Detection method of abnormal sugar chain glycoprotein

Abstract

The invention relates to the technical field of biological detection, and discloses a method for detecting abnormal sugar chain glycoprotein, which comprises the following steps: by taking lectin modified magnetic nanoparticles as a capture probe, constructing a double-recognition system by combining a fluorescently-labeled sugar chain recognition antibody; the accurate quantification of the abnormal sugar chain glycoprotein is realized through magnetic separation and enrichment, fluorescence signal amplification and fluorescence spectrophotometry detection. The method comprises the steps of capture probe preparation, sample pretreatment, targeted capture and magnetic separation, fluorescence labeling, signal detection and result analysis. The detection limit can reach 0.05 ng / mL, the recovery rate is high, normal sugar chain glycoprotein and abnormal sugar chain glycoprotein can be effectively distinguished, and the method has the advantages of being high in specificity, high in sensitivity, easy and convenient to operate, rapid in detection and the like, is suitable for clinical disease screening, prognosis monitoring and batch detection of abnormal sugar chain glycoprotein in biological samples, and has wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a method for detecting abnormal glycan glycoproteins, which is particularly suitable for the accurate detection and batch screening of abnormal glycan glycoproteins related to clinical diseases. Background Technology

[0002] Glycan glycoproteins are an important class of biomolecules in living organisms, widely involved in various physiological processes such as cell recognition, signal transduction, and immune responses. As a crucial component of glycoproteins, abnormal modifications to the glycan structure (such as alterations in glycosylation sites, abnormal glycan length, and increases or decreases in glycan types) are often closely related to the occurrence and development of various diseases, including cancer, autoimmune diseases, and cardiovascular diseases. Therefore, abnormal glycan glycoproteins are considered important disease biomarkers, and their accurate detection has significant clinical implications for early disease screening, diagnosis, prognostic monitoring, and treatment efficacy evaluation.

[0003] Currently, the main methods for detecting abnormal glycan glycoproteins include high-performance liquid chromatography (HPLC), mass spectrometry (MS), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. HPLC and MS offer advantages such as high resolution and the ability to analyze glycan structures, but they suffer from drawbacks including expensive equipment, complex operation, long detection cycles, and cumbersome sample pretreatment, making them unsuitable for large-scale clinical testing. ELISA is simple to operate and low-cost, but its sensitivity is limited, with a detection limit typically around 1 ng / mL, and it is easily affected by sample matrix interference, requiring further improvement in specificity. Immunohistochemistry is suitable for qualitative detection of tissue samples but cannot achieve quantitative analysis, and its detection efficiency is relatively low.

[0004] Magnetic nanoparticles, due to their large specific surface area, strong magnetic responsiveness, and ease of surface modification, are widely used in the field of bioseparation and detection. Lectins, as proteins that specifically recognize glycan structures, can bind specifically to the glycans of glycoproteins; while glycan recognition antibodies can further specifically recognize the specific structures of abnormal glycans. Combining these two can construct a dual-recognition system, improving the specificity and sensitivity of detection. However, there is currently no mature method for the efficient detection of abnormal glycan glycoproteins by combining lectin-modified magnetic nanoparticles with fluorescently labeled antibodies. Optimizing probe preparation processes, controlling reaction conditions, reducing background signals, and improving detection performance are pressing technical problems that need to be solved in this field.

[0005] Therefore, developing a method for detecting abnormal glycan glycoproteins that is highly specific, sensitive, easy to operate, and cost-effective is of great significance for promoting the development of clinical disease diagnostic technology. Summary of the Invention

[0006] Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for detecting abnormal glycan glycoproteins. This method constructs a dual recognition system consisting of a lectin-modified magnetic nanoparticle capture probe and a fluorescently labeled glycan recognition antibody detection probe. By combining magnetic separation enrichment and fluorescence signal amplification techniques, it achieves accurate quantitative detection of abnormal glycan glycoproteins, aiming to solve the problems of poor detection performance and difficulty in meeting the needs of large-scale clinical testing in existing technologies.

[0007] Technical solution To achieve the above objectives, the present invention provides the following technical solution: a method for detecting abnormal glycan glycoproteins, which uses lectin-modified magnetic nanoparticles as capture probes and fluorescently labeled glycan recognition antibodies as detection probes to construct a dual-recognition detection system, specifically including the following steps: (1) Preparation of capture probe: Magnetic nanoparticles were dispersed in buffer solution, activated by an activator, and then lectin was added for coupling reaction. After the reaction was completed, magnetic separation and washing were performed to obtain lectin-modified magnetic nanoparticle capture probes. (2) Sample pretreatment: Take the biological sample to be tested, add protein extraction solution to extract total protein, and obtain sample pretreatment solution after centrifugation and filtration; (3) Targeted capture and magnetic separation: Add the capture probe prepared in step (1) to the sample processing solution, incubate at 25-37℃ for 30-60 min, perform magnetic separation by external magnetic field, remove the supernatant, and wash the precipitate 2-3 times with washing buffer; (4) Fluorescent labeling: Add fluorescently labeled glycan recognition antibody to the washed precipitate, incubate at 25-37℃ in the dark for 20-40 min, then magnetically separate and wash the precipitate 2-3 times to remove unbound free antibody; (5) Signal detection: Eluent was added to the final precipitate, and after ultrasonic dispersion, the fluorescence signal intensity was detected using a fluorescence spectrophotometer; (6) Results analysis: Based on the pre-drawn standard curve, the intensity of the detected fluorescence signal is converted into the concentration of abnormal glycan glycoprotein in the sample to be tested.

[0008] Further, in step (1), the magnetic nanoparticles are Fe3O4 magnetic nanoparticles with a particle size of 50-200 nm and surface modified with amino or carboxyl groups; the lectin is selected from one or more of canavalia lectin A, malt lectin, and pea lectin, and the coupling mass ratio of the lectin to the magnetic nanoparticles is 1:5-1:20.

[0009] Further, in step (1), the activator is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, the molar ratio of EDC to NHS is 1:1-2:1, the activation time is 20-40 min, the coupling reaction temperature is 25-37℃, and the coupling reaction time is 2-4 h.

[0010] Further, in step (2), the biological sample is selected from one of serum, plasma, tissue homogenate, and urine; the protein extraction solution is a buffer solution containing 1-3% sodium dodecyl sulfate and 50-100 mmol / L Tris-HCl (pH 7.4), the extraction temperature is 4℃, the extraction time is 30-60 min, the centrifugation speed is 8000-12000 r / min, and the centrifugation time is 10-20 min.

[0011] Further, in step (4), the fluorescently labeled glycan recognition antibody is fluorescein isothiocyanate, rhodamine B or carbon quantum dot-labeled anti-abnormal glycan specific antibody, the antibody concentration is 0.1-1 μg / mL, the incubation temperature is 30-37℃ in the dark, and the incubation time is 25-35 min.

[0012] Furthermore, the buffer solutions involved in steps (1), (3), and (4) are all phosphate buffer solutions with a pH of 7.2-7.6, containing 0.05-0.1% Tween-20 and 0.5-1% bovine serum albumin; the elution buffer in step (5) is a buffer solution containing 0.5-1% sodium dodecyl sulfate and 50 mmol / L Tris-HCl (pH 8.0), and the sonication time is 5-10 min.

[0013] Further, in step (5), the excitation wavelength of the fluorescence spectrophotometer is 480-520nm, the emission wavelength is 530-580nm, and the detection slit width is 5-10nm; in step (6), the standard curve is plotted after detecting the fluorescence signal intensity using a series of abnormal glycan glycoprotein standards of known concentrations according to the methods in steps (3)-(5), and the linear range of the standard curve is 0.1-100ng / mL, with a correlation coefficient R2≥0.995.

[0014] Furthermore, the detection limit of this method is ≤0.05 ng / mL, the sample spike recovery rate is 92%-106%, the relative standard deviation (RSD) is ≤3.5%, and the cross-reactivity with normal glycan glycoproteins is ≤5%.

[0015] Beneficial technical effects This invention constructs a dual-recognition system of lectin and glycan recognition antibody. The lectin first specifically captures glycoproteins, and the glycan recognition antibody then specifically recognizes abnormal glycan structures. This dual screening effectively avoids cross-reaction with normal glycan glycoproteins, with a cross-reaction rate ≤5%, significantly improving detection specificity and reducing false positive rate. Combining the efficient enrichment effect of magnetic nanoparticles and the signal amplification technology of fluorescent labeling, this invention achieves a detection limit of 0.05 ng / mL, enabling accurate detection of low concentrations of abnormal glycan glycoproteins and meeting the needs of early disease screening. The magnetic separation step requires no complex equipment, is simple and quick to operate, effectively removes sample matrix interference, and shortens sample pretreatment time. Compared with traditional methods, it significantly improves detection efficiency and is suitable for batch sample testing. Optimized probe preparation process and reaction conditions ensure repeatability of detection results, with a small relative standard deviation, high recovery rate, and high detection accuracy, providing reliable data support for clinical diagnosis. The method for detecting abnormal glycan glycoproteins of the present invention can be used to detect abnormal glycan glycoproteins in various biological samples such as serum, plasma, tissue homogenate, and urine. It is suitable for early screening, prognostic monitoring, and treatment effect evaluation of various diseases such as cancer and autoimmune diseases, and has broad clinical application prospects and industrialization value. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] 1. Preparation of capture probe (1) Pretreatment of magnetic nanoparticles: Select Fe3O4 magnetic nanoparticles with a particle size of 50-200nm, whose surface is modified with amino or carboxyl groups, disperse them in phosphate buffer solution at pH 7.4, and ultrasonically disperse for 10-15min to obtain a magnetic nanoparticle dispersion with a concentration of 1-5mg / mL. (2) Activation of magnetic nanoparticles: Add activator EDC and NHS to the above dispersion. The molar ratio of EDC to NHS is 1:1-2:1 and the final concentration of activator is 50-100 mmol / L. Stir and activate at 25°C for 20-40 min to activate the carboxyl or amino groups on the surface of the magnetic nanoparticles. (3) Lectin coupling: Add lectin to the activated magnetic nanoparticle dispersion. The coupling mass ratio of lectin to magnetic nanoparticles is 1:5-1:20. The coupling is carried out at a constant temperature of 25-37℃ for 2-4 hours. The lectin is selected from one or more of canavalia brevicornu lectin A (ConA), malt lectin (WGA), and pea lectin (PSA). The corresponding lectin can be selected according to the structural characteristics of the target abnormal sugar chain. (4) Purification and washing: After the coupling reaction is completed, magnetic separation is performed by an external magnetic field to remove the supernatant. The precipitate is washed 2-3 times with PBS buffer containing 0.05% Tween-20 and 1% BSA. Magnetic separation is performed after each wash. Finally, the precipitate is dispersed in PBS buffer to obtain the lectin-modified magnetic nanoparticle capture probe, which is stored at 4°C for later use.

[0018] 2. Sample pretreatment (1) Sample acquisition: Take biological samples to be tested, including serum, plasma, tissue homogenate, urine, etc. Serum or plasma samples need to be centrifuged at 2-8℃ after collection to remove blood cells; tissue homogenate samples need to be ground with liquid nitrogen before adding protein extraction solution. (2) Protein extraction: Add protein extraction solution to the sample to be tested. The volume ratio of sample to extraction solution is 1:5-1:10. The protein extraction solution is a buffer solution containing 1-3% SDS and 50-100 mmol / L Tris-HCl (pH 7.4). Stir and extract at 4°C for 30-60 min. During the extraction, use ultrasound to assist extraction 1-2 times, each time for 5 min. (3) Purification and filtration: Centrifuge the extracted sample at 8000-12000r / min for 10-20min, take the supernatant, filter it through a 0.22μm filter membrane to remove impurities and macromolecular aggregates, and obtain the sample processing solution, which is then stored at 4℃ for later use.

[0019] 3. Targeted capture and magnetic separation Add the capture probe prepared in step 1 to the sample processing solution. The amount of capture probe added is 50-100 μL of capture probe with a concentration of 1 mg / mL per 1 mL of sample processing solution. Incubate at 25-37℃ for 30-60 min, gently shaking once every 10 min during the incubation period to ensure that the capture probe binds specifically and fully to the abnormal glycan glycoprotein in the sample. After incubation, place the reaction system in a magnetic field and let it stand for 5-10 min for magnetic separation to remove the supernatant (containing unbound contaminants). Wash the precipitate 2-3 times with PBS buffer containing 0.05% Tween-20 and 1% BSA, and perform magnetic separation after each wash to remove residual contaminants and free components.

[0020] 4. Fluorescent labeling Add fluorescently labeled glycan recognition antibody to the washed precipitate at a final concentration of 0.1-1 μg / mL. The fluorescently labeled glycan recognition antibody is a FITC, Rhodamine B, or carbon quantum dot-labeled antibody specifically targeting abnormal glycans. This antibody can only bind to the specific abnormal glycan structure of abnormal glycan glycoproteins and does not bind to normal glycan glycoproteins. Incubate at 25-37°C in the dark for 20-40 min, gently shaking 2-3 times during the incubation period to ensure that the antibody fully binds to the captured abnormal glycan glycoproteins. After incubation, perform magnetic separation again, remove the supernatant, and wash the precipitate 2-3 times with PBS buffer to completely remove unbound free fluorescently labeled antibody and reduce background signal.

[0021] 5. Signal Detection Add 50-100 μL of elution buffer to the final precipitate after washing. The elution buffer is a buffer solution containing 0.5-1% SDS and 50 mmol / L Tris-HCl (pH 8.0). Sonicate the solution for 5-10 min to fully elute the abnormal glycan glycoprotein-fluorescent antibody complex bound to the capture probe into the elution buffer. Transfer the elution buffer to a fluorescence detection cuvette and detect the fluorescence signal intensity using a fluorescence spectrophotometer. Set the excitation wavelength to 480-520 nm, the emission wavelength to 530-580 nm, and the detection slit width to 5-10 nm. Record the fluorescence intensity value.

[0022] 6. Results Analysis (1) Standard curve plotting: Prepare a series of abnormal glycan glycoprotein standards with known concentrations (0.1 ng / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL). Detect the fluorescence signal intensity of each standard according to steps 3-5. Plot a standard curve with the standard concentration as the abscissa and the corresponding fluorescence signal intensity as the ordinate. Use linear regression analysis to obtain the regression equation, and require the correlation coefficient R0 to be... 2 ≥0.995; (2) Sample quantification: Substitute the fluorescence signal intensity of the sample to be tested into the regression equation of the standard curve to calculate the concentration of abnormal glycan glycoprotein in the sample to be tested; if the sample detection signal intensity exceeds the linear range of the standard curve, the sample processing solution needs to be appropriately diluted and retested.

[0023] Key process parameter optimization basis (1) Selection of lectin and coupling ratio of capture probe: Different lectins have different specificities for recognizing glycans. Selecting the corresponding lectin in combination with the structural characteristics of the abnormal glycan of the target can improve the specificity of capture. The coupling mass ratio of lectin to magnetic nanoparticles is controlled at 1:5-1:20. If the ratio is too high, it will cause excessive aggregation of lectins on the surface of magnetic nanoparticles, affecting the recognition activity. If the ratio is too low, the capture efficiency is insufficient and the sensitivity decreases. (2) Incubation temperature and time: The incubation temperature for targeted capture and fluorescent labeling is controlled at 25-37℃. This temperature range can ensure the spatial structure stability of proteins (lectins, antibodies, glycoproteins) and avoid protein denaturation due to excessively high temperature or decreased binding efficiency due to excessively low temperature. The incubation time is optimized according to the rate of binding reaction to ensure sufficient binding while shortening the detection cycle. (3) Optimization of buffer composition: Adding 0.05-0.1% Tween-20 to the buffer can reduce non-specific adsorption, and adding 0.5-1% BSA can block the non-specific binding sites on the surface of magnetic nanoparticles and glycoproteins, further reducing background signal and improving detection specificity; (4) Selection of fluorescent labeling antibodies: Using antibodies that specifically recognize abnormal glycan structures can avoid cross-reaction with normal glycan glycoproteins. Combined with highly efficient fluorescent labels such as fluorescein, rhodamine B or carbon quantum dots, signal amplification can be achieved and detection sensitivity can be improved.

[0024] EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS: N-hydroxysuccinimide; FITC: fluorescein isothiocyanate; PBS: phosphate buffer; BSA: bovine serum albumin; RSD: relative standard deviation.

[0025] Fluorescent label: Thermo Fisher Scientific. Magnetic nanoparticles, lectins, antibodies, datura stramonium lectin (DSA), peanut lectin (PNA), lentil lectin (LCA), wheat germ lectin (WGA), E-type red kidney bean lectin (E-PHA), and L-type red kidney bean lectin (L-PHA) were all purchased from Vector Laboratories.

[0026] Example 1 Preparation of the capture probe: 10 mg of carboxyl-modified Fe3O4 magnetic nanoparticles with a particle size of 100 nm were dispersed in 10 mL of PBS buffer at pH 7.4 and ultrasonically dispersed for 10 min; 0.5 mmol each of EDC and NHS were added and activated by stirring at 25 °C for 30 min; 1 mg of canavagin A was added and coupled by stirring at 37 °C for 3 h; after magnetic separation, the sample was washed three times with PBS buffer containing 0.05% Tween-20 and 1% BSA, and finally dispersed in 10 mL of PBS buffer to obtain a capture probe of 1 mg / mL, which was then stored at 4 °C for later use. Sample pretreatment: Take 1 mL of serum from healthy individuals and add 0.1 ng / mL of abnormal glycan glycoprotein standard to prepare a serum simulant sample; add 5 mL of protein extraction buffer (containing 2% SDS, 80 mmol / L Tris-HCl, pH 7.4) to the simulant sample, stir and extract at 4℃ for 45 min, with ultrasonic-assisted extraction once (5 min); centrifuge at 10000 r / min for 15 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the sample processing solution; Targeted capture and magnetic separation: Take 1 mL of sample processing solution, add 80 μL of capture probe, incubate at 37 °C for 45 min, shaking once every 10 min during the period; remove the supernatant by magnetic separation, and wash the precipitate 3 times with washing buffer; Fluorescent labeling: Add FITC-labeled anti-abnormal glycan glycoprotein antibody to a final concentration of 0.5 μg / mL, incubate at 37°C in the dark for 30 min; wash the precipitate three times after magnetic separation; Signal detection: Add 80 μL of elution buffer and sonicate for 8 min; use a fluorescence spectrophotometer to detect the fluorescence signal intensity at an excitation wavelength of 490 nm and an emission wavelength of 525 nm, recording the fluorescence signal intensity as 852. Results Analysis: Based on the standard curve regression equation y = 835.8x + 51.7 (R²), 2 =0.998), the concentration of abnormal glycan glycoprotein in the sample was calculated to be 0.102 ng / mL, the recovery rate was 102.0%, and the relative standard deviation was 2.1%.

[0027] Example 2 Preparation of the capture probe: 10 mg of carboxyl-modified Fe3O4 magnetic nanoparticles with a particle size of 100 nm were dispersed in 10 mL of PBS buffer at pH 7.4 and ultrasonically dispersed for 12 min; 0.6 mmol of EDC and 0.3 mmol of NHS were added and activated by stirring at 25 °C for 25 min; 0.67 mg of malt lectin was added and coupled by stirring at 30 °C for 4 h; after magnetic separation, the nanoparticles were washed 3 times and dispersed in 10 mL of PBS buffer to obtain a capture probe of 1 mg / mL. Sample pretreatment: Take 1 mL of serum from healthy individuals, add 50 ng / mL of abnormal glycan glycoprotein standard to prepare a serum simulant sample; add 6 mL of protein extraction buffer (containing 1.5% SDS, 100 mmol / L Tris-HCl, pH 7.4), stir and extract at 4℃ for 50 min, and perform ultrasonic-assisted extraction twice; centrifuge at 12000 r / min for 12 min, and filter to obtain the sample pretreatment solution; Targeted capture and magnetic separation: Take 1 mL of sample processing solution, add 100 μL of capture probe, and incubate at 30 °C for 60 min; wash 3 times with magnetic separation. Fluorescent labeling: Add rhodamine B-labeled anti-abnormal glycan glycoprotein antibody to a final concentration of 0.8 μg / mL, incubate at 30°C in the dark for 35 min; wash three times with magnetic separation. Signal detection: Add 100 μL of elution buffer and sonicate for 10 min; use a fluorescence spectrophotometer to detect the fluorescence signal intensity at an excitation wavelength of 510 nm and an emission wavelength of 550 nm. Results Analysis: Substituting the values ​​into the standard curve regression equation y = 833.1x + 27.9 (R²) 2 =0.997), the calculated concentration was 49.8 ng / mL, the recovery rate was 99.6%, and the relative standard deviation was 1.8%.

[0028] Example 3 Preparation of the capture probe: 10 mg of carboxyl-modified Fe3O4 magnetic nanoparticles with a particle size of 100 nm were dispersed in 10 mL of PBS buffer at pH 7.4 and ultrasonically dispersed for 15 min; 0.8 mmol of EDC and 0.4 mmol of NHS were added, and the mixture was stirred at 25 °C for 40 min to activate the nanoparticles; 0.83 mg of canavalialin A and 0.83 mg of malt lectin were added, and the nanoparticles were stirred at 37 °C for 2.5 h to couple the nanoparticles; the nanoparticles were magnetically separated and washed 3 times, and dispersed in 10 mL of PBS buffer to obtain a capture probe of 1 mg / mL. Sample pretreatment: Take 1 mL of serum from a patient with clinical disease, add 5 mL of protein extraction buffer (containing 3% SDS, 50 mmol / L Tris-HCl, pH 7.4), stir and extract at 4℃ for 30 min, and extract twice with ultrasonic assistance; centrifuge at 8000 r / min for 20 min, and filter to obtain the sample pretreatment solution; Targeted capture and magnetic separation: Take 1 mL of sample processing solution, add 90 μL of capture probe, and incubate at 37 °C for 50 min; wash 3 times with magnetic separation. Fluorescent labeling: Add carbon quantum dot-labeled anti-abnormal glycan glycoprotein antibody to a final concentration of 0.3 μg / mL, incubate at 37°C in the dark for 25 min; wash three times with magnetic separation. Signal detection: Add 90 μL of elution buffer and sonicate for 6 min; use a fluorescence spectrophotometer to detect the fluorescence signal intensity at an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Results Analysis: Substituting the values ​​into the standard curve regression equation y = 841.2x + 32.5 (R²) 2 =0.996), the calculated concentration of abnormal glycan glycoprotein in the sample was 36.2 ng / mL, with a relative standard deviation of 2.5%, which meets the requirements for clinical testing.

[0029] Six gradients were set up within the linear range of 0.1-100 ng / mL, and the regression equation for the detection data was supplemented:

[0030] Example 1 (FITC label): Linear regression yields the equation y = 835.8x + 51.7 (R²). 2 =0.998), slope 835.8; Example 2 (Rhodamine B label): Regression equation y = 833.1x + 27.9 (R 2 =0.997), slope 833.1; Example 3 (Carbon Quantum Dot Labeling): Regression Equation y = 841.2x + 32.5 (R²) 2 =0.996), slope 841.2.

[0031] Comparative Examples 1-2 are shown in the table below.

[0032] As shown in the table, Comparative Example 1, using a single lectin magnetic separation method, suffered from a detection concentration deviating from the actual value (58.6 ng / mL compared to the actual 50 ng / mL) due to the lack of secondary specific recognition by antibodies, resulting in a recovery rate of only 88.7%, indicating errors caused by non-specific binding. Comparative Example 2, lacking a magnetic separation step, experienced significant sample matrix interference and high background signal, leading to even greater deviations in detection results and a recovery rate of only 82.1%. Example 3, using serum samples from patients with clinical diseases, yielded stable and reliable results with a relative standard deviation of 2.5%. In contrast, Comparative Example 2, when detecting the same clinical sample, exhibited significant deviations due to severe matrix interference, with a relative standard deviation reaching 5.2%. This demonstrates that the method of the present invention has strong resistance to matrix interference and is suitable for the detection of actual biological samples.

[0033] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0034] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0035] Those skilled in the art should understand that the above descriptions are merely several specific embodiments of the present invention, and not all embodiments.

Claims

1. A method for detecting abnormal glycan glycoproteins, characterized in that, A dual-recognition detection system was constructed using lectin-modified magnetic nanoparticles as capture probes and fluorescently labeled glycan recognition antibodies as detection probes. The specific steps include: (1) Preparation of capture probe: Magnetic nanoparticles were dispersed in buffer solution, activated by an activator, and then lectin was added for coupling reaction. After the reaction was completed, magnetic separation and washing were performed to obtain lectin-modified magnetic nanoparticle capture probes. (2) Sample pretreatment: Take the biological sample to be tested, add protein extraction solution to extract total protein, and obtain sample pretreatment solution after centrifugation and filtration; (3) Targeted capture and magnetic separation: Add the capture probe prepared in step (1) to the sample processing solution, incubate at 25-37℃ for 30-60 min, perform magnetic separation by external magnetic field, remove the supernatant, and wash the precipitate 2-3 times with washing buffer; (4) Fluorescent labeling: Add fluorescently labeled glycan recognition antibody to the washed precipitate, incubate at 25-37℃ in the dark for 20-40 min, then magnetically separate and wash the precipitate 2-3 times to remove unbound free antibody; (5) Signal detection: Eluent was added to the final precipitate, and after ultrasonic dispersion, the fluorescence signal intensity was detected using a fluorescence spectrophotometer; (6) Results analysis: Based on the pre-drawn standard curve, the intensity of the detected fluorescence signal is converted into the concentration of abnormal glycan glycoprotein in the sample to be tested.

2. The method for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, In step (1), the magnetic nanoparticles are Fe3O4 magnetic nanoparticles with a particle size of 50-200 nm and surface modified with amino or carboxyl groups; the lectin is selected from one or more of canavalia lectin A, malt lectin, and pea lectin, and the coupling mass ratio of the lectin to the magnetic nanoparticles is 1:5-1:

20.

3. The method for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, In step (1), the activator is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, the molar ratio of EDC to NHS is 1:1-2:1, the activation time is 20-40 min, the coupling reaction temperature is 25-37℃, and the coupling reaction time is 2-4 h.

4. The method for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, In step (2), the biological sample is selected from one of serum, plasma, tissue homogenate, and urine; the protein extraction solution is a buffer solution containing 1-3% sodium dodecyl sulfate and 50-100 mmol / L Tris-HCl, the extraction temperature is 4℃, the extraction time is 30-60 min, the centrifugation speed is 8000-12000 r / min, and the centrifugation time is 10-20 min.

5. The method for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, In step (4), the fluorescently labeled glycan recognition antibody is fluorescein isothiocyanate, rhodamine B or carbon quantum dot-labeled anti-abnormal glycan specific antibody, the antibody concentration is 0.1-1 μg / mL, the incubation temperature is 30-37℃ in the dark, and the incubation time is 25-35 min.

6. The method for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, The buffer solutions involved in steps (1), (3), and (4) are all phosphate buffer solutions with a pH of 7.2-7.6, containing 0.05-0.1% Tween-20 and 0.5-1% bovine serum albumin; the elution buffer in step (5) is a buffer solution containing 0.5-1% sodium dodecyl sulfate and 50 mmol / L Tris-HCl, and the sonication time is 5-10 min.

7. The method for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, In the step (5), the excitation wavelength of the fluorescence spectrophotometer is 480-520 nm, the emission wavelength is 530-580 nm, and the detection slit width is 5-10 nm; in the step (6), the standard curve is drawn by a series of known concentrations of abnormal glycan glycoprotein standards, and the fluorescence signal intensity is detected according to the method of steps (3)-(5), the linear range of the standard curve is 0.1-100 ng / mL, and the correlation coefficient R 2 ≥0.

995.

8. The method for detecting abnormal glycan glycoproteins according to any one of claims 1-7, characterized in that, The detection limit of this method is ≤0.05 ng / mL, the sample spike recovery rate is 92%-106%, the relative standard deviation is ≤3.5%, and the cross-reactivity with normal glycan glycoproteins is ≤5%.

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