 seconds.

[0024] The detection process of this invention: Add 50 μL of the human serum sample to be tested and 50 μL of the immunomagnetic bead suspension to the reaction tube, and incubate at 37°C with shaking for 15 min. Magnetic separation was performed, the supernatant was discarded, and 200 μL of 50 mM Tris-HCl buffer (pH 7.4) containing 0.20 wt% Tween-20 was added. After mixing, magnetic separation was performed again, and the washing was repeated 3 times. Add 50 μL of HRP-labeled detection antibody solution and incubate at 37°C with shaking for 10 min; Perform magnetic separation again, and wash three times as above; Add 200 μL of chemiluminescent substrate solution containing 0.5 mM luminol, 0.20 mM p-iodophenol, and 0.05 wt% H2O2, dissolved in 80 mM Tris-HCl buffer (pH 8.5). 0.5 seconds after substrate addition, relative light units (RLU) were measured using a chemiluminescence analyzer, and the adiponectin concentration in the sample was calculated based on a standard curve fitted by a four-parameter logistic equation.

[0025] Comparative Example 1: The same magnetic beads and antibody pair were used, but the detection antibody was labeled acridinium ester. The trigger solution was 0.1M NaOH + 0.3% H2O2. The rest of the operation followed the conventional CLIA procedure for magnetic microparticles.

[0026] The testing process is as follows: Add 50 μL of the human serum sample to be tested and 50 μL of the immunomagnetic bead suspension to the reaction tube, and incubate at 37°C with shaking for 15 min. Magnetic separation was performed, the supernatant was discarded, and the sample was washed three times with PBS (pH 7.4) containing 0.1 wt% Tween-20. Add 50 μL of acridinium ester-detection antibody and incubate at 37°C with shaking for 10 min; Perform magnetic separation again, and wash three times as above; Add 200 μL of triggering solution (0.1 M NaOH + 0.3 wt% H2O2); Relative light units (RLU) were measured using a chemiluminescence analyzer 0.3 seconds after substrate addition.

[0027] Comparative Example 2: Except for the following conditions, the rest of the operation is the same as in Example 2: The washing buffer is 50 mM Tris-HCl (pH 7.4) and does not contain Tween-20; The chemiluminescent substrate solution contains 0.5 mM luminol, 0.5 mM p-iodophenol, and 0.05 wt% H2O2.

[0028] I. Linear Range Test Adiponectin enterprise reference samples with different concentrations of 0.01, 0.10, 1.00, 10.00, 50.00, and 100.00 mg / L were used for testing. Each enterprise reference sample was tested three times and the average value of each concentration was calculated. The linear equation y = a + bx (y: the test value of the reagent, x: the actual value of the enterprise reference sample) and the correlation coefficient r were calculated according to formulas (1), (2), and (3).

[0029] In the formula: The slope of the b-regression line; |a| - the absolute value of the intercept of the regression line; r - correlation coefficient; Xi - Actual value of the enterprise reference product; The mean of three repeated measurements of the Yi-enterprise reference product; i-1, 2, 3, ..., n; n - Number of samples measured.

[0030] The results are as follows: Example 2: Linear equation y = 0.982x + 0.008, r = 0.9995, linear range 0.01–100.00 mg / L; Comparative Example 1: Linear equation y = 1.02x – 0.15, r = 0.998, linear range 0.05 – 50.00 mg / L; Comparative Example 2: Signal saturation at >80 mg / L, linear range 0.05–80.00 mg / L (r=0.996).

[0031] II. Detection Limit Test According to CLSI EP17-A2 guidelines, perform 60 tests on blank samples (without adiponectin matrix): Example 2: Mean=120RLU, SD=48RLU, LoD=120+1.645×48≈199RLU (corresponding concentration 0.009mg / L). Comparative Example 1: LoD = 0.05 mg / L; Comparative Example 2: The background was as high as 1250 RLU, and the signal of the 0.01 mg / L sample was overwhelmed and could not be accurately quantified.

[0032] III. Repeatability Testing Under repeatability conditions, the 0.05 mg / L and 80.0 mg / L reference samples were measured 10 times each, and the mean (X) and standard deviation (S) of the measured values ​​were calculated respectively. The coefficient of variation (CV) was calculated according to formula (4).

[0033] CV = S / X 100%............................................ (4); The results are as follows: IV. Accuracy Test for High-Concentration Samples The reference standard was tested at 60.0 mg / L. The reference standard was tested three times. The mean of the sample test results was calculated, and the relative deviation (Bias%) was calculated according to formula (5).

[0034] Bias%=(XX) / X 100% ............................................ (5); X - Mean value of sample measurement results; X-Determine the theoretical value of the sample.

[0035] The results are as follows: Example 2: Measured concentration was 59.2 mg / L, with a deviation of -1.3%; Comparative Example 1: Measured at 49.4 mg / L, with a deviation of -17.7% (obvious hook effect). Comparative Example 2: 57.1 mg / L was measured, with a deviation of -4.8%.

[0036] V. Low-value sample detection capability test Testing of 0.03 mg / L clinical samples (n=10): Example 2: All were detected, with a mean value of 0.031 mg / L; Comparative Example 1: 7 cases reported "<0.05 mg / L" (not detected); Comparative Example 2: Due to the high background, the results fluctuated greatly (0.01–0.08 mg / L) and were unreliable.

[0037] Based on the above results, the following conclusions can be drawn: This method significantly improves sensitivity: by employing HRP-catalyzed cascade amplification and an optimized luminol enhancement system, the lower limit of detection (LoD) can reach 0.01 mg / L, which is far superior to conventional methods (usually ≥0.05 mg / L), meeting the needs of early metabolic risk screening for ultra-low concentration detection.

[0038] This method has a wide linear range: the linear detection range covers 0.01 mg / L to 100 mg / L, effectively avoiding the "hook effect" common in high-concentration samples, ensuring the accuracy of results, and is especially suitable for high-value sample detection in obese or severely metabolic patients.

[0039] This method significantly reduces the background signal: by rationally selecting phenolic enhancers (such as p-iodophenol) and combining them with a thorough magnetic bead washing step, the nonspecific background signal is controlled below 200 RLU, the signal-to-noise ratio (S / N) is greatly improved, and the reliability of low-value sample detection is guaranteed.

[0040] This method has strong anti-interference ability: thanks to the efficient separation ability of the magnetic microparticle solid-phase carrier, this method has excellent tolerance to common clinical interfering substances (such as heterophilic antibody HAMA, high lipid, hemolyzed or jaundiced samples), with intra-batch / inter-batch coefficients of variation (CV) of less than 5%.

[0041] This method is compatible with mainstream instrument platforms: by adjusting the luminescence reaction kinetics (such as substrate pH and reading time window), the signal peak appears within 0.3 to 1.0 seconds after substrate addition, perfectly adapting to the detection programs of existing mainstream chemiluminescence analyzers (such as New Industries, Mindray, etc.), and achieving high-precision detection without hardware modification.

[0042] 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 method for detecting adiponectin based on chemiluminescence, characterized in that, Includes the following steps: S1. Provide magnetic microspheres with an MQ silicone resin layer on their surface, wherein the magnetic microspheres have Fe3O4 as the core, the molar ratio of monofunctional siloxane units M to tetrafunctional siloxane units Q in the MQ silicone resin is 1:2 to 1:5, and the average particle size of the microspheres is 0.8 to 1.5 μm. S2. Immobilize the anti-adiponectin capture antibody on the surface of the magnetic microspheres to form immunomagnetic beads; S3. Mix the human serum or plasma sample to be tested with the immunomagnetic beads and incubate to allow adiponectin to bind with the capture antibody; S4. After magnetic separation and washing, add horseradish peroxidase (HRP)-labeled anti-adiponectin detection antibody and incubate to form a sandwich immune complex. S5. After magnetic separation and washing again, a chemiluminescent substrate solution is added, wherein the chemiluminescent substrate solution contains luminol, an enhancer and hydrogen peroxide, and the enhancer is a phenolic compound; S6. Detect the intensity of the generated chemiluminescence signal and calculate the concentration of adiponectin in the sample to be tested based on the standard curve.

2. The adiponectin detection method based on chemiluminescence according to claim 1, characterized in that: Both the washing steps in S4 and S5 use a buffer solution containing a nonionic surfactant for washing.

3. The adiponectin detection method based on chemiluminescence according to claim 2, characterized in that: The nonionic surfactant is Tween-20, and its concentration in the washing buffer is 0.15–0.30 wt%.

4. The adiponectin detection method based on chemiluminescence according to claim 1, characterized in that: The reinforcing agent is selected from p-iodophenol, 4-hydroxyphenylacetic acid, or vanillic acid.

5. The adiponectin detection method based on chemiluminescence according to claim 4, characterized in that: The enhancer is p-iodophenol, and the concentration of p-iodophenol in the chemiluminescent substrate solution is 0.15–0.25 mM.

6. The adiponectin detection method based on chemiluminescence according to claim 1, characterized in that: The incubation temperature in steps S3 and S4 is 37±1℃, the incubation time in S3 is 10-20 minutes, and the incubation time in S4 is 8-15 minutes.

7. The adiponectin detection method based on chemiluminescence according to claim 1, characterized in that: In step S6, the intensity of the chemiluminescent signal is detected within 0.3 to 1.0 seconds after the chemiluminescent substrate solution is added.

8. The adiponectin detection method based on chemiluminescence according to claim 1, characterized in that: The concentration range of the standard curve covers 0.01 mg / L to 100 mg / L.

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